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Editorial Board

Editor-in-chief Assoc. Prof. G. Pascalev, MD, PhD

Deputy Editor-in-chief Prof. T. Tsvetkova, MD, PhD, DMSc

Science Editor Prof. I. Karnolski, MD, PhD, DMSc Prof. St. Kostianev, MD, PhD, DMSc

Executive Secretary V. Ouroumova

Members: Prof. V. Anastassov, MD, PhD, DMSc Prof. G. Baltadjiev, MD, PhD, DMSc Prof. B. Chilova, MD, PhD, DMSc Prof. D. Dimitrakov, MD, PhD, DMSc Prof. D. Getova-Spasova, MD, PhD, DMSc Prof. L. Grigorov, MD, PhD, DMSc Prof. I. Dimitrov, MD, PhD Prof. E. Hadjipetrova, MD, PhD, DMSc Prof. D. Iluchev, MD, PhD, DMSc Prof. N. Ivanov, PhD, DChSc Prof. I. Karnolski, MD, PhD, DMSc Prof. P. Mandulova, MD, PhD, DMSc Prof. T. Mihailov, MD, PhD Prof. N. Milchev, MD, PhD, DMSc Prof. F. Mitov, MD, PhD, DMSc

Prof. A. Nedeva, MD, PhD, DMSc Prof. N. Popivanova, MD, PhD Prof. P. Rashkov, PhD, DTSc Prof. P. Solakov, MD, PhD, DMSc Prof. P. Stavrev, MD, PhD, DMSc Prof. M. Stoycheva, MD, PhD, DMSc Prof. P. Uchikov, MD, PhD, DMSc Prof. K. Velkova, MD, PhD, DMSc Prof. M. Yaneva, MD, PhD, DMSc Prof. Z. Zahariev, MD, PhD, DMSc Assoc. Prof. G. Ivanov, MD, PhD, DMSc Assoc. Prof. M. Kukleva, MD, PhD Assoc. Prof. V. Ozanian-Sarafian, MD, PhD, DMSc Assoc. Prof. St. Vladimirov, MD, PhD Assoc. Prof. I. Yovchev, MD, PhD, DMSc

Prof. Bernard Amor, MD, PhD, DMSc (Paris, France) Prof. George Anastassov, MD, PhD, DDSc (NYC, USA) Prof. Lidia Benevolenscaya, MD (Moscow, Russia) Prof. John Christakis, MD (Thessaloniki, Greece) Prof. Patrick Dhellemmes, MD, PhD (Lille, France) Prof. Konstantinos Kouskoukis, MD (Alexandroupolis, Greece) Prof. Mikhail Mikhailovsky, MD, PhD, DMSc (Novosibirsk, Russian) Prof. Philippe Pellerin, MD, PhD (Lille, France) Prof. Yves Poumay, MD, PhD, DMSc (Namur, Belgique) Prof. Constantinos Simopoulos, MD (Alexandroupolis, Greece) Prof. Greg Skalkeas, MD, Academician (Athens, Greece) Prof. Francisco Soriano, MD, PhD (Madrid, Spain) Prof. Eberhard Wieland, MD, PhD, DMSs (Göttingen, Germany) Translated by: O. Obretenov, Ass. Prof. S. Sivkov, MD, PhD, F. Vassileva, MD, B. Vladimirov, MD, D. Dimitrova Technical Editor & Proof-reader: O. Obretenov Technical Secretary: V. Ouroumova (e-mail: [email protected]) Typeset by Medical University - Plovdiv, Centre for educational and scientific materials Printed in Bulgaria ISSN 0204-8043 Editorial

Correspondence

FOLIA MEDICA, Tel. ++359 32 / 602 224 MU, Library and Information Center, 15 A Vassil Aprilov St., 4002 Plovdiv, Bulgaria e-mail: [email protected]

XL, 1

2008

C o n t e n t s Reviews Maria G. Ganeva, Damianka P. Getova, Vesselina G. Gadjeva Adverse Drug Reactions and Reactive Oxygen Species .............................................................................5 Ljudmila G. Vladimirova-Kitova Asymmetric Dimethylarginine - Mechanisms and Targets Fortherapeutic Management ......................12

Original Articles Clinical Investigations Ljudmila G. Vladimirova-Kitova, Ivan Hr. Manukov, Ivanka Sirakova , Tania Iv. Deneva Evaluation of the Relationship between Flow-mediated Vasodilation and Some Atherogenic Risk Markers in Severe Hypercholesterolemia .........................................................................................22 Regina H. Khater, Pepa K. Atanassova, Yourii K. Anastassov Autologous Fat Transplantation in the Facial Region - Clinical and Morphological Studies of 30 Cases . .................................................................................................................................................29 Marin A. Atanassov, Marieta Iv. Konareva-Kostianeva Central Corneal Thickness Measurements in Ocular Hypertension, Primary Open Angle Glaucoma, Glaucoma Suspectsand Control Subjects ................................................................................35 Yavor P. Enchev, Rumen V. Popov, Kiril V. Romansky, Marin B. Marinov, Ventzeslav A. Bussarsky Role of Neuronavigation in the Resection of Intracranialarteriovenous Malformations . .....................40

Dental Investigations Neshka A. Manchorova, Stoyan B. Vladimirov Clinical Evaluation оf Restorations with Self-etch Adhesive аnd Nanofilled Composite in Class I аnd Class II Cavities .....................................................................................................................46

Experimental Investigations Milena N. Draganova-Filipova, Milena G. Georgieva, Ekaterina N. Peycheva, George A. Miloshev, Victoria S. Sarafian, Lyudmil P. Peychev Effects of Propolis and САРЕ on Proliferation and Apoptosis of McCoy-Plovdiv Cell Line . .........53

Case Reports Ivan D. Doichinov, Yonka А. Doichinova, Svetlozar S. Spasov, Nikolai D. Marinov Suicide by Unusual Manner of Hanging. Case Report ...........................................................................60 Ali Mojallal, Christo Shipkov, Fabienne Braye Breast Reconstruction in Poland Anomaly with Endoscopically-assisted Latissimus Dorsi Muscle Flap and Autologous Fat Tissue Transfer: a Case Report and Review of the Literature ....63

In Memoriam

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С ОД Е РЖ А Н И Е Îáçîð М. Ганева, Д. Гетова, В. Гаджева Нежелательные лекарственные реакции и реактивные кислородные виды................................................11 Л. Владимирова-Китова Асимметрический диметиларгинин - механизмы и таргеты для терапевтического влияния...............20

Îðèãèíàëüíûe ñòàòüè Êëèíè÷åñêèå èññëåäîâàíèÿ Л. Владимирова, И. Мануков, И. Сиракова , Т. Денева Оценка взаимосвязи между поток-индуцированной вазодилатацией и маркерами атерогенного риска при выраженной гиперхолестеролемии.............................................................................28 Р. Хатр, П. Атанасова, Ю. Анастасов Аутологическая трансплантация жировой ткани в области лица - клинико-морфологические исследования 30 случаев.......................................................................................................................................................34 М. Атанасов, М. Конарева-Костянева Центральная толщина роговицы при глазной гипертензии, первичной открытоугольной глаукоме, глаукоме под сомнением и у лиц в качестве контрольной группы...............................................39 Я. Енчев, Р. Попов, К. Роминский, М. Маринов,В. Бусарский Место нейронавигации при резекции интракраниальных артериовенозных мальформаций............45

Ñòîìàòîëîãè÷åñêèå èññëåäîâàíèÿ Н. Манчорова, С. Владимиров, З. Доненчева, И. Драшкович, С. Манолов, В. Неделчева Клиническая оценка обтураций из self-etch adhesive и нанофильного композита при I и II классах кавитетов.............................................................................................................................................................51

Ýêñïåðèìåíòàëüíыå èññëåäîâàíèÿ М. Драганова-Филипова, М. Георгиева, Е. Пейчева, Г. Милошев, В. Сарафян, Л. Пейчев Эффекты прополиса и САРЕ на пролиферацию и апоптоз клеточной линии МсСоу-Plovdiv...............................................................................................................................................................................59

Êàçóèñòèêà И. Дойчинов, Й. Дойчинова, Св. Спасов, Н. Маринов Самоубийство через повешение необычайным способом. Сообщение об одном случае.....................62 A. Mojallal, Хр. Шипков, F. Braye Реконструкция грудной железы при синдроме Poland с помощью эндоскопически ассистированного мышечного лоскута (ламбо) от latissimus dorsi и аутотрансплантации жировой ткани - клинический случай и обзор литературы.......................................................................................................69

In Мemoriam ....................................................................................................................................................................70

Folia Medica, XL, 1/2008

REVIEWS ADVERSE DRUG REACTIONS AND REACTIVE OXYGEN SPECIES Maria G. Ganeva, Damianka P. Getova1, Vesselina G. Gadjeva2

Section of General and Clinical Pharmacology, Department of Physiology, Pathophysiology and Pharmacology, Thracian University, Stara Zagora, 1Department of Pharmacology and Clinical Pharmacology, Medical University, Plovdiv, 2Department of Chemistry and Biochemistry, Faculty of Medicine, Thracian University, Stara Zagora, Bulgaria ABSTRACT Adverse drug reactions represent a major source of morbidity in hospitalized- and outpatients. Adverse drug reactions may affect the function of any organ or system. Due to their clinical nonspecificity adverse drug reactions are often difficult to be recognized. The clinical manifestation, the type and severity of adverse drug reactions vary a great deal depending on patient- and drug-related factors. In recent years growing evidence has emerged implicating involvement of free radicals and reactive oxygen species in chronic degenerative diseases and in acute conditions, such as stroke and infection. The review focuses on the role of reactive oxygen species in the pathogenesis of drug-induced disease with special reference to hepatotoxicity, nephrotoxicity and allergic disease. The participation of reactive oxygen species in the development of adverse drug reactions induced by special drug classes (non-steroidal anti-inflammatory drugs, anti-infective agents, cytostatics) is described. Results from studies on animals and humans reveal the putative implication of oxidative stress in drug-induced disease states. Key words: adverse drug reactions, reactive oxygen species INTRODUCTION

An adverse drug reaction (ADR) is defined as any noxious, unintended and undesired effect of a drug that can occur at doses used for prophylaxis, diagnosis or therapy of a disease, or for modification of physiological function.1 The terms “adverse reaction” and “adverse effect” refer to the same phenomenon, the former seen from the point of view of the patient, while the latter - from the point of view of the drug. The science and activities relating to the detection, assessment, understanding and prevention of adverse effects or any other drug-related problem are defined by the World Health Organization as “pharmacovigilance”. ADRs contribute significantly to patient morbidity, mortality and health care expenditure. The incidence of serious ADRs in hospitalized patients in the US is 6.7%. ADRs are estimated to be the fourth to sixth leading cause of death according to a meta-analysis of 39 prospective studies over a period of 32 years.2 A systematic review of prospective and retrospective studies on ADRs in hospital patients, published from 1966 to

1999 worldwide establishes a higher weighted mean ADR rate of European studies in comparison with studies from other geographical regions.3 There are few large-scale pharmacoepidemiological studies on ADRs in Bulgaria. In the 80s the International Agranulocytosis and Aplastic Anaemia Study (IASS), which included Bulgaria, found no evidence of increased risk of agranulocytosis related to the use of dipyrone in this country.4 The incidence only of drug dermatoses in hospital-treated patients for a 10-year period (1970 - 1979) was examined at the Research Institute of Dermatology and Venereology in Sofia, Bulgaria.5 Our pharmacovigilance study of 806 hospitalized patients at the Clinic of Dermatology and Venereology in Stara Zagora revealed the importance of patient-related factors for the development of ADRs.6 ADRs present with diverse clinical symptoms and nosological entities. The classification proposed by Rawlins and Thompson (1977) defines ADRs as type A if thought to be related to the known pharmacological properties of the suspected drug or type B if otherwise. Type A ADRs often

Correspondence and reprint request to: M. Ganeva, Section of General and Clinical Pharmacology, Department of Physiology, Pathophysiology and Pharmacology, Faculty of Medicine, Thracian University, Stara Zagora 11 Armeiska Str, 6000 Stara Zagora, Bulgaria  Received 14 December 2006; Accepted for publication 27 June 2007

ADVERSE DRUG REACTIONS AND REACTIVE OXYGEN SPECIES

present as an augmented response to the drug; they are dose-related, predictable, and associated with low mortality. Type A reactions may also arise from secondary pharmacological effects of a drug. The mechanisms underlying these reactions are related to variations in the pharmaceutical, pharmacokinetic or pharmacodynamic properties of the drug due to the presence of certain diseases or to specific pharmacogenetic characteristics of the patients. Type B reactions are neither doserelated nor predictable and may be associated with considerable mortality. Type B reactions are usually caused by immunological or pharmacogenetic mechanisms. Reactive oxygen species (ROS) – superoxide radical (O2 ), nitric oxide (NO ), hydrogen peroxide (H2O2), hydroxyl radical (OH ), etc. in low concentrations are essential for normal cell physiology. The term “free radicals” has been equated with the term “reactive species or oxidants” although not all free radicals are oxidants. Cellular homeostasis can be regarded as a balance between oxidant formation and oxidant elimination. When the production of ROS exceeds the capacity of endogenous antioxidant defense mechanisms oxidative stress occurs. The term “oxidative stress” has been defined as a “disturbance in the pro-oxidant-antioxidant balance in favour of the former, leading to potential damage”.7 The main sources of ROS in humans are the mitochondrial respiratory chain, inflammatory cells (NAD(P)H oxidase – a membrane-bound enzyme of phagocytes transfers electrons from NADH or NADPH to extracellular oxygen forming O2 in the process of killing pathogens), vascular cells (they contain a similar enzyme NAD(P)H oxidase), cytoplasmic xanthine oxidase and enzymatic reactions catalysed by cyclooxygenase, lipooxygenase, cytochrome P450 and nitric oxide synthase. ROS play an essential role in many vital processes such as signal transduction and defense against pathogens. There is evidence suggesting that ROS may be involved in pathological processes and adverse side effects.8 The generation of ROS has been associated with inflammatory response; they have been found to contribute to the damaging effects of inflammatory reactions.9 It is presumed that ROS may cause toxic damage via lipid peroxidation of polyunsaturated fatty acids in cell membrane phospholipids, disturbance of calcium homeostasis, DNA fragmentation, covalent binding to proteins, etc. The short half-life of most ROS does not permit their direct detection and quantification.10



Oxidative stress is therefore indirectly detected upon measuring the changes in the biological targets of ROS – lipids, proteins, DNA. For example the end product malondialdehyde is measured as a marker of lipid peroxidation. Antioxidant defense capacity of the cell is assessed by the levels of certain enzymes (superoxide dismutase, catalase) and substances like glutathione. In recent years, there has been substantial interest in oxidative stress and its potential role in the development of diverse conditions and diseases. ADRs, on the other hand, may affect the function of any body organ and system. The molecular mechanisms underlying a drug-induced disease have not been fully clarified. There is emerging evidence that ROS may be implicated in the development of ADRs to various drug classes. ROS AND DRUG-RELATED ORGAN TOXICITY

Drug-induced organ toxicity may be related to higher drug concentrations (type A ADRs) or to immunologic reactions (type B ADRs). The basic biochemical mechanisms underlying cell injury include the loss of energy (ATP depletion), mitochondrial damage, calcium homeostasis breakdown, defects in plasma membrane permeability and generation of ROS and other free radicals. Drug-induced liver injury (DILI) is very important for drug development and drug safety. Although most hepatotoxic compounds are detected in preclinical studies, hepatotoxicity remains the leading cause of drug withdrawal from the market.11,12 There are more than 800 drugs and herbal remedies that have been implicated as causing DILI.13 Some drugs cause hepatotoxicity directly by targeting organelles such as mitochondria or nuclei. In most of the cases metabolism to reactive metabolites and free radicals by products is required. These reactive metabolites and free radicals can bind to proteins, lipids and DNA causing lipid peroxidation and oxidative stress resulting in dysfunction of organelles and cell injury.13 Alternatively, toxic metabolites may lead to inhibition of kinases, transcription factors and gene expression profiles which may sensitize hepatocytes to the toxic effects of the innate immune system, or induce immune response to the hepatocytes as a result of hapten formation (Fig. 1). DILI depends on the effects of toxic metabolites and the participation of inflammation, cytokines and repair processes. Cell death results through apoptosis or necrosis depending on the drug, duration of exposure and a variety of

Folia Medica, XL, 1/2008

Drug metabolite haptenization massive

Covalent binding Oxidative stress

Innate immune response

subtle Mitochondria

Adaptive immune response

-Altered signal transduction and gene expression - Organelle stress

Necrosis

Mitochondria

Apoptosis

Figure 1. Pathogenesis of drug hepatotoxicity (adapted from Gunawan and Kaplowitz, 2004).

environmental and genetic factors.13 Drugs may damage the kidney by different mechanisms. Vasoconstriction is the main mechanism of nephrotoxicity for vasopressors. Altered glomerular hemodynamics is responsible for renal dysfunction from non-steroidal anti-inflammatory drugs (NSAIDs) and angiotensin-converting enzyme inhibitors. Tubular cell toxicity has been found to be the cause of kidney injury associated with aminoglycosides, amphotericin, cisplatin, methotrexate, antiviral agents, etc. Crystal deposition is another factor for the development of druginduced nephrotoxicity (acyclovir, sulfonamides). ROS have been associated with some biological processes potentially important in glomerular diseases.14 Experimental evidence suggests that oxidative stress plays an important role in drug-induced nephrotoxicity. Antioxidants reduce nephrotoxicity of adriamycin15 or protect experimental animals from cyclosporine-induced nephrotoxicity16,17. In vivo studies demonstrate that gentamicin-induced glomerular dysfunction is related to ROS generation and the application of antioxidants diminishes glomerular filtration reduction.18,19 Toxic renal damage induced by diclofenac in mice is also

probably associated with the production of ROS and oxidative stress.20 Recently, oxidative stress has been implicated in the pathogenesis of some allergic diseases. Adverse drug reactions with an immunological basis may involve activation of concomitant, nonspecific mechanisms like the generation of ROS. Analysis of peripheral blood mononuclear cells from patients with non-immediate adverse drug reactions (urticaria, maculopapular exanthema and toxic epidermal necrolysis) shows increased activity and expression of superoxide dismutase while in urticaria and maculopapular exanthema thiobarbituric acid reactive substances as markers of oxidative damage have also been increased.21 Human immunodeficiency virus (HIV) infected individuals have been found to be more susceptible to non-immediate cutaneous drug reactions. A lowered antioxidant status in HIV-infected individuals has been suggested as a predisposing factor for cutaneous drug reactions in these patients.22 ROS seem to be engaged in several of the critical steps (maturation of dendritic cells, release of cytokines, T-cell activation) in the development of the skin immune response.23 Tissue damage in inflamma-



ADVERSE DRUG REACTIONS AND REACTIVE OXYGEN SPECIES

tion has been linked to the release of various pro-oxidants24, which may either directly affect dendritic cell maturation and/or activation 25 or act as a signal to induce expression of different immune regulatory molecules26. ROS AND ADRs ACCORDING TO DRUG CLASS

Non-steroidal anti-inflammatory drugs Non-steroidal anti-inflammatory drugs are commonly used for the management of rheumatological diseases, and as analgesics and antipyretics. Hepatotoxicity is an uncommon, but potentially lethal complication of NSAID therapy. It can occur with all NSAIDs, but appears to be more common with diclofenac and particularly sulindac.27 Selective cyclooxygenase-2 inhibitors, a new class of NSAIDs are generally well tolerated. However, there is evidence that nimesulide, a selective cyclooxygenase-2 inhibitor can cause rare but serious cases of liver injury, and for that reason it was temporarily withdrawn from the market in some countries in 2002. In vitro animal models have been used to explore the mechanisms of NSAID-related hepatotoxicity. Studies using rat liver mitochondria and freshly isolated rat hepatocytes showed that diphenylamine, which is common in the structure of NSAIDs, uncouples oxidative phosphorylation, decreases hepatic ATP content and induces hepatocyte injury.28 Incubation of mitochondria with diphenylamine, mefenamic acid or diclofenac caused mitochondrial swelling. In addition, loss of mitochondrial membrane potentials (one of the characteristics of uncoupling of oxidative phosphorylation) has been demonstrated. In diclofenac-induced toxicity in hepatocytes, no significant oxidative stress (decrease in glutathione and lipid peroxidation) or increase in intracellular calcium concentration was seen.28 Other studies have shown that ferrous iron release from rat liver microsomes contributes to naproxen-induced microsomal lipid peroxidation.29 Furthermore, diclofenac is more cytotoxic to drugmetabolizing cells than to non-metabolizing cell lines.28 Toxicity thus relates both to impairment of ATP synthesis by mitochondria and to drug metabolism, and is reduced by the addition of cytochrome P450 inhibitors (prothiaden and ketoconazole) to culture medium.28 The in vitro cytotoxicity in this study correlated well with the formation of 5-hydroxydiclofenac and particularly the n,5-dihydroxydiclofenac metabolite - the latter in



particular, can inhibit ATP synthesis. Mitochondrial permeability transition (MPT) has also been shown to be important in diclofenac-induced liver injury, resulting in generation of ROS, mitochondrial swelling and oxidation of NADP and protein thiols.30 The molecular and cellular mechanisms of NSAID-associated hepatotoxicity are incompletely known. Although hepatotoxicity is a class effect, it is more likely that some compound-selective mechanisms act together.31 Three basic mechanisms have been emerging: mitochondrial injury, induction of cholestasis and protein adduct formation by reactive drug metabolites. There is experimental evidence that mitochondrial injury is due to uncoupling of oxidative phosphorylation but most studies have used isolated mitochondria and cultured cells, so the relevance in vivo is not clear. The fact that some NSAIDs induce intracellular oxidative stress which can cause NAD(P)H oxidation and protein thiol oxidation is an attractive hypothesis to alternatively explain mitochondrial injury.31 A recent study of drug-induced hepatotoxicity in rats concerning diclofenac, the antimycotic agent ketoconazole and sodium diethyldithiocarbamate32 showed that hepatotoxic changes correlated with massive liver DNA fragmentation and increase in lipid peroxidation. The authors postulate that calcium may be a mediator of drug-induced oxidative stress, associated with hepatotoxicity. Cytostatics The cytotoxic activity of various anticancer agents (anthracyclines, bleomycin, vincristin, cyclophosphamide, nitrosoureas, etc) is associated with the generation of ROS.33,34 Methotrexate belongs to the group of antimetabolites and acts by inhibiting the synthesis of RNA, DNA and proteins. The cytotoxicity of methotrexate is dependent on its property to inhibit dihydrofolate reductase and prevent generation of tetrahydrofolate from dihydrofolate therefore inhibiting purine and pyrimidine synthesis. The anti-inflammatory and immunosuppressive activities of methotrexate are not likely to be related to this mechanism. Phillips, et al. put forward the hypothesis that the generation of ROS is an important mechanism in the immunosuppressive effects of methotrexate.35 There are several hypotheses regarding the mechanism of toxicity of chemotherapeutic agents like methotrexate, among which oxidative stress is noticeable. The intestinal damage in rats induced by methotrexate administration is primar-

Folia Medica, XL, 1/2008 ily associated with oxidative stress which precedes neutrophil infiltration of intestinal mucosa.36 In rats following a single dose of methotrexate (20 mg/kg) malondialdehyde (MDA) activity increased and glutathione (GSH) levels decreased in liver, ileum and kidney. These changes were reversed by taurine a potent free radical scavenger.37 In another study on rats melatonin, an effective antioxidant and free radical scavenger prevented methotrexate-induced hepatorenal oxidative injury.38 Hepatotoxicity induced by the alkylating agent azathioprine in rats has also been associated with oxidative stress32 and liver injury and markers of oxidative stress were prevented to a different extent by herbal plants with hepatoprotective activity. Anti-infective agents ROS and the reactive nitrogen species (RNS) have been implicated in the renal damage induced by the aminoglycoside antibiotic gentamycin.39,40 Tubular cell toxicity is a well known effect of gentamycin on the kidney. However, chronic treatment with gentamycin also modifies glomerular hemodynamics. There is evidence that gentamycin-induced glomerular and tubular dysfunction may be associated with increased generation of ROS.41 Gentamycin induces superoxide anion, hydrogen peroxide, and hydroxyl radical production from renal mitochondria.39 In addition, lipoperoxidation is increased and that of reduced glutathione is diminished40 in renal cortex from gentamycin-treated rats. Administration of several compounds with antioxidant properties, ROS scavengers, and/or antioxidant enzymes is able to ameliorate the severity of gentamycin-induced renal damage.40 Some beta-lactam antibiotics can cause acute proximal tubular necrosis. Significant renal toxicity is rare with penicillins and cephalosporins, but penems present a greater risk. Lipid peroxidation initiated by ROS is discussed as one of the mechanisms of renal injury induced by cephaloridine and other beta-lactams.42 Fluoroquinolones produce ROS in phagocytic cells.43 Some adverse effects of this group of drugs like phototoxicity and cartilage damage may be attributed to the production of ROS.44,45 Renal dysfunction caused by the glycopeptide antibiotic vancomycin has also been associated with the presence of oxidative stress.46 Calcium-channel blockers ROS and RNS can play the role of messengers in normal cell functions. However, at oxidative stress levels they can disrupt normal physiological

pathways and cause cell death. Such a switch is largely mediated through Ca2+ signaling. Oxidative stress causes Ca 2+ influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum or sarcoplasmic reticulum (ER/SR) through the cell membrane and the ER/SR channels, respectively. Rising Ca2+ concentration in the cytoplasm causes Ca2+ influx into mitochondria and nuclei. In mitochondria Ca2+ accelerates and disrupts normal metabolism leading to cell death. In nuclei Ca2+ modulates gene transcription and nucleases that control cell apoptosis. Both in nuclei and cytoplasm Ca2+ can regulate phosphorylation/ dephosphorylation of proteins and can modulate signal transduction pathways as a result.47 In a study on perfused rat hepatocytes Farghali et al.48 concluded that in hepatocyte oxidative injury, calcium channel blockers exhibited hepatoprotective properties. Recently Allanore et al. found that dihydropyridines (nifedipine or nicardipine) significantly decrease oxidative stress in systemic sclerosis patients, in both the short and long term.49 In conclusion, ROS have been implicated in the etiology of a number of diseases including cardiovascular disease, diabetes, cancer, Alzheimer’s disease and other neurodegenerative disorders. The role played by oxidative stress in disease pathogenesis varies in different diseases. ADRs, via diverse mechanisms and depending on their severity, may lead to substantial tissue injury and organ dysfunction (drug-induced hepatitis, drug-induced diabetes, drug-induced nephritis, etc.). Withdrawal of the incriminated drug is not always possible or it may not suffice to achieve recovery from drug-induced injury. The presence of oxidative stress in druginduced disease is worth investigating in various clinical situations because this may provide basis for additional treatment options of ADRs. REFERENCES

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ADVERSE DRUG REACTIONS AND REACTIVE OXYGEN SPECIES

5. Berova N, Cankov N, Pramatarov K, et al. Drug dermatoses. Dermatol i venerol 1983;XXII 3:8-15 (Bulgarian). 6. Ganeva M, Gancheva T, Lazarova R, et al. Adverse drug reactions in hospitalized patients - evaluation of predisposing factors. Stara Zagora Union of Scientists. Scientific conference with international participation Stara Zagora, Volume IV Human Medicine 2005:355-60 (Bulgarian). 7. Sies H. Oxidative Stress, oxidants and antioxidants. New York, NY: Academic Press; 1991. 8. Georgieva NV. Oxidative stress as a factor of disrupted ecological oxidative balance in biological systems – a review. Bulg J Vet Med 2005;8(1):1-11. 9. Cuzzocrea S, Thiemermann C, Salvemini D. Potential therapeutic effect of antioxidant therapy in shock and inflammation. Curr Med Chem 2004;11(9):1147‑62. 10. Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest 1982;47(5):412‑26. 11. Lasser KE, Allen PD, Woolhandler SJ, et al. Timing of new black box warnings and withdrawals for prescription medications. JAMA 2002;287(17):2215‑20. 12. Temple RJ, Himmel MH. Safety of newly approved drugs: implications for prescribing. JAMA 2002;287:2273-5. 13. Gunawan B, Kaplowitz N. Clinical perspectives in xenobiotic-induced hepatotoxicity. Drug Metabol Rev 2004;36(2):301-12. 14. Shah SV. Effect of enzymatically generated reactive oxygen metabolites on the cyclic nucleotide content in isolated glomeruli. J Clin Invest 1984;74:393‑401. 15. Washio M, Nanishi F, Onoyama K, et al. Alpha tocopherol improves focal glomerulosclerosis in rats with adriamycin induced progressive renal failure. Nephron 1994;68:347-52. 16. Naidu MUR, Kumar KV, Shifow AA, et al. Lacidipine protects against cyclosporine-induced nephrotoxicity in rat. Nephron 1999;81:60-6. 17. Kumar KV, Naidu MUR, Shifow AA, et al. Melatonon: an antioxidant protects against cyclosporine-induced nephrotoxicity in rats. Transplantation 1999;67:1065-8. 18. Ali BH. Gentamycin nephrotoxicity in humans and animals: Some recent research. Gen Pharmacol 1995;26:1477-87. 19. Nakajima T, Hishida A, Kato A. Mechanism for protective effects of free radical scavengers on gentamicin-mediated nephropathy in rats. Am J Physiol 1994;266:F425-F431. 20. Hickey ЕJ, Raje RR, Reid VE, et al. Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death. Free Radic Biol Med

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2001;31:139-52. 21. Cornejo-Garcia JA, Mayorga C, Torre MJ, et al. Antioxidant enzyme activities and expression and oxidative damage in patients with non-immediate reactions to drugs. Clin Exp Immunol 2006;145(2):287-95. 22. B uhl R, Jaffe HA, Holroyd KJ, et al. Systemic glutathione deficiency in symptom-free HIVseropositive individuals. Lancet 1989;2:1294-8. 23. Fuchs J, Zollner TM, Kaufmann R, et al. Redoxmodulated pathways in inflammatory skin diseases. Free Radic Biol Med 2001;30:337-53. 24. Briganti S, Picardo M. Antioxidant activity, lipid peroxidation and skin diseases: what’s new. J Eur Acad Dermatol Venereol 2003;17:663-9. 25. Chain BM. Current issues in antigen presentation: focus on the dendritic cell. Immunol Lett 2003; 89:237‑41. 26. Hubbard AK, Rothlein R. Intercellular adhesion molecule-1 (ICAM-1) expression and cell signaling cascades. Free Radic Biol Med 2000;28:1379-86. 27. O’Connor N, Dargan PI, Jones AL. Hepatocellular damage from non-steroidal anti-inflammatory drugs. Q J Med 2003;96:787-91. 28. Bort R, Ponsoda X, Jover R, et al. Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. J Pharmacol Exp Ther 1999;288:65-72. 29. Ji B, Masubuchi Y, Horie T. A possible mechanism of naproxen-induced lipid-peroxidation in rat liver microsomes. Pharmacol Toxicol 2001;89:43–8. 30. M asubuchi Y, Nakayama S, Horie T. Role of mitochondrial permeability transition in diclofenacinduced hepatotoxicity in rats. Hepatology 2002; 35:544–51. 31. B oelsterli UA. Mechanisms of NSAID-induced hepatotoxicity. Focus on Nimesulide. Drug Saf 2002;25(9):633-48. 32. Amin A, Hamsa A. Oxidative stress mediates druginduced hepatotoxicity in rat: a possible role of DNA fragmentation. Toxicology 2005;208:367-75. 33. Reszka K, Kolodziejczyk P, Tsoungas PG, et al. Photosensitization by antitumor agents-6. Production of superoxide radical and hydrogen peroxide during illumination of diaminoanthracenediones in the presence of NADH in aqueous solutions: an EPR study. Photochem Photobiol 1988;47(5):625-33. 34. Gadzheva V, Ichimori K, Nakazawa H, et al. Superoxide scavenging activity of spin-labeled nitrosourea and triazene derivatives. Free Radic Res 1994;21(3):177‑86. 35. Phillips DC, Woollard KJ, Griffiths HR. The antiinflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species. Br J Pharmacol 2003;138:501–11. 36. Miyazono Y, Gao F, Horie T. Oxidative stress contribu-

Folia Medica, XL, 1/2008 tes to methotrexate-induced small intestinal toxicity in rats. Scand J Gastroenterol2004;39(11):1119‑2. 37. C etiner M, Sener G, Sehirli AO, et al. Taurine protects against methotrexate-induced toxicity and inhibits leukocyte death. Toxicol Appl Pharmacol 2005;209(1):39-50. 38. Jahovic N, Cevik H, Sehirli AO, et al. Melatonin prevents methotrexate-induced hepatorenal oxidative injury in rats. J Pineal Res. 2003;34 (4):282-7. 39. Walker PD, Barri Y, Shah SV. Oxidant mechanisms in gentamycin nephrotoxicity. Ren Fail 1999;21(3‑4): 433-42. 40. M orales AI, Buitrago JM, Santiago JM, et al. Protective effect of trans-resveratrol on gentamycininduced nephrotoxicity. Antioxid Redox Signal 2002; 4:893‑8. 41. Martinez-Salgado C, Eleno N, Tavares P, et al. Involvement of reactive oxygen species on gentamycininduced mesangial cell contraction and proliferation. Kidney Int 2002;6:1682-92. 42. Cojocel C, Laeschke KH, Inselmann G, et al. Inhibition of cephaloridine-induced lipid peroxidation. Toxicology 1985;35:295-305. 43. Matsumoto T, Takahashi K, Nagafuji T, et al. Fleroxacin enhancement of superoxide production by

polymorphonuclear leukocytes: the role of protein kinases. Chemotherapy 1996;42: 280–5. 44. Thuong-Guyot M, Domarle O, Pocidalo JJ, et al. Effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production J Pharmacol Exper Ther 1994; 271(3):1544-9. 45. Bulera SJ, Theiss JC, Festerling TA, et al. In vitro photogenotoxic activity of Clinafloxacin: A paradigm predicting photocarcinogenicity. Tox Appl Pharm 1999;156: 222-30. 46. N ishino Y, Takemura S, Minamiyama Y, et al. Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat. Redox Rep 2002;7(5):317-9. 47. Ermak G, Davies KJA. Calcium and oxidative stress: from cell signaling to cell death. Mol Immunol 2002;38:713-21. 48. Farghali H, Kmonickova E, Lotkova H, et al. Evaluation of calcium channel blockers as potential hepatoprotective agents in oxidative stress injury of perfused hepatocytes. Physiol Res 2000;49:261-8. 49. Allanore Y, Borderie D, Lemarechal H, et al. Acute and sustained effects of dihydropyridine-type calcium channel antagonists on oxidative stress in systemic sclerosis. Am J Med 2004;116(9): 595-600.

Нежелательные лекарственные реакции и реактивные кислородные виды

За последние годы накопились доказательства относительно роли свободных радикалов в развитии хронических дегенеративных заболеваний и острых состояний (инсульты и инфекции). Обзор рассматривает роль реактивных кислородных видов в патогенезе лекарственно-вызванной болезни и конкретнее в патогенезе гепатотоксичности, нефротоксичности и аллергических заболеваний. Описывается участие реактивных кислородных видов в развитии нежелательных лекарственных реакций, вызванных определенными лекарственными группами (нестероидные противовоспалительные средства, антиинфекционные средства, цитостатики). Результаты исследования на подопытных животных и на людях показывают возможное участие оксидативного стресса в развитии болезненных состояний, вызванных лекарствами.

М. Ганева, Д. Гетова, В. Гаджева

Резюме Нежелательные лекарственные реакции способствуют увеличению заболеваемости как госпитализированных, так и амбулаторных пациентов. Они могут повредить функцию каждого органа или системы. Распознавание побочных лекарственных реакций часто затрудняется их неспецифической клинической картиной. Клиническое проявление, вид и тяжесть этих реакций варьируют в большом диапазоне в зависимости от факторов со стороны пациента и от факторов со стороны лекарственного препарата.

11

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management Ljudmila G. Vladimirova-Kitova

Clinic of Cardiology, “St.George” University Hospital, Medical University, Plovdiv, Bulgaria Abstract Evidence has been accrued recently that chronic high levels of asymmetric dimethylarginine (ADMA) can be directly beneficial to the treatment of atherosclerotic vascular disorders thus making the substance a promising new therapeutic target. A therapeutic target can be theoretically each stage in the process of generation and elimination of asymmetric dimethylarginine. The methylation of L-arginine residues is a universal biological process involving hundreds of proteins but still with unknown effects. Interference with these mechanisms can generate ambiguous and speculative discussions. Supplementation with L-arginine seems to be the most natural way to reverse the detrimental effect of ADMA on the endothelium. The enzymatic activity of endogenous nitric oxide synthase is regulated by the ratio between the concentrations of L-arginine (the natural substrate) and that of ADMA (the endogenous inhibitor): in the presence of normal L-arginine levels, any elevation of ADMA levels may cause relative L-arginine deficiency thus attenuating the activity of the endogenous nitric oxide synthase. Target replacement therapy with L-arginine to increase the L-arginine plasma levels results in the normalisation of the L-arginine/ADMA ratio in the presence of higher levels of the latter. There is still some controversy about the effects of the most frequently used drugs on asymmetric dimethylarginine. Most of the relevant studies show that statins do not affect the ADMA levels. On the other hand, patients with high levels of ADMA are resistant to statin therapy - to improve the endothelium-dependent vasodilation they need a combined therapy with L-arginine. The angiotensin-converting enzyme inhibitors and the angiotensin receptor blockers are the most extensively studied substances, the studies predominantly centring on confirming their ADMA reducing effect. Until the specific ADMA-reducing therapy becomes readily available, it is the therapies of modification of the risk factors causing the increase of ADMA or the depletion of L-arginine, and the L-arginine replacement therapy that are the most realistic therapeutic solutions for patients with high plasma levels of ADMA because the synthesis of nitric oxide correlates with the L-arginine/ADMA ratio. A study was conducted in the Surgery of Preventive Cardiology with the Clinic of Cardiology in Plovdiv which included 40 patients with pronounced hypercholesterolemia (HC) – it was found that a one-month therapy with 40 mg simvastatin did not change statistically significantly ADMA plasma levels in spite of the optimal lipid regulation. Key words: hypercholesterolemia, asymmetric dimethylarginine, endogenous nitric oxide synthase, flow-induced vasodilation, endothelial dysfunction Introduction

Evidence has been emerging that asymmetric dimethylarginine (ADMA) as inhibitor of the endogenous nitric oxide synthase (eNOS) not only serves as a marker but also is involved in the pathophysiological process of atherosclerosis. ADMA infusion impairs the endothelium-dependent vasodilation, increases the systemic and renal vascular resis-

tance and reduces the cardiac flow. This evidence suggests that the chronic elevation of ADMA can be directly supportive of atherosclerotic vascular disorders which makes ADMA a promising new therapeutic target.1 Theoretically, each stage of the generation and elimination of ADMA can be a therapeutic target.1-3 The figure below presents the eight stages of this process (Fig. 1).

Correspondence and reprint request to: L. Vladimirova-Kitova, Clinic of Cardiology, Medical University, Plovdiv, “St.George” University Hospital, Plovdiv 66 Peshtersko shosse, 4002 Plovdiv, Bulgaria 12 Received 6 August 2007; Accepted for publication 17 October 2007

Folia Medica, XL, 1/2008 The endogenous formation of ADMA starts with the asymmetric demethylation of L-arginine residues within various cellular proteins by the specific protein L-arginine methyltransferase (PRMTs). The product of this reaction is S-adenosyl-homocysteine (PRMT’s inhibitor) which hydrolyses to L-homocysteine and then remethylates back to L-methionine. This reaction, which closes the cycle of methylation, is dependent on folic acid and vitamin B12. Each step of the methylation-demethylation cycle is a potential substrate for therapeutic targeting to decrease the concentration of S-adenosyl-methionine. A number of considerations of practical and

clinical significance render these targets unsuitable for now. The methylation of L-arginine residues is a universal biologic process involving hundreds of proteins. The effects of the process of methylation are still poorly studied. Any interference with these mechanisms can lead to ambiguous and speculative discussions. Knowing what the role of the homocysteine-methionine cycle is, it is logical to assume that replacement therapy with folic acid and vitamin B12 would intensify the synthesis of S-adenosyl-homocysteine and hence lead to the generation of ADMA. Most of the studies in this field have shown that folic acid and vitamin B12

Figure 1. Stages in the process of generation and release of ADMA (according to Böger).

13

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management

have no effect on the ADMA plasma level in the presence of elevated levels of homocysteine, while other studies have found that folic acid decreases ADMA levels.4,5 The next stage of proteolysis of asymmetric dimethylarginine residues and generation of ADMA is also not suitable for a therapeutic target. Developing a rather specific inhibitor of the proteosomes involved in the degradation of these proteins would lead to the accumulation of proteins and their fragments which clearly opens a possibility for health impairment.3 The next stage of ADMA degradation from dimethylaminohydrolase (DDAH) is not only unsuitable to be therapeutically targeted but also possibly dangerous. ADMA selectively is degraded by DDAH to citrulline and dimethylamine. Two large isoforms of DDAH (DDAH-1, and DDAH-2) have been found in men controlling the process of breaking down in the kidneys, liver and small intestines. It is unknown what percentage of the endogenously formed ADMA is metabolised directly in the cell, where it derives from and how much of it is released into other tissues. It is not known which of the isoforms predominates in which body organ.3 It has been established though that it is the endothelium that is the important site for the metabolisation of ADMA.6 There is still little known about the release of ADMA from DDAH. Furthermore, any therapeutically induced increase of DDAH expression and activity leads to intensified tumour growth and angiogenesis.3 As it is in patients with chronic renal failure that increased levels of ADMA were first found, it is far from appropriate to talk about renal filtration, tubular excretion and readsorption as therapeutic targets. As much as 16% of ADMA have been found to take part in this process. It is still impossible to determine exactly what role is played by each of these mechanisms.3 The most appropriate method for compensating the damaging effect of ADMA on the endothelium seems to be the addition of L-arginine. As ADMA is a competitive inhibitor of eNOS, its inhibitory action can be overcome by increasing the concentration of the enzyme’s substrate, L-arginine.8-11 The enzyme activity of eNOS is regulated by the ratio between the concentrations of L-arginine (natural substrate) and ADMA (endogenous inhibitor). Any elevation of ADMA can cause a relative L-arginine deficiency even in the presence of normal L-arginine levels in plasma thus decreasing the eNOS activity. Target replacement therapy with L-arginine leading to the elevation of its plasma levels will

14

normalise the L-arginine/ADMA ratio against the background of the higher levels of the latter. Larginine should be received regularly to achieve the therapeutic objective – decrease of ADMA and improvement of the endothelium-dependent vasodilation. It is well known that the protein Larginine methylation is a universal biologic process occurring in bacteria, plants and animals. The free and protein-bound ADMA can be associated with the modulation of a suitable diet or generated by appropriate bacterial strains. These are just some possible therapeutic targets that can be investigated in future to reduce ADMA levels but they are still not studied.3 Arginine paradox L-arginine used in people has been found to exert anti-atherogenic effects by improving the endothelium-dependent vasodilation. 12-15 These are findings of in vivo studies which are contrary to what some experimental studies suggest, namely that supplementation with L-arginine does not improve the endothelium-dependent vasodilation, or that exogenous amounts of L-arginine have no effect on the activity of eNOS. This discrepancy is known as L-arginine paradox.16 In physiologically normal conditions the eNOS enzyme activity is reduced in comparison with the maximal, although the L-arginine concentration is such that eNOS can act at maximal rate. eNOS functions at only 50% of its maximally possible activity. In pathological conditions the elevated intracellular concentration of eNOS inhibitors causes additional reduction of the activity of eNOS, which, in its turn, reduces the generation of nitric oxide (NO). In such pathological conditions in which the eNOS inhibitors are elevated even a small change in the concentration of L-arginine leads to a considerable change in the activity of eNOS. Supplementation with L-arginine leads to increased NO formation. This effect is dependent on the amount of L-arginine; also, chronic administration of L-arginine is required. Therefore, chronic supplementation of L-arginine increases the production of nitric oxide. The Role of Adma for the Successful Therapy with L-arginine Of all hypotheses about the L-arginine paradox the most logical one that provides the most convincing evidence is currently the hypothesis which implicates ADMA.16 The in vivo supplementation with L-arginine increases the L-arginine/ADMA ratio and this is the major factor that controls the

Folia Medica, XL, 1/2008 activity of eNOS. When L-arginine is elevated, this ratio increases thus reducing the inhibitory effect of ADMA on eNOS. This hypothesis is valid when ADMA is elevated at baseline.16 Initial

clinical evidence for the effect of some

drugs on

ADMA

plasma levels

1. Hydroxymethylglutaryl-CoA reductase inhibitors (statins) Some of the lipid-independent favourable effects of statins are due to their ability to upregulate the gene expression of eNOS which leads to increased bioavailability of NO (Fig. 2A).1,17,18 Elevated LDL levels in animals and humans have been found to correlate with the elevated ADMA levels in a few studies.19 There are two antipodal explanations as to what causes the effect of statins on the endotheliumdependent vasodilation – in some clinical studies the statins have been shown to improve the flow-induced vasodilation, while in others they have been found to cause no improvement. This discrepancy could be explained by the different levels of ADMA. The accepted hypothesis now is that it is ADMA that modifies the statins’ therapeutic effect. Few are the studies that investigate the statins’ effect on ADMA plasma levels (Table 1). In a monotherapy with different doses of statins ADMA is reduced statistically significantly only when rosuvastatin is given in a dose of 10 mg.20 Pravastatin, the most widely studied statin for its pleiotropic effect, was not able to reduce the ADMA levels at a dose of 40 mg/d in two separate studies.21,22 Simvastatin at low and high dosage had no effect on the plasma levels of ADMA; the same refers to the mean therapeutic dose of 40 mg of atorvastatin.23,24 Most of the studies in this field have demonstrated that

statins have no effect on ADMA levels.18 High ADMA levels inhibit eNOS activity; generation of nitric oxide is also inhibited with all its antiatherogenic traits suppressed (Fig. 2B).1,3 Perhaps ADMA can account for all these contradictory facts. Functionally, it is probably no use upregulating the gene expression of statins when the enzyme catalytic activity is inhibited by ADMA. Janatuinen, et al. found that pravastatin improved the myocardial blood flow (measured by positron emission tomography) only in patients with low ADMA levels and had no effect on those with elevated ADMA levels.22 They investigated the hypothesis that ADMA could inhibit eNOS against the background of statin therapy, regardless of the improvement of the gene expression and that this inhibition could be overcome with supplementation with L-arginine.1 It was found in a randomized controlled study that simvastatin improved the endothelium-dependent vasodilation in patients with elevated ADMA levels only when it was used in combination with L-arginine.24,25 There is evidence that reject the positive effect of the combination on ADMA (Fig 2C). In view of the contradictory data reported in the literature about the different effects of the statins on the endothelium-dependent vasodilation and ADMA, further studies are needed to find any causal relationship. The currently accepted view is that patients with high ADMA levels resist any statin therapy and in order to improve the endothelium-dependent vasodilation we should use a combined therapy with L-arginine. This has been confirmed by the reports in the literature for the past month.26 2. Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers

Table 1. Effect of statins on plasma ADMA levels Statin

Weeks

n

% change

P

Pravastatin 40 mg/d (Eid, et al. 2003)21

8

32

-9

n.s.

Pravastatin 40 mg/d (Janatuinen, et al. 2001)22

26

21

No changes

n.s.

Simvastatin 80 mg/d (Päivä, et al. 2003)23

8

24

No changes

n.s.

Atorvastatin 40 mg/d (Päivä, et al. 2003)23

8

24

No changes

n.s.

Simvastatin 20 mg/d (Periera, et al. 2003)24

8

25

No changes

n.s.

Rosuvastatin 10 mg/d (Lu, et al. 2004)20

6

23

- 18

< 0.001

n.s. - nonsignificant

15

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management

Figure 2A. Vasculoprotective effect of statins, regulation of the gene expression of eNOS.

Figure 2B. High concentration of ADMA inhibits eNOS in spite of the administration of statin.

Figure 2C. Supplementation with Larginine leads to increase of eNOS activity.

It has been established that in some pathological conditions such as hypertension, diabetes and ischemic heart disease, the angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) improve the endothelium-dependent vasodilation and NO bioavailability. There are only

16

a few studies on the effect of ACE inhibitors and ARB on ADMA plasma levels in patients with these diseases.27-29 In hypertension, diabetes and X syndrome ACE inhibitors have been found to reduce ADMA levels by 12-20% while they do not change significantly L-arginine concentrations.30 ARB re-

Folia Medica, XL, 1/2008 duce the plasma ADMA levels by only 10-16%.28 These studies, however, are short (1 to 12 weeks) and include only small study samples (Table 3). It has been established in an experimental model that ADMA has NO-independent effects which can be influenced by ACE and are associated with oxidative stress through AT1-independent pathways. 3. Hypoglycemic drugs In type II diabetes patients a three-month treatment with 0.5 - 1.0 d/g of metformin as a monotherapy (n = 16) or in combination with a sulfonylurea drug (n = 15) decreases the plasma ADMA levels by 28.5% and 32.0%, respectively.30 No correlation has been currently found between the ADMA reduction and glycemic control. Most probably, the ADMA reduction is mediated by glucose-independent mechanisms. It is worth noting in this respect that metformin is structurally similar to ADMA.31 4. Hormone replacement therapy It is well known that a hormone replacement therapy improves the endothelium function and the NO synthesis. ADMA levels decrease during normal pregnancy. The precise mechanisms, however, estrogens utilise to reduce ADMA are still not clarified. The effects of different forms of hormone replacement therapy on ADMA are studied in some small randomized case-controlled studies.32-34 In these studies ADMA levels were reduced by 5-21% with 17-beta estradiol given in a monotherapy or in a combination with cyclogesterone or trimegestone. Simultaneously L-arginine levels declined resulting in no change or even a decrease in the L-arginine/ADMA ratio. The decrease in L-arginine was assumed to be the

result of increased consumption in NO synthesis, and not because of upregulation of arginase activity. So it remains questionable how much benefit can be conferred by hormone replacement therapy on patients with an elevated ADMA. There are widely divergent reports in the available literature about the effects of different drugs on the plasma ADMA levels; not all of these drugs have been shown to act on the endothelial function.1 Whether these effects are dose-dependent remain still to be found in further studies. The answer to the question of how much the changes in АДМА levels influence the endothelial function and hence the cardiovascular risk will determine whether ADMA will be considered a cardiovascular risk factor. Recently, ADMA was reported to be a predictor of total and cardiovascular mortality in patients with angiographically confirmed ischemic heart disease.35 Conclusions

There is still no specific ADMA-reducing therapy. The results of pilot studies with such widely used drugs as ACE, ARB, hypoglycated drugs and hormone replacement therapy are promising. There should be more extensive research aimed at finding a more specific therapy targeted at the mechanisms of increasing the plasma levels of ADMA. As ADMA elevation is usually chronic patients should receive a long-term therapy. The problematic aspects of such therapies are associated with the acceleration of angiogenesis and tumour growth. Until a specific ADMA-reducing therapy becomes available, it is the

Table 2. Effect of ACE and ARB on ADMA plasma concentrations Weeks

n

% change

P

Enalapril 20 mg/d (Delles, et al. 2002)

1

20

- 17

< 0.05

Enalapril 10 mg/d (Chen, et al. 2002)

8

10

- 20

< 0.01

Enalapril 20 mg/d (Napoli, et al, 2004)

12

38

- 16

< 0.05

Zofenopril 15-30 mg/d (Napoli, et al. 2004)

12

38

- 24

< 0.01

Perindopril 4 mg/d (Ito, et al. 2001)

4

7

- 12

< 0.05

Perindopril 4 mg/d (Ito, et al. 2002)

1

11

- 16

0.02

Eprosartan 600 mg/d (Delles, et al. 2002)

1

20

- 16

< 0.05

Eprosartan 300 mg/d + enalapril 20 mg/d (Delles, et al. 2002)

1

20

- 18

< 0.05

Losartan 50 mg/d (Ito, et al. 2001)

4

7

- 10

< 0.05

Drug

17

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management

Table 3. Effect of some novel or some widely used drugs on ADMA plasma concentrations and endothelial function Therapy

L-arginine

ACE inhibitors (perindopril, enalapril)

Clinical condition Hypercholesterolemia, peripheral arterial disease, congestive heart failure, ischemic heart disease, syndrome X, hyperhomocycteinemia Type 2 diabetes, syndrome X, hypertension

ADMA

Endothelial function



+/-; n.a.



n.a.

AT1-receptor blockers (losartan, eprosartan)

Hypertension



n.a.

ACE + AT1 (enalapril, eprosartan)

Hypertension



n.a.

Statins (lovastatin, pravastatin, simvastatin, atorvastatin)

Hypercholerestolemia



n.a.

Probucol

Experimental LDL injection

↓ (с LDL) monotherapy

+n.a.

Antioxidants (PDTS, daviditin A, vitamin E)

Experimental LDL or LPC injection/incubation

↓ (с LDL) monotherapy

+n.a.

Antidiabetic (rosiglitasone, metformin)

Healthy individuals, Type 2 diabetes

Hormone replacement therapy (Conjugated oestrogen, raloxifene)

Hysterectomized women Postmenopausal women

β-blocker (bisoprolol)

Hypertension

Folic acid ± vitamins B

Hyperhomocysteinemia



n.a.

↓(Conjugated) ↔(raloxifene)

n.a.



n.a.

Just folic acid ↔

n.a. ↔

n.a. - not available

therapies of modification of the risk factors causing ADMA increase or L-arginine depletion, and L-arginine replacement therapy that are the most realistic therapeutic solutions for patients with high plasma levels of ADMA because the synthesis of nitric oxide correlates with the L-arginine/ADMA ratio. We conducted a study in the Surgery of Preventive Cardiology at the Clinic of Cardiology of the Medical University, Plovdiv on the effect of simvastatin in a moderate dose (40 mg) on ADMA plasma levels in a pronounced HC. The t-test showed no statistically significant changes

18

in ADMA – the plasma levels of ADMA did not change significantly in spite of the optimal lipid regulation. 36-39 This finding is consistent with other reports in the literature reporting no effect on ADMA both at a low dose (20 mg), and at a high dose (80 mg) of simvastatin.23,24 It is logical to expect reduction of ADMA plasma levels when the lipids are regulated optimally. The fact that they did not decline suggests the presence of other, unknown mechanisms increasing ADMA in HC. Therefore, it is quite likely that statins bear no relation to ADMA metabolic pathways and the pathophysiological abnormalities associated with

Folia Medica, XL, 1/2008 them. The solution of this problem is a therapeutic challenge in patients with pronounced CH and elevated levels of ADMA which require chronic administration of statins. The resistance of the statin therapy in moderate doses is a therapeutic challenge so that alternatives and combined treatments can be sought. Reference

1. Böger RH. Asymmetric dimethylarginine (ADMA) modulates endothelial function - therapeutic implications. Vasc Med 2003;8:149-51. 2. Bełtowaski J, Kędra A. Asymmetric dimethylarginine (ADMA) as a target for pharmacothepary. Pharmacological Report 2006;58:159-78. 3. Maas Renke. Pharmacotherapies and their influence on asymmetric dimethylarginine (ADMA) Vascular Medicine 2005;10:S49-57. 4. Sydow K, Schwedhelm E, Arakawa N. ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocysteinemia: effects of L-arginine and B vitamins. Cardiovascular Research 2003;57:244-52. 5. Holven KB, Haugstad TS, Holm T, et al. Folic acid treatment reduced elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects. Br J Nutr 2003;89:359-63. 6. McCarty MF. Vascular endothelium is the organ chiefly responsible for the catabolism of plasma asymmetric dimethylarginine – an explanation for the elevation of plasma ADMA in disorders by endothelial dysfunction. Med Hypotheses 2004;63:699708. 7. Stroes ES, Koomans HA, de Bruin TW, Rabelink TJ. Vascular function in the forearm of hypercholesterolaemic patients off and on lipid-lowering medication. Lancet 1995;346:467-71. 8. Azuma H, Sato J, Hamasaki H, et al. Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L-arginine in regenerated endothelial cells. Br J Pharmacol 1995;115:1001‑4. 9. Clarkson P, Adams MR, Powe AJ, et al. Oral Larginine improves endothelium-dependent dilation in hypercholesterolemic young adults. J Clin Invest 1996;97:1989-94. 10. Cooke JP, Dzau VJ. Derangement of the nitric oxide synthase pathway, L-arginine and cardiovascular disease. Circulation 1997;96:379-82. 11. Böger RH. Association of asymmetric dimethylarginine and endothelial dysfunction. Clin Chem Lab Med 2003;41:1467-72. 12. Bode-Böger S, Böger RH, Galland A, et al. L-arginine-induced vasodilatation in healthy humans:

pharmacokinetic-pharmacodynamic relationship. Br J Clin Pharmacol 1998;46:489-97. 13. Böger RH, Bode-Böger SM, Kienke S, et al. Dietary L-arginine decreases myointimal cell proliferation and vascular monocyte accumulation in cholesterolfed rabbits. Atherosclerosis 1998;136:67-77. 14. B öger RH, Bode-Böger SM, Brandes RP, et al. Dietary L-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits. Circulation 1997;96:1282-90. 15. Creager MA, Gallagher SJ, Girerd XJ, et al. L-arginine improves endothelium-dependent vasodilatation in hypercholesterolemic humans. J Clin Invest 1992;90:1248-53. 16. Tsikas D, Böger RH, Sandmann J, et al. Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox. FEBS Lett 2000;478:1-3. 17. Laufs U, La F, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129-35. 18. Böger RH. Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med 2006;38(2):126-36. 19. Böger RH, Bode-Böger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 1998;98:1842-7. 20. Lu TM, Ding YA, Leu HB. Effect of rosuvastatin on plasma levels of asymmetric dimethylarginine in patients with hypercholesterolemia. Am J Cardiol 2004;94(2):157-61. 21. Eid HM, Eritsland J, Larsen J, et al. Increased levels of asymmetric dimethylarginine in population at risk for atherosclerotic disease. Effects of pravastatin. Atherosclerosis 2003;166:279-84. 22. Janatuinen T, Laakso J, Laaksonen R, et al. Plasma asymmetric dimethylarginine modifies the effect of pravastatin on myocardial blood flow in young adults. Vasc Med 2003;8:185-9. 23. Päivä H, Laakso J, Lehtimäki T, et al. Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors. J Cardiovasc Pharmacol 2003;41:219-22. 24. Pereira EC, Bertolami MC, Faludi AA, et al. Effects of simvastatin and L-arginine on vasodilatation, nitric oxide metabolites and endogenous NOS inhibitors in hypercholesterolemic subjects. Free Radical Research 2003;37(5):529-36. 25. Böger GI, Maas R, Schwedhelm E, et al. Improvement of endothelium-dependent vasodilation by simvastatin is potentiated by combination with L-arginine in patients with elevated asymmetric dimethylarginine levels. J Am Coll Cardiol 2004;43(suppl):525A.

19

Asymmetric dimethylarginine – mechanisms and targets for therapeutic management

26. Böger G, Rudolph T, Maas R. Asymmetric dimethylarginine determines the improvement of endothelium-dependent vasodilatation by Simvastatin. J Am College Cardiology 2007;49:2274-82. 27. Delles C, Schneider MP, John S, et al. Angiotensin converting enzyme inhibition and angiotensin II AT 1-receptor blockade reduce the level of asymmetrical N (G),N(G)-dimethylarginine in human essential hypertension. Am J Hypertens 2002;15:590-3. 28. Chen JW, Hsu NW, Wu TC, et al. Long-term angiotensin-converting enzyme inhibition reduced plasma asymmetric dimethylarginine and improves endothelial nitric oxide bioavailability and coronary microvascular function in patients with syndrome X. Am J Cardiol 2002;90:974-82. 29. Ito A, Egashira K, Narishige T, et al. Angiotensinconverting enzyme activity is involved in the mechanism of increased endogenous nitric oxide synthase inhibitor in patients with type 2 diabetes mellitus. Circ J 2002;66:811-5. 30. Asagami T, Abbasi F, Stühlinger M, et al. Metformin treatment lowers asymmetric dimethylarginine concentration in patients with type 2 diabetes. Metabolism 2002;51:843-6. 31. Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 2004;24:1023-30. 32. Holden DP, Cartwright JE, Nussey SS, Whitley GS. Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine. Circulation 2003;108:1575-8.

33. Post MS, Verhoeven MO, van der Mooren MJ, et al. Effect of hormone replacement therapy on plasma levels of the cardiovascular risk factor asymmetric dimethylarginine: randomized, placebo-controlled 12-week study in healthy early postmenopausal women. J Clin Endocrinol Metab 2003;88:4221-6. 34. Teerlink T, Neele SJ, de Jong S, et al. Oestrogen replacement therapy lowers plasma levels of asymmetric dimethylarginine in healthy postmenopausal women. Clin Sci (Lond) 2003;105:67-71. 35. Meinitzer A, Seelhorst U, Wellnitz B, et al. Asymmetric dimethylarginine independently predicts total and cardiovascular mortality in individuals with angiographic coronary artery disease (The Ludwigrshafen risk and cardiovascular health study). Clinical Chemistry 2007;53,2:273-83. 36. Vladimirova-Kitova L, D. Terzieva. Asymmetric dimethylarginine in hypercholesterolemic subjects. Journal of Cardiology 2007, in press. 37. Vladimirova-Kitova L, Terzieva D. Asymmetric dimethylarginine and flow-mediated vasodilatation in hypercholesterolemic subjects. Atherosclerosis Suppl. 2007;119 38. Juonala M, Viikari J, Alfthan G, Raitakari O. Asymmetrical dimethylarginine is inversely associated with brachial flow-mediated dilatation in young adults. The cardiovascular risk in young Finns study. Atherosclerosis Suppl. 2007;119 39. Vladimirova-Kitova L, Terzieva D. Effect of highdose simvastatin on plasma concentration of asymmetric dimethylarginine in hypercholesterolemic subjects. Atherosclerosis Suppl. 2007;201

Асимметрический диметиларгинин – механизмы и таргеты для терапевтического влияния

может привести к недвусмысленным и спекулятивным обсуждениям. Самый логический способ противодействия поражающему эффекту АДМА на эндотелий - это прибавление Л-Аргинина. Энзимная активность эндогенной азотной окисьсинтетазы регулируется отношением между концентрацией Л-Аргинина (естественный субстрат) и АДМА (эндогенный ингибитор). При нормальных Л-Аргининовых уровнях каждое повышение АДМА может вызвать относительный функциональный дефицит Л-Аргинина даже при наличии нормальных уровней Л-Аргинина. Это приводит к уменьшению активности эндогенной азотной окись-синтетазы. Таргетное заместительное лечение Л-Аргинином, приводящее к повышению плазменных уровней Л-Аргинина, будет нормализовать отношение ЛАргинин/АДМА на фоне более высоких уровней последнего. Данные о влиянии очень часто использованных медикаментов на асимметрический диметиларгинин противоречивы. Большинство

Л. Владимирова-Китова

Резюме На настоящем этапе ряд событий показывает, что хроническое повышение асимметрического диметиларгинина (АДМА) может непосредственно способствовать лечению атеросклеротических сосудистых заболеваний, что делает его обещающим новым терапевтическим таргетом. Теоретически каждый этап образования и элиминирования асимметрического диметиларгинина может быть таргетом. Метилирование Л-Аргининовых остатков представляет универсальный биологический процесс, вовлекающий сотни белков. Эффекты этого метилирования все еще неизвестны. Взаимодействие с этими механизмами

20

Folia Medica, XL, 1/2008 исследований показывает, что статины не влияют на уровни АДМА. С другой стороны, пациенты с высокими уровнями АДМА нечувствительны к терапии статинами, и в целях улучшения эндотел-зависимой вазодилатации приходится применять комбинированную терапию Л-Аргинином. Ингибиторы конвертирующего энзима и ангиотензинрецепторные блокеры подвергнуты множеству исследований, доказывающих понижающий их эффект на АДМА. Пока специфическая асимметрическая диметиларгинин-понижающая терапия станет реальностью, лечение модифицированием факторов риска, приводящих к повышению АДМА или исчер-

пыванию Л-Аргинина, как и заместительное лечение Л-Аргинином, по всей вероятности является самым реальным решением для пациентов с высокими плазматическими уровнями АДМА, так как синтез окиси азота коррелирует с отношением Л-Аргинин/ АДМА. В кабинете превентивной кардиологии к Кардиологической клинике проведено исследование на 40 пациентах с выраженной гиперхолестеролемией. Установлено, что лечение препаратом Simyastatin (40 mg) статистически значимо не изменяет плазматические уровни АДМА, несмотря на оптимальное липидорегулирование (Р > 0.05).

21

flow-mediated vasodilation in severe hypercholesterolemia

Original Articles Clinical Investigations Evaluation of the relationship between flow-mediated vasodilation and some atherogenic risk markers in severe hypercholesterolemia Ljudmila G. Vladimirova, Ivan H. Manukov, Ivanka Sirakova1 , Tania Iv. Deneva1

Clinic of Cardiology, 1Clinic of Invasive Cardiology, 2Department of Clinical Laboratory, Medical University, Plovdiv, Bulgaria Abstract Background: In recent years, there has been a shift of interest in preventive cardiology towards primary prevention in high risk patients such as the patients with severe hypercholesterolemia. There is scanty information available in the respective literature on the levels of asymmetric dimethylarginine in severe hypercholesterolemia and on the correlation of flow-mediated vasodilation with some atherogenic risk biomarkers. Aim: To assess the relationship of flow-mediated vasodilation with lipids, apoproteins and some serum markers of endothelial activation in severe hypercholesterolemia. Patients and methods: The study population consisted of 58 patients with severe hypercholesterolemia (> 7.5 mmol/l) as categorised by Simon-Broome’s criteria for clinical definite and probable diagnosis of familial hypercholesterolemia. All patients were asymptomatic. The study included also 50 control subjects that had no evidence of hypercholesterolemia. Results: The analysis of the lipid profile, the atherogenic lipid indices, and the apoproteins of patients and controls supports the alternative hypothesis: there is a statistically significant difference determined by the higher values of these parameters in the patients group (P < 0.0001). The mean values of asymmetric dimethylarginine calculated at baseline in both groups differed significantly (1.64 ± 0.04 µmol/l vs. 0.47 ± 0.02 µmol/l for patients and controls, respectively, P < 0.001). There was also a significant difference in the mean values of flow-mediated vasodilation calculated at baseline between the two groups (4.49 ± 0.62% vs. 8.64 ± 0.61% for patients and controls, respectively, P < 0.001). We found a very strong negative correlation, which reached statistical significance, between the flow-mediated vasodilation and the asymmetric dimethylarginine, apoprotein B and the apoprotein B/apoprotein A-I ratio (rxy = - 0.687 with apoprotein B, p < 0.0001; rxy = - 0.518 with apoprotein B/apoprotein A-I ratio, p < 0.0001; and rxy = -0.895 with asymmetric dimethylarginine, p < 0.0001). Conclusions: Univariate analysis showed that 80% of all variations in the values of flowmediated vasodilation can be explained by variations in the values of asymmetric dimethylarginine. The significant negative correlation between the flow-mediated vasodilation and apoprotein B, the apoprotein B/apoprotein A-I ratio and the asymmetric dimethylarginine indicates that these biomarkers are more strongly associated with the endothelial dysfunction (the earliest functional abnormality of the vascular wall) than with the lipid profile components that are usually used in clinical practice. Key words: hypercholesterolemia, apoproteins, asymmetric dimethylarginine, flow-mediated vasodilation

Correspondence and reprint request to: L. Vladimirova-Kitova, Clinic of Cardiology, Medical University, Plovdiv, “St.George” University Hospital Plovdiv 66 Peshtersko shosse, 4002 Plovdiv, Bulgaria 22 Received 6 August 2007; Accepted for publication 30 January 2008

Folia Medica, XL, 1/2008 Introduction

High serum cholesterol levels is the only risk factor that can single-handedly trigger atherosclerosis (AS) in people and animals even in the absence of any other known risk factors.1 The apoproteins in dyslipidemia are more informative than the routine lipid profile because they indicate not only the number of the cholesterol particles, but also the ratio of their number to their density. The ratio of apoprotein B to apoprotein A-I (Apo B/Apo A‑I) is a strong predictor of future coronary artery disease.2 In asymptomatic patients with severe hypercholesterolemia (HC) the endothelial dysfunction, which is the earliest functional abnormality of the vascular wall, is reported to be assessed by both markers of endothelial activation and markers of the functional condition of the peripheral arteries. They provide indirect information on the activity of the endogenous nitric oxide synthase (eNOS). There are two reasons, according to the literature, that may cause to change its level – it is either disrupted generation of eNOS or abnormal release. The former is mostly associated with the elevated levels of eNOS inhibitors. It is now accepted that there are two endogenous inhibitors of eNOS - the asymmetric dimethylarginine (ADMA) and the symmetric dimethylarginine (SDMA). As the plasma levels of ADMA are 10-fold higher than those of SDMA it is ADMA that focuses the interests of most research.3-7 Hypercholesterolemia leads to reduced activity of dimethylaminohydrolase (DDAH) which causes the high levels of ADMA. The low density cholesterol (LDL) causes upregulation of the expression of methylated arginine residues from which ADMA is derived.8 Accumulation of ADMA leads to cholesterol-induced endothelium-dependent dysfunction. It has been proved that the oxidized form of LDL increases ADMA faster than the native LDL does. The eNOS activity is reduced in HC, which leads to disruption in the endothelium-dependent vasodilation, reduction of the thrombocyte aggregation and monocytic adhesion.9-12 The endothelial function is also assessed using functional tests in which the peripheral arteries are investigated.13-18 Because AS is a systemic process the brachial artery is preferred although the femoral and the radial arteries can also be used for the purpose. For routine clinical practice the most convenient method in this respect is evaluating the percentage increase of the flow-mediated vasodilation (FMD%), which is a simple, easily available, informative, and repeatable method. In

1955, Anderson et al. reported that the coronary and peripheral circulations were correlated.19 There is very little information in the literature about the levels of ADMA in severe HC and about the relationship of flow-mediated vasodilation with some atherogenic risk biomarkers. The emphasis currently in preventive cardiology is placed on primary prevention in high risk patients such as the patients with severe HC.20 Aim

The aim of the study was to assess the relationship between flow-mediated vasodilation with lipids, apoproteins and some serum markers for endothelial activation in severe hypercholesterolemia. Patients and methods

Fifty eight patients were included in the study, all with severe hypercholesterolemia (> 7.5 mmol/l) as categorized by Simon-Broome’s criteria for a clinical definite and probable diagnosis of familial hypercholesterolemia.21 All patients had a negative stress echocardiographic test which means that they had no evidence of ischemic heart disease. Fifty subjects without any evidence of hypercholesterolemia were also included as controls. Laboratory studies were performed in the Department of Clinical Laboratory in Plovdiv Medical University. Total cholesterol, triglycerides, the cholesterol in HDL and the cholesterol in LDL were determined with direct automatic analysis. Apoproteins were determined using the immunoturbidimetric assay. The biochemical analyser Konelab 60i (Thermo Electron-Co, USA) was used to measure all lipids and apoproteins. ADMA was determined by direct immunoenzyme assay (ELISA).22 The flow-mediated vasodilation was measured on the brachial artery using a high frequency 7.5 MHz transducer of a Hewlett Packard echocardiograph. The brachial artery is at a depth of one to two centimetres. It is in the cubital fossa, lateral to the medial humeral epicondyle and medial to the tendon of the biceps. The diameter of the brachial artery was measured by marking the leading edge of the intima-lumen surface of the proximal wall to the lumen-intima surface of the distal wall.23-25 Measuring was done with an automatic computer programme MedicalSoft. IMT.lab by fixing with marker the initial point of the leading edge of the intima lumen surface of the proximal wall and the terminal point 10 mm away from the

23

flow-mediated vasodilation in severe hypercholesterolemia

first and measuring automatically the distance to the lumen intima surface of the distal wall at the same distance. The automatic measurement of the diameter of the brachial artery is more precise and has better reproducibility than manual measurement26 (Fig. 1).

Results

The calculated mean values of the components of a routine lipid profile in both studied groups differed significantly. The results are given in Table 1. The abnormalities in the routine lipid profile affected the values of LDL which is a generalising lipid param-

Figure 1. Automatic measurement of brachial artery diameter.

The patient is positioned supine and his nondominating arm is investigated. The blood pressure is taken using a mercury apparatus for arterial blood pressure. The patient’s hand is fixed in a splint to limit the movement of the artery during measurement. The algorithm of the study is presented in Diagram 1.27-29 ALGORITHM OF FLOW-MEDIATED VASODILATION I. BASELINE PHASE 1. DIAMETER OF THE BRACHIAL ARTERY (1-2 minutes) 2. RATE OF THE BRACHIAL ARTERY (1-2 minutes) II. CUFF PRESSURE PHASE DIAMETER OF THE BRACHIAL ARTERY AND RATE OF BRACHIAL ARTERY AT 1, 2, 3, 4, 5 MINUTES (each phase lasts 30 sec)

Diagram 1. Phases of the study of flow-mediated vasodilation.

Statistical analysis was performed using the following statistical methods: 1. The Student t-test was used to test for the significance of difference of quantifiable parameters with normal distribution. 2. Correlation analysis was used to test whether there was a valid statistically significant correlation between quantifiable variables read on the relative scale. The coefficient of linear correlation r (Pearson’s coefficient) was calculated.

24

eter. The LDL cholesterol was determined directly and the error resulting from the Friedewald formula was eliminated. The analysis of the atherogenic lipid indices of the two groups - patients and controls supports the alternative hypothesis (H1), meaning that there was a significant difference because of the higher values measured in the patients. The error in the derivative atherogenic indices of LDL was minimal because they were determined directly. We found a significant difference in the levels of apoproteins between patients and controls. In all patients the Apo-B/Apo A-I ratio was greater than 1, that is, the patients were at very high risk of heart disease.2 The mean values of ADMA calculated at baseline in the groups differed significantly. There was no significant difference in the values of the blood glucose between the two groups: 4.5 ± 0.01 mmol/l in patients versus 4.4 ± 0.04 mmol/l in controls (p > 0.05). The mean FMD% at baseline differed significantly in the two groups (Table 2). The values for FMD% at baseline were lower than the values in HC reported in literature. This finding is connected with the fact that in our study sample the patients had severe HC and this is related with the reduction of FMD%. The results of the present study of FMD% are consistent with those reported in the literature: other authors have also found statistically significant differences between the patients with HC and controls with respect to FMD%.9 We found a significant very strong negative correlation between FMD% and ADMA, Apo B and Apo B/Apo A-I ratio. The univariate analysis

Folia Medica, XL, 1/2008 Table 1. Lipid profile, atherogenic lipid indices, apoproteins and ADMA in patients and controls

x ± Sx

Sx

t

58 50

8.76 ± 0.14 4.45 ± 0.04

1.54 0.43

29.08

< 0.0001

58 50

1.52 ± 0.49 0.69 ± 0.18

0.46 0.18

3.95

< 0.001

58 50

0.95 ± 0.02 1.42 ± 0.03

0.24 0.29

12.86

< 0.0001

58 50

6.78 ± 0.13 2.54 ± 0.04

1.42 0.41

31.34

< 0.0001

Не-HDL = LDL+0.78 Patients Controls

58 50

7.66 ± 0.11 3.32 ± 0.04

1.28 0.40

34.90

< 0.0001

Total cholesterol / HDL Patients Controls

58 50

8.28 ± 0.21 3.21 ± 0.05

2.34 0.57

22.65

< 0.0001

Triglycerides / HDL Patients Controls

58 50

1.75 ± 0.75 0.50 ± 0.07

0.80 0.17

9.45

< 0.0001

LDL / HDL Patients Controls

58 50

6.42 ± 0.18 1.86 ± 0.04

2.05 0.47

23.47

< 0.0001

58 50

1.20 ± 0.16 1.83 ± 0.18

0.01 0.01

26.88

< 0.0001

58 50

2.07 ± 0.35 1.17 ± 0.11

0.03 0.01

26.14

58 50

1.75 ± 0.38 0.64 ± 0.10

0.03 0.01

139.64

58 50

1.64 ± 0.04 0.47 ± 0.02

0.49 0.12

Lipid profile Total cholesterol Patients Controls

(mmol/l)

Triglycerides Patients Controls

(mmol/l)

HDL cholesterol Patients Controls

(mmol/l)

LDL cholesterol Patients Controls

(mmol/l)

Apoprotein A-I Patients Controls

(g/l)

Apoprotein B Patients Controls

(g/l)

Apo B/Apo A-I ratio Patients Controls ADMA Patients Controls

(µmol/l)

number

coefficient shows that 80% of the variations in the Аpо-В/Аpо-А1 ratio can be explained by variations in the values of ADMA (Fig. 2). There was also mild correlation between other studied biomarkers of the atherogenic risk that are

24.59

P

< 0.0001

< 0.0001

< 0.001

used routinely in clinical practice: total cholesterol (rxy = -0.233; P = 0.038), atherogenic lipid indices [TC/HDL (rxy = -0.313; P = 0.025); LDL/HDL (rxy = -0.322; P = 0.027)], and the flow-mediated vasodilation.

25

flow-mediated vasodilation in severe hypercholesterolemia

Table 2. FMD% in patients and controls FMD% numindices ber FMD (%) Patients Controls

58 50

x ± Sx

Sx

Discussion t

P

4.49 ± 0.62 0.05 < 0.001 - 49.21 8.64 ± 0.61 0.06

Hypercholesterolemic patients have an abnormal endothelial function; the cholesterol-induced endothelial dysfunction is reported in literature to be associated with LDL oxidation rather than with the concentration of LDL. This explains the statistically significant, though weak negative correlation between FMD% and TC and also the significant negative correlation with Apo B. It is well known that Apo B is more informative than the routine lipid profile. The significant negative correlation between FMD% and Apo B, the Apo B/Apo A-I ratio, and ADMA shows that these biomarkers are more strongly associated with the endothelial dysfunction than with the lipid profile components that are usually used in clinical practice. The pathophysiological relation between ADMA and eNOS (or FMD% accordingly) is the likely reason for the strong correlation between the two parameters. This finding suggests that there should be further studies to establish the causal relationship between them. Conclusions

We found statistically significantly higher values of ADMA in patients with severe HC in comparison with the controls. Of all studied atherogenic risk biomarkers in severe HC (routine lipid profile, apoproteins, ADMA) 80% of the variations in FMD% can be explained by changes in ADMA. Currently, there is enough evidence that shows that ADMA, as eNOS inhibitor, is not only a marker but also a participant in the pathophysiological process of atherosclerosis. References

Figure 2. Correlations between FMD% and some markers of the atherogenic risk in HC.

26

1. Vita JA, Keaney JF Jr. Endothelial function: a barometer for cardiovascular risk? Circulation 2002;106:640-2. 2. Walldius G, Jungner I. The apo B/apo A-I ratio: a strong, new risk factor for cardiovascular disease and a target for lipid-lowering therapy – a review of the evidence. J Intern Med 2006;259:493-519. 3. Böger RH, Vallance P, Cooke JP. Asymmetric dimethylarginine (ADMA): a key regulator of nitric oxide synthase. Atherosclerosis 2003;Suppl.4:1-3. 4. Böger RH. Asymmetric dimethylarginine (ADMA) modulates endothelial function-therapeutic implications. Vasc Med 2003;8:149-51. 5. Böger RH. Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med 2006;38(2):126-36. 6. Böger RH. The emerging role of asymmetric di-

Folia Medica, XL, 1/2008 methylarginine as a novel cardiovascular risk factor. Cardiovasc Res 2003;59(4):824-33. 7. Verma S, Buchanan MR, Anderson TJ. Endothelial function testing as a biomarker of vascular disease. Circulation 2003;108:2054-9. 8. Böger RH, Sydow K, Borlak J, et al. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells. Circ Res 2000;87:99-105. 9. Böger RH, Bode-Böger SM, Szuba A, еt al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 1998;98:1842-7. 10. Chowienczyk PJ, Watts GF, Cockgroft JR, et al. Impaired endothelium-dependent vasodilatation of forearm resistance vessels in hypercholesterolemia. Lancet 1992;340:14302. 11. Denke MA. Weighing in before the fight. Lowdensity lipoprotein cholesterol and non-high-density lipoprotein cholesterol versus apolipoprotein B and the best measure of therapy. Circulation 2005;112:3368-70. 12. Drexler H, Zeiher AM, Koster W, et al. Endothelial dysfunction in the coronary circulation in hypercholesterolemia. Circulation 1992;86 (Suppl I):117. 13. Bots ML, Westerink J, Rabelink TJ, De Koning EJP. Assessment of flow-mediated vasodilatation (FMD) of the brachial artery: effects of technical aspects of the FMD measurement on the FMD response. Eur Heart J 2005;26:363-8. 14. Kitamoto Sh, Egashira K. Endothelial dysfunction and coronary atherosclerosis. Curr Drug Targets Cardiovasc Haematol Disord 2004 Mar;4(1):13‑22. 15. Kobayeshi K, Akishita M, Yu W, et al. Interrelationship between non-invasive measuments of atherosclerosis: flow-mediated dilatation of brachial artery, carotid intima-media thickness and pulse wave velocity. Atherosclerosis 2004;173:13-8. 16. Mombouli JV, Vanhoutte PM. Endothelial dysfunction; from physiology to therapy. J Mol Cell Cardiol 1999;31:61-74. 17. Cooke JP. The endothelium: a new target for therapy. Vasc Med 2000;5:49-53. 18. Cooke JP. Therapeutic intervention in endothelial dysfunction: endothelium as a target organ. Clin Cardiol 1997;20 (Suppl II):45-51.

19. Anderson TJ, Uehata A, Gernard MD, et al. Close relation of endothelial function in the human coronary and peripheral circulation. J Am Coll Cardiol 1995;26:1235-41. 20. Favre A, Monpere C, Voger C, et al. How to improve primary prevention in asymptomatic high risk subjects. Eur Heart J 2004 (Suppl J) J59-J63. 21. Scientific Steering Committee of the Simon Broome Register Group. Risk of fatal coronary heart disease in familial hypercholesterolemia. BMJ 1991;303:893-6. 22. Schulze F, Wesemann R, Schwedhelm E, et al. Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay. Clin Chem Lab Med 2004;42:(12):1377-83. 23. Corretti MC, Anderson TJ, Benjamin EJ, et al. Guidelines for the ultrasound assessment of endothelialdependent flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol 2002;39:257‑­65. 24. Bots ML, Westerink J, Rabelink TJ, De Koning EJP. Assessment of flow-mediated vasodilatation (FMD) of the brachial artery: effects of technical aspects of the FMD measurement on the FMD response. Eur Heart J 2005;26:363-8. 25. Mannion TC, Vita JA, Keaney JF Jr, Benjamin EJ, Hunter L, Polak JF. Non-invasive assessment of brachial artery endothelial vasomotor function: the effect of cuff position on level of discomfort and vasomotor responses. Vascular Medicine 1998;3:263‑7. 26. Fritz HF, Juzy RV, Roy V, et al. Validation of automated computerized analysing system for measuring common carotid artery intima-media thickness by brightness mode ultrasound. Journal for Vascular Ultrasound 2005;29(1):21-6. 27. Barth J. Which tools are in your cardiac workshop? Carotid ultrasound, endothelial function and magnetic resonance imaging. Am J Cardiol 2001;87:8A‑14A. 28. Vladimirova-Kitova L. Non-invasive techniques for assessment of atherosclerosis. Bulgarian Cardiology 2006;12:12-20 (Bulgarian). 29. Vladimirova-Kitova L, Manukov I, Stefanov R, Nikolov F. Determination of intima-media thickness of the carotid artery by manual and automatic method. Bulgarian Journal of Cardiology 2006;2:63‑8.

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flow-mediated vasodilation in severe hypercholesterolemia

Оценка взаимосвязи между потокиндуцированной вазодилатацией и маркерами атерогенного риска при выраженной гиперхолестеролемии Л. Владимирова, И. Мануков, И. Сиракова , Т. Денева

Резюме Введение: В последние годы внимание в превентивной кардиологии направлено к первичной превенции у высоко рисковых пациентов, какими являются пациенты с выраженной гиперхолестеролемией. Имеющиеся в литературе данные об уровнях асимметрического диметиларгинина при выраженной гиперхолестеролемии, как и о зависимости потокиндуцированной вазодилатации биомаркерами атерогенного риска недостоточны. Цель: Работа ставит себе целью исследовать поток-индуцированную вазодилатацию липидами, апопротеинами и некоторыми сывороточными маркерами (эндотелиальное активирование) при выраженной гиперхолестеролемии. Материал и методы: Обследовано 58 пациентов с выраженной гиперхолестеролемией ( > 7.5 μmol/l) по критериям Simon-Broome на клинически «достоверный» и «вероятный» диагноз – «семейная гиперхолестеролемия». Все пациенты асимптоматичны. Обследовано также и 50 лиц без данных о гиперхолестеролемии в качестве контрольной группы. Результаты: Анализ элементов липидного профиля, атерогенных липидных индексов, аполипопротеинов

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обеих сопоставляемых групп подтверждает альтернативную гипотезу, т.е. налицо выраженная статистическая разница, определяющаяся более высокими стоимост ями при первой группе (Р < 0.0001). Вычисленные средние стоимости исходного уровня асимметрического диметиларгинина в обеих обследованных группах существенно различаются (пациенты – 1.64 ± 0.04 μmol/ l; контрольная группа – 0.47 ± 0.02 μmol/l; Р < 0.001). Вычисленные средние стоимости исходного уровня поток-индуцированной вазодилатации в обеих обследованных группах существенно различаются (пациенты – 4.49 ± 0.62%; контрольная группа – 8.64 ± 0.61%; Р < 0.001). Установлена статистически значимая очень сильная обратная корреляция потокиндуцированной вазодилатации с асимметрическим диметиларгинином, алипопротеином-В и индексом алипопротеин-В/алипопротеин-А1 (алипопротеин-В - rху = - 0.687; Р < 0.0001; индекс алипопротеинВ/алипопротиен-А1 - r ху = 0.518; Р < 0.0001; асимметрический диметиларгинин – rху = -0.895; Р < 0.0001). Заключение: Вычисленный коэффициент показывает, что 80% вариаций в стоимостях потокиндуцированной вазодилатации могут быть объяснены вариациями в стоимостях асимметрического диметиларгинина. Значительная обратная связь потокиндуцированной вазодилатации с алипопротеиномВ, индексом алипопротеин-В/алипопротеин-А1 и асимметрическим диметил-аргинином доказывает факт, что эти биомаркеры более сильно ассоциированы с эндотелиальной дисфункцией (самая ранняя функциональная абнормность сосудистой стенки), чем применяемые в ежедневной клинической практике элементы липидного профиля.

Folia Medica, XL, 1/2008

Autologous fat transplantation in the facial region – clinical and morphological studies of 30 cases Regina H. Khater, Pepa К. Atanassova¹, Yourii K. Anastassov

Unit of Plastic and Craniofacial Surgery, “St. George” University Hospital, Plovdiv, ¹Department of Anatomy, Histology and Embryology, Medical University, Plovdiv, Bulgaria ABSTRACT Objective: This study presents the results of a clinicomorphological study of autologous fat transplantation in the face region. The aim was to investigate and compare adipose tissue morphology after it was purified by two methods - centrifugation and serum lavage, and to find a correlation between the histological outcomes and the postoperative results. Patients and methods: The evaluation was performed on a series of 30 patients assigned into two groups of 15 patients. Centrifuged adipose tissue was transplanted in one of the groups, and noncentrifuged (saline solution washed) in the other group. In all cases fat was obtained by syringe liposuction of subgluteal zones, processed by one of the two methods and then transplanted. Part of the material was fixed between vitaline membranes and embedded in paraffin. Histological analysis was performed after hematoxylin eosin staining of the slides. Post-operative effect was assessed by comparison of the pre- and postoperative photos on a 4-grade scale. Results: The morphological analysis revealed that adipose tissue was damaged by neither of the purification methods but the serum lavage preserved greater number of non-differentiated fat cells and connective tissue fragments. Furthermore, our clinical results suggested clearly that the noncentrifuged adipose tissue transplantation was more advantageous than the other type of transplantation. Conclusion: The present study is an attempt to make a contribution towards a better understanding of the mechanisms involved in fat graft preservation. Key words: lipofillng, lipostructure, autologous fat grafting INTRODUCTION

One of the main goals of plastic surgery is reconstruction or/and refinement of appearance by acting on the morphology, volume and shape of tissues, all these done in full conformity with the established anthropometric criteria of beauty. In the past, a great variety of synthetic materials and allomaterials were used to achieve this goal, but today they have been replaced by natural products. The autologous fat grafting is an example of this modern tendency. The method was reported for the first time in 1893 by Neuber who used little pieces of fat for cicatrix refinements1 and afterwards it became an object of many clinical and morphological evaluations.2,3 The founder of modern lipofilling is SR Coleman who introduced his Lipostructure® technique in 1994.4,5 The author presented its three basic stages – syringe lipoaspiration, graft purification by centrifugation and reimplantation, introducing a method of free

fat cells transplantation and not en block adipose tissue transfer. Although the method has been proved to have a number of advantages such as immunological safety, simple realization, good repeatability, viability of the transplant and comparatively good results,2,3 a few controversial issues still remain unsolved. One of the major problems is the unpredictable rate of post-operative resorption and hence the variability of late results. According to literature data resorption varies between 30% and 70% 6,7 depending on various, still largely unspecified factors which later prompted a number of histological and clinical studies and a modification of the Coleman’s algorithm. It has been firmly established that atraumaticity at each stage of surgery and creation of optimal conditions for future revascularization are the factors of utmost significance for graft survival.6 Furthermore, the

Correspondence and reprint request to: R. Khater, Unit of Plastic and Craniofacial Surgery, “St. George” University Hospital, Medical University, Plovdiv, Bulgaria 15A Vassil Aprilov St., 4002 Plovdiv, Bulgaria Received 05 June 2007; Accepted for publication 30 January 2008 29

Autologous fat transplantation in the facial region ...

supremacy of syringe lipoaspiration over vacuum liposuction has now been scientifically proved to be the best way to achieve cell preservation.7-9 All that made us search for the possible causes of resorption in the second stage of Coleman’s algorithm – the transplant processing for reimplantation. We therefore decided to investigate in the present study the morphological characteristics of adipose tissues purified by centrifugation or by washing in saline solution (without centrifugation) and to assess the clinical results after transplantation of this tissue in the facial region. The aim was to compare morphologically the transplant materials prepared by the two methods and find any correlation with the final clinical effect of the surgery. PATIENTS AND METHODS

A total of 30 patients were recruited for the clinical and morphological evaluation of lipofilling which was jointly developed by the Unit of Plastic and Craniofacial Surgery and the Department of Anatomy, Histology and Embryology. In all cases autologous fat transplantation was performed in the facial region – 10 superior lip augmentations in combination with rhinoplasty, 13 cases of facial rejuvenation, 5 cases of correction of facial asymmetry and 2 of secondary correction of superior lip thickness in a bilateral cleft lip. The age of patients ranged between 15 and 47 years; only four patients were male. In all 30 cases we pursued improvement of the esthetical appearance. Interventions included the three stages of modern lipofilling (fat harvesting, purification and reimplantation) described above; the surgery was performed under general anesthesia. The first stage was bilateral deep liposuction from the subgluteal regions. No local infiltration was applied. Liposuction was performed with 10 cc Luer-lock syringes and blunt cannulas (2.6 mm internal diameter) with two wide apertures allowing free passage of adipocytes. In the next stage the harvested aspirate was divided into two parts in all 30 patients, then purified using two different methods – centrifugation or saline solution washing (without centrifugation). Centrifugation was performed according to Coleman’s technique: 3000 revolutions per minute for 3 minutes, resulting in sedimentation and formation of three layers – oil from the adipocytes degradation products at the top, blood products at the bottom and medium layer consisting of fat

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cells. Purification was considered complete after removal of the blood and oil. Washing with saline solution was performed until we obtained a blood-free solution. The last stage was reimplantation. Fifteen of the patients received serum adipose tissue washed in saline solution and the remaining 15 – centrifuged fat tissue. The mean average quantity varied according to patients’ needs from 3 cc to 25 cc. There were no complications during the postoperative period. Recipient site swelling and slight bruising of the donor site were noted. They disappeared by day 14. In all thirty cases samples of the materials processed by the two methods as described above were studied histologically. The adipose tissue was included between two vitaline membranes, fixed in 10% formalin and embedded in paraffin. The 5-µm-thick paraffin slices were stained with hematoxylin eosin. RESULTS

The follow-up period was one year. Clinical outcomes were documented by preoperative and postoperative photos taken at the regular visits during the first, third, and sixth month and one year. The images were obtained at a distance of one meter against a monochrome background by a 5-mega-pixel digital camera (2592х1944) with macro mode and automatic focus adjustment. In all patients there was evident subjective and objective improvement of the appearance at the end of week 4. After 14 weeks we observed various degrees of resorption regardless of the purification method applied. The analysis of clinical results was based on both the subjective assessment and the objective assessment of the postoperative effect. The latter was done by comparison of the pre- and post-operative photos, which were standardized by size, contrast, projection and illumination using Adobe Photoshop 8.0 (Adobe Systems Inc., San Jose, CA). Both patients and surgeons assessed the results using a 4-grade scale: 1 – excellent (the effect as anticipated), 2 – good (effect present but not as anticipated), 3 – without any changes and 4 worse. At the end of the first year subjective and objective analyses defined the results as excellent (1st grade) in 12 patients (Figs 1,2) and good (2nd grade) in 3 patients with noncentrifuged adipose tissue transplantation. The others to whom lipofill-

Folia Medica, XL, 1/2008 ing was performed with centrifuged adipose tissue received the following assessments – without any changes (3rd grade) – four cases (Fig. 3) and good (2nd grade) in the other 9 (Fig. 4). The histological study of the centrifuged adipose tissue samples (Fig. 5А) showed that the fat tissue had a typical lobular structure composed of mature adipocytes with normal size (40-60 µm) and outlines. These were unilocular cells with one big lipid drop occupying the whole cell volume and pushing the cytoplasm to the periphery. Nuclei were flatten, with no signs of picnosis and also located peripherally. Between them we observed single multilocular fat cells. They contained a few

tiny little droplets and a centrally located roundish nucleus. According to their configuration they were referred to as preadipose cells with their adipocyte differentiation still incomplete. The noncentrifuged adipose tissue (Fig. 5B) showed the same typical characteristics of lobules of mature (unilocular) adipocytes and single multilocular adipose cells as those described above. We also observed little parts of connective tissue preserved because the fat tissues were not centrifuged. They were made up of collagen fibres, fibrocytes and capillary vessels. Among them there were single multilocular cells – preadipocytes.

Figure 1. A patient with superior lip augmentation with noncentrifuged adipose tissue and rhinoplasty: А – preoperatively; B – 1 year postoperatively.

Figure 2. A patient with noncentrifuged fat grafting in the right temporal region: А - preoperatively – after operative treatment of retinoblastoma in childhood; B – 1 year postoperatively.

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Autologous fat transplantation in the facial region ...

DISCUSSION

The present study reports the clinical results of lipofilling in the facial region and the morphological characteristics of the graft purified by two different methods – this is the first research of this kind in the country. Many authors worldwide have done research on the technique of autologous fat grafting using different methods of investigation.3,10 They have all reached the same conclusion as we have – that the major problem is the variable grade of resorption and hence the unpredictability of the

postoperative effect. The factors widely believed to be of crucial importance in this respect are the traumatic fat harvesting and purification, the presence of blood and degradation products in the transplant provoking phagocytosis and the implantation of great quantities of fat which hinders the fast revascularisation of the transplant. The technique developed by Coleman in 1994 (lipostructure®) devises an algorithm that takes into account each of the factors we mentioned above. In spite of the achieved recognition and success, the technique has

Figure 3. A patient with superior lip augmentation with centrifuged adipose tissue and rhinoplasty: А – preoperatively; B – 1 year postoperatively.

Figure 4. A patient with superior lip augmentation and nasolabial fold correction with centrifuged adipose tissue and rhynoplasty: А – preoperatively; B – 1 year postoperatively.

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Folia Medica, XL, 1/2008 not solved the problem of resorption. That is why we addressed the problem of the different methods of adipose tissue processing. The comparison of the two sets of results (clinical and experimental) shows that graft washing with saline solution is clearly the more advantageous of the two methods. The histological analysis of the noncentrifuged adipose tissue shows presence of more preadipose cells (both between mature adipose cells and in the

connective tissue fragments), intact capillary vessels and connective tissue elements. According to the mechanisms of graft development in the recipient site, this finding should be conducive to the better survival of the graft - by future differentiation of preadipocytes, the positive effect of endothelial cells on adipocytes11 and the bigger chances of revascularisation. This was also confirmed by the clinical results which were in favour of the noncentrifuged

Figure 5. Adipose tissue. Hematoxylin eosin staining, magnification x 200: А – centrifuged; B – noncentrifuged.

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Autologous fat transplantation in the facial region ...

adipose tissue grafting. The post operative effect in this case was considered very good. CONCLUSIONS

In conclusion both methods do not damage the adipose tissue but flushing with saline solution leads to the preservation of more connective tissue fragments and preadipocytes and increases the chances of graft survival. The immature adipose cells could even lead to de novo adipose tissue accumulation in the recipient site. As the clinical results are in favor of washing with saline solution we believe that this type of purification is better than the one done by centrifugation. REFERENCES

1. Neuber GA. Fett transplantation. Verl Dtsch Ges Chir 1893;22:66. 2. Mojallal A, Foyatier J.-L. Historique de l’utilisation du tissu adipeux comme produit de comblement en chirurgie plastique. Ann chir plast esthet 2004;(49):419-25. 3. Mojallal A, Breton P, Delay E, Foyatier J.-L. Greffe d’adipocytes: applications en chirurgie plastique et esthetique. In: Foyatier J.-L. Encyclopedie MedicoChirurgicale: Techniques chirurgicales. 1st ed. Elsevier SAS; 2004:45-125.

Аутологическая трансплантация жировой ткани в области лица – к л ини к о - м ор ф ол о г и ч ес к ие исследования 30 случаев Р. Хатр, П. Атанасова, Ю. Анастасов,

Резюме Цель: Настоящая работа представляет результаты клинико-морфологического исследования аутологической трансплантации жировой ткани в области лица и ставит себе целью исследовать морфологию жировой ткани, обработанной центрифугированием или сывороточным промыванием (lavage), при чем сравниваются результаты обоих методов воздействия и их корреляция с конечным эффектом интервенции. Методы и материалы: Эксперимент поставлен на 30 пациентах, разделенных на две группы по 15. Первой группе пациентов трансплантирована центрифугированная жировая ткань, а другой – нецентрифугированная (промытая физиологической

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4. Coleman SR. Long-term survival of fat transplants: controlled demonstrations. Aesthetic Plast Surg 1995;19:421-5. 5. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg 2001; 28:111-9. 6. Carpeneda CA, Ribeiro MT. Study of the histological alterations and viability of the adipose graft in humans. Aesthetic Plast Surg 1993;17:43-7. 7. Har-Shai, Lindenbaum ES, Gamliel-Lazarovich A, Beach D, Hirshowitz B. An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects. Plast Reconstr Surg 1999;104:945-54. 8. Mojallal A, Foyatier J.-L. Facteurs influenсant la survie de la greffe d’adipocytes. Annales de chirurgie plastique esthetique 2004;49:426–36. 9. Lalikos JF, Li YQ, Roth TP, Doyle JW, Matory WE, Lawrence WT. Biochemical assessment of cellular damage after adipocyte harvest. J Surg Res 1997;70:95. 10. Jauffret JL. Utilisation de la graisse autologue en chirurgie plastique et esthétique: la technique de S. R. Coleman. [thèse de doctorat d’État en médecine], Marseille: Universite de la mediterranee: faculte de medicine de Marseille;1998. 11. Aoki S, Toda S, Sakemi T. Culture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro. Cell structure and function 2003;28:55-60.

сывороткой). Во всех случаях материал получен посредством липоаспирации (с помощью спринцовки) от пелвеотрохантерной зоны, подвергнут одному из двух типов обработки и трансплантирован. Часть материала включена в желточные (вителинные) мембраны и парафин. На парафиновых срезах, окрашенных гематоксилином и еозином, проведен гистологический анализ, а постоперативный эффект оценен по 4-степенной шкале при сравнении фотодокументации. Р езультаты : Морфологическое исследование установило, что и оба метода обработки не поражают жировую ткань, но при сыворочном лаваже сохраняется бóльшее количество недифференцированных жировых клеток и соединительнотканных элементов. С другой стороны клинические результаты показали тенденцию в пользу липофилинга нецентрифугированной жировой тканью. Заключение: Своим исследованием авторы хотели бы способствовать более углубленному пониманию механизмов сохранения трансплантата.

Folia Medica, XL, 1/2008

Central corneal thickness measurements in ocular hypertension, primary open angle glaucoma, glaucoma suspects and control subjects Marin A. Atanassov, Marieta Iv. Konareva-Kostianeva

Department of Ophthalmology, Medical University, Plovdiv, Bulgaria ABSTRACT Purpose: To compare central corneal thickness (CCT) in patients with ocular hypertension (OH), primary open-angle glaucoma (POAG), glaucoma suspects and control subjects and to determine if there is a correlation between CCT and age. Design: Prospective study. Patients and methods: CCT was evaluated in 50 eyes of 25 OH patients (mean age 52.4 ± 1.6 yrs, x ± Sx ) who were allocated into group 1, in 26 eyes of 13 glaucoma patients (mean age 64.1 ± 1.0 yrs) – group 2, 46 eyes of 23 glaucoma suspects (mean age 60.1 ± 1.3 yrs) – group 3, and 144 eyes of 72 control subjects (mean age 61.4 ± 0.9 yrs) – group 4. CCT was measured using an ultrasonic pachymeter (Pach IV, Accutome). Results: The OH patients had a mean CCT of 582.88 ± 5.51 µm. The mean CCT for the glaucoma patients was 552.38 ± 5.90 µm, for the glaucoma suspects – 551.04 ± 4.23 µm and for the controls – 549.47 ± 3.07 µm. There was significant difference in age between group 1 and group 2 patients (P = 0.007) and between group 1 and group 4 patients (P = 0.001). CCT in the eyes with OH was significantly greater than that in POAG eyes (P = 0.003), in glaucoma suspects eyes (P < 0.001) and in control eyes (P < 0.001) (ANOVA, with Bonferroni correction). Conclusions: The comparative evaluation of CCT in patients with OH, POAG, glaucoma suspects and controls shows that CCT is the thickest in patients with OH. There is a correlation between CCT and age - younger patients possess thicker corneas. Our results suggest that CCT should be taken into account when assessing the risk for the development of glaucoma in OH patients. Key words: central corneal thickness, ocular hypertension, glaucoma, intraocular pressure Introduction

The results from the Ocular Hypertension Treatment Study (OHTS) made it possible to identify the basic risk factors for development of glaucoma in patients with ocular hypertension.1,2 The central corneal thickness is among the established significant factors determining the progression of OH into POAG. Ocular hypertension is defined as a condition of increased intraocular pressure (IOP), normal appearance of the optic disks, with no asymmetry and no change in visual acuity and visual fields. IOP in ocular hypertension is in the range of 21 to 29 mm Hg (for USA: 22-29 mm Hg - mean 25.5 mm Hg, and for Europe: 21-27 mm Hg - mean 24.0 mm Hg). It has been estimated that between 4% and 7% of the population over 40 years of age have elevated IOP without any detectable structural

or functional changes. In any case of ocular hypertension one should take into consideration the individual basic risk factors (age, IOP, vertical cup/disk ratio, pattern standard deviation (PSD) from Humphrey visual field, ССТ), in order to determine the necessity for topical hypotensive therapy. Patients with OH needing IOP lowering treatment are those with: - IOP ≥ 25 mm Hg, even in the absence of other risk factors; - IOP < 25 mm Hg, with cup-disc ratio > 0.48; - IOP < 25 mm Hg and CCT < 553 µm. The central corneal thickness in the general population varies in a broad range (450-750 µm) and shows a Gaussian distribution with a peak between 551 µm and 575 µm.

Correspondence and reprint request to: M. Atanassov, Department of Ophthalmology, Medical University, Plovdiv, Bulgaria 15A Vassil Aprilov St., 4002 Plovdiv, Bulgaria Received 11 December 2007; Accepted for publication 30 January 2008

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Central corneal thickness measurements ...

Central corneal thickness influences the IOP measurement.3 In the cases of ССТ below or over the mean (approx. 555 µm), there are obvious errors in the measured IOP. This is due to the fact that the Goldmann applanation tonometer (GAT) has been designed and calibrated to produce accurate measurements in eyes with established ССТ, radius of corneal curvature and corneal rigidity. In thin corneas the measured IOP is low while in thick corneas it is high. This is the reason nomograms have been designed to compensate the corneal thickness effect on GAT by recalculating the GAT readings according to the CCT. In thick corneas a value (mm Hg) is subtracted depending on the measured CCT over the mean. In OH patients without changes in the optic disk, retinal nerve fiber layer or visual field, the measurement of CCT is absolutely necessary.4 It is perhaps the thick corneas in some patients without any structural or functional glaucomatous changes that causes the elevated IOP in them.

acuity, GAT, slit lamp examination, evaluation of the optic disc with + 90 D lens biomicroscopy and automated computed perimetry with Humphrey field analyzer (program 30-2 with SITA). The data were statistically analysed using Statistical Package for Social Science (SPSS) version 11.0.1 (SPSS Inc., Chicago, IL, USA) with ANOVA with Bonferroni correction. Results

The mean values of CCT in the studied groups were: in the OH group (1) - 582.88 ± 5.51 µm ( x ± Sx ), in the POAG group (2) - 552.38 ± 5.90 µm, in the glaucoma suspects group (3) - 551.04 ± 4.23 µm and in the control group (4) - 549.47 ± 3.07 µm. Fig. 1 presents the CCT distribution in the four groups in a box plot. The eyes with OH had significantly thicker central corneas compared with the glaucoma eyes (t = 3.48, P = 0.003). The difference was also significant between group 1 (OH) and group 3 (POAG suspects) (t = 4.53, P < 0.001) and between group 1 (OH) and group 4 (controls) (t = 5.44, P < 0.001) (ANOVA, with Bonferroni correction). Only 8 eyes (16%) with OH had CCT values under 550 µm as shown in the histogram of CCT for group 1 (Fig. 2). The IOP was significantly higher in the OH, POAG, and glaucoma suspects groups compared to the control group (Р < 0.001). There was a significant difference in the visual acuity between the glaucoma group and the other three groups (P = 0.001). The mean age of the OH patients was lower in comparison with the other three groups and significantly different from the POAG (P = 0.007) and control groups (P = 0.001). There was a negative correlation between CCT and age (r = -0.18, P = 0.003). Fig. 3 presents the CCT distribution by age with the regression line. Thicker corneas are associated with younger age – the correlation is weak, but statistically significant.

Aim

To compare the central corneal thickness (CCT) in patients with ocular hypertension, primary open angle glaucoma, suspected primary open angle glaucoma (sPOAG), and healthy controls and to find if there is a correlation between CCT and age. Patients and Methods

The present study included 25 patients with OH (50 eyes, mean age 52.4 ± 1.6 yrs, x ± Sx , group 1), 13 POAG patients (26 eyes, mean age 64.1 ± 1.0 yrs, group 2), 23 patients with suspected POAG (normal IOP and no visual field changes, but vertical C/D ratio > 0.4) (46 eyes, mean age 60.1 ± 1.3 yrs, group 3), and 72 control subjects (144 eyes, mean age 61.4 ± 0.9 yrs, group 3). The CCT of the eyes of all groups was measured with an ultrasonic pachymeter (Pach IV, Accutome).5 All study subjects (n = 113) were given a complete ophthalmic examination, including visual

Table 1. Mean values of IOP and visual acuity of the studied groups Group Ocular hypertension POAG Glaucoma suspects Control group

36

Eyes (n) 50 26 46 144

IOP

Visual acuity

x

Sx

x

Sx

19.98 21.53 20.35 16.51

0.51 1.05 0.72 0.12

0.95 0.69 0.95 0.80

0.02 0.07 0.02 0.03

Folia Medica, XL, 1/2008 800

700

700

600 600

500

CCT

500

400 Glaucoma suspects Glaucoma

CCT

OHT

Controls

400 20

GROUPS

Figure 1. Box plot of ССТ for the studied groups. 12

30

40

50

60

70

80

AGE

Figure 3. Distribution of CCТ by age with a regression line.

10

8

6

4

2

0 520

540

560

580

600

620

640

660

680

CCT

Figure 2. Histogram of ССТ in group 1 (eyes with OH). Discussion

Despite the fact that CCT is highly variable in normal subjects, in our study we found higher values of CCT in the OH group. Our results are consistent with the results of other studies: Velten IM (586 µm), Phillips LJ (595 µm), Wu LL (582 µm), Bechmann M (593 µm).6-9 Kniestedt C, et al. and Aghaian F, et al. have also found increased CCT in OH patients and Dave H, et al. have reported

even a mean CCT value of 624 µm in their OH group.10-12 As IOP measured by GAT is higher in eyes with thick corneas, pachymetry is indicated in all patients with OH in order to acquire a “corrected” IOP. The IOP value corrected by CCT may influence a physician’s decision to initiate or modify a treatment.13 Some sources recommend combined measurement of IOP and CCT for calculating the exact IOP9, while others do not advise the routine “correction” of IOP by an algorithm.14 Dave H. points out that in the future CCT measurements will be considered in glaucoma patients along with IOP measurement and computed perimetry.12 Brandt JD defines ССТ as part of the complete ophthalmic examination which provides information about the risk of glaucoma.15 There is still controversy about the need to correct IOP reading according to CCT in all patients with established or suspected glaucoma. Even those who oppose the “corrected” IOP as routine practice point out the need of pachymetry in cases of suspected glaucoma.14 The analysis of risk factors in the Ocular Hypertension Treatment Study shows that patients with thin corneas and elevated IOP are particularly at risk for developing glaucoma – even more than patients with thick corneas and high cup/disk ratio.1 Some authors claim that CCT has little influence on the diagnosis of POAG8 and consider it necessary to take into account other factors besides CCT, like corneal and 37

Central corneal thickness measurements ...

scleral rigidity6. It has been established that young individuals have thicker corneas16 while corneas in older people are thinner11. The group of consecutive patients with OH had a smaller mean age than the other studied groups which can account for the negative correlation between CCT and age. Our results for CCT are consistent with those found by other Bulgarian authors.17 Petkova N, et al. have found by ultrasonic pachymetry a mean CCT of 554.3 µm for a group of initial glaucoma patients and a mean CCT of 554.3 µm for a group with advanced glaucoma patients. In Bulgarian ophthalmic literature several authors point out the importance of CCT and recommend that CCT measurement should be made a mandatory part of the glaucoma diagnostic work–up – in patients with suspected glaucoma, after corneal refractive surgery, ocular hypertension or normal tension glaucoma.18-21 Conclusions

The comparative evaluation of CCT in patients with OH, POAG, suspected glaucoma and healthy controls shows that CCT is greater in patients with OH. There is correlation between CCT and age – in younger patients thicker corneas are observed. Our results show that CCT should be taken into consideration in the analysis of risk factors for the development of glaucoma in OH patients. References

1. Gordon MO, Beiser JA, Brandt JD, et al. for the Ocular Hypertension Treatment Study Group. The Ocular Hypertension Treatment Study: baseline factors that predict the onset of primary open-angle glaucoma. Arch Ophthalmol 2002;120:714-20. 2. Kass MA, Heuer DK, Higginbotham EI, et al, for the Ocular Hypertension Treatment Study Group. The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch Ophthalmol 2002;120:701-13. 3. Brandt JD, Beiser JA, Kass MA, Gordon MO. Central corneal thickness in the Ocular Hypertension Treatment Study (OHTS). Ophthalmology 2001;108:1779-88. 4. European Glaucoma Society. Terminology and Guidelines for Glaucoma. IInd Edition. Dogma Srl: Savona; 2003. 5. Realini T, Lovelace K. Measuring central corneal thickness with ultrasound pachymetry. Optom Vis Sci 2003;80(6):437-9.

38

6. Velten IM, Bergua A, Hom FK, Junemann A, Korth M. Central corneal thickness in normal eyes, patients with ocular hypertension, normal-pressure and openangle glaucomas – a clinical study. Klin Monatsbl Augenheilkd 2000;217 (4):219-24. 7. Phillips LJ, Cakanac CJ, Eger MW, Lilly ME. Central corneal thickness and measured IOP: a clinical study. Optometry 2003;74(4):218-25. 8. Wu LL, Suzuki Y, Araie M. Central corneal thickness and intraocular pressure in cases with ocular hypertension and glaucoma. Zhonghua Yan Ke Za Zhi 2000;36 (6):438-41. 9. Bechmann M, Thiel MJ, Roesen B, Ullrich S, Ulbig MW, Ludwig K. Central corneal thickness determined with optical coherence tomography in various types of glaucoma. Br J Ophthalmol 2000;84 (11):1233-7. 10. Kniestedt C, Lin S, Choe J, Nee M, Bostrom A, Sturmer J, Stamper RL. Correlation between intraocular pressure, central corneal thickness, stage of glaucoma, and demographic patient data: prospective analysis of biophysical parameters in tertiary glaucoma practice populations. J Glaucoma 2006;15:91-7. 11. Aghaian F, Choe JE, Lin S, Stamper RL. Central corneal thickness of Caucasians, Chinese, Hispanic, Filipinos, African Americans, and Japanese in glaucoma clinic. Ophthalmology 2004;111:2211-19. 12. Dave H. Kutschan A, Pauer A, Wiegand W. Measurements of corneal thickness in glaucoma patients. Ophthalmology 2004; 101(9): 919-24. 13. Detry-Morel M. Use of corneal pachymetry in ocular hypertension and chronic glaucoma. Bull Soc Belge Ophthalmol 2004; 293: 35-43. 14. Sullivan-Mee M, Halverson KD, Saxon GB, Saxon MC, Shafer KM, Sterling JA, Sterling MJ, Qualls C. The relationship between central corneal thickness – adjusted intraocular pressure and glaucomatous visual-field loss. Optometry 2005; 75 (4): 228-38. 15. Brandt JD. Corneal thickness in glaucoma screening, diagnosis, and management. Curr Opin Ophthalmol 2004; 15(2): 85-9. 16. Nemesure B, Wu SY, Hennis A, Leske MC, for the Barbados Eye Study Group. Corneal thickness and intraocular pressure in the Barbados eye studies. Arch Ophthalmol 2004; 122(3):425-6. 17. Petkova N, Kostova S, Rankova Ch, Marinov A. Central corneal thickness in patients with primary open angle glaucoma. Bulgarian Ophthalmologic Review 2005;1:3-5 (Bulgarian). 18. Guguchkova P, Dokov S, Trendafilova E. Measurement of central corneal thickness in diagnosing glaucoma. Bulgarian Ophthalmologic Review 2004;2:6-11 (Bulgarian).

Folia Medica, XL, 1/2008 19. Dokov S, Angelov B. Measuring central corneal thickness with ultrasound biometry in primary open angle glaucoma. Ophthalmologic Reference Bulletin 2004;6:22-8. 20. Atanasov M, Chilova-Atanasova B. Central corneal thickness – a risk factor for the development of

glaucoma or a source of errors in IOP measurement. Bulgarian Ophthalmologic Review 2005;1:48-51. (Bulgarian) 21. Kostova S, Petkova N. Central corneal thickness in patients with advance open angle glaucoma. Bulgarian Ophthalmologic Review 2006;1:13-18. (Bulgarian)

Центральная толщина роговицы при глазной гипертензии, первичной открытоугольной глаукоме, глаукоме под сомнением и у лиц в качестве контрольной группы

Для измерения ССТ использован ультразвуковый пахиметр (Pach ІV, Accutome). Результаты: Пациенты с ГГ показали среднюю стоимость ССТ 582.88 ± 5.51 μm. Соответственно средняя ССТ у больных с глаукомой была 552.38 ± 5.90 μm, у пациентов с глаукомой под сомнением – 551.04 ± 4.23 μm и у лиц контрольной группы – 549.47 ± 4.23 μm. По отношению возраста первая группа статистически значимо различается от второй группы (Р = 0.007) и от ІV группы (Р = 0.001). Глаза с гипертензией отличаются значимо бόльшей толщиной роговицы по сравнению с глазами с ПОУГ (Р = 0.003), с глазами с глаукомой под сомнением (Р < 0.001) и с глазами контрольной группы (Р < 0.001) (ANOVA, with Вonferroni correction). З аключение : Сравнительная оценка ССТ у пациентов с ГГ, ПОУГ, глаукомой под сомнением и у лиц контрольной группы показывает, что ССТ самая большая у пациентов с ГГ. Отмечена связь между ССТ и возрастом - у более молодых пациентов наблюдаестя бóльшая толщина роговицы. Полученные авторами результаты показывают, что ССТ следует принимать во внимание при анализе факторов риска развития глаукомы при глазной гипертензии.

М. Атанасов, М. Конарева-Костянева

Резюме Цель: Сравнить центральную роговичную толщину – central corneal thickness (ССТ) у пациентов с глазной гипертензией (ГГ), первичной открытоугольной глаукомой (ПОУГ), с глаукомой под сомнением и у здоровых лиц в качестве контрольной группы, а также и определить существует ли корреляция ССТ и возрастом. Дизайн: Проспективное исследование. Методы: ССТ определена у 25 больных (50 глаз) с ГГ (средний возраст 52.4 ± 1.6 г.) – І группа; у 13 больных с глаукомой (26 глаз; средний возраст 64.1 ± 1.0 г) – ІІ группа; у 26 пациентов с глаукомой под сомнением (46 глаз; средний возраст 60.1 ± 1.3 г.) – ІІІ группа и у 72 лиц контрольной группы (144 глаза; средний возраст 61.4 ± 0.9 г.) – ІV группа.

39

Role of neuronavigation in the resection of intracranial arteriovenous malformations

Clinical Investigations Role of neuronavigation in the resection of intracranial arteriovenous malformations Yavor P. Enchev, Rumen V. Popov, Kiril V. Romansky, Marin B. Marinov, Ventzeslav A. Bussarsky Clinic of Neurosurgery, “St. I. Rilsky” University Hospital, Medical University, Sofia, Bulgaria

Abstract Aim: The aim of this study was to investigate the effect of neuronavigation on the following parameters: “skin incision”, “craniotomy”, “intraoperative anatomical orientation”, “dissection guiding”, “localization of the pathological formation”, “assessment of the degree of resection” and “duration of surgical procedure” in the resection of intracranial arteriovenous malformations (AVMs) and to specify the indications for application of neuronavigation in the surgical treatment of AVMs. Methods: Five neuronavigation-assisted resections of intracranial AVMs were performed between March 2003 and December 2005 at the Clinic of Neurosurgery of St. I. Rilsky University Hospital, Medical University, Sofia. The female/male ratio in the series was 2:3 (40%:60%). The patients’ mean age was 28.2 ± 18.27 (range 10 to 56) years. The study was prospective in design. Patients were examined and followed in a standardized manner. Results: Arteriovenous malformations were totally removed in all 5 cases of neuronavigationassisted resections. We did not observe any morbidity or mortality associated with the method. Neuronavigation allowed precise localization and individual design of the skin incision and craniotomy. Neuronavigation facilitated the surgeon during intraoperative anatomical orientation. Dissection guiding, localization of the formation and assessment of the degree of resection were assessed as markedly useful. This resulted in reduced duration of surgery compared to conventional neurosurgery. Conclusions: In AVMs resection neuronavigation optimizes surgical approach by visualizing the relationship of AVMs to the skull and various critical anatomical structures. Deep vessel components and nidus margins, especially in the vicinity of the ventricles can be identified precisely. Neuronavigation can improve the early post-operative results in cerebral AVMs reducing operating time and blood loss. Key words: AVMs, neuronavigation, frameless stereotaxy, image-guided resection, minimally invasive neurosurgery. Introduction

There is sparse information in the literature on the application of neuronavigation in the surgery treatment of AVMs.1-8 Muacevic et Steiger1 described a method of AVMs resection using neuronavigation (VectorVision 2) for the localization and clipping of the feeding arteries aiming at intranidal decompression before the formal nidal resection. Russell, et аl.2 were the first to report their clinical study on a control group with the aim to determine the effect of MRI-based frameless stereotaxy on the surgical results in AVMs. In their series of MR image

data transferred to the navigation system all the feeding arteries over 1.25 mm in diameter were easily identified. That information helped the surgeon to get an idea of the most crucial nidal segments. Russell et аl. reported that neuronavigation was most useful in the resection of deeply localized AVMs with minimal or without cortical representation.2 They found significant reduction of the intraoperative blood loss and the operating time in their navigation-assisted interventions (22/44 cases). In a series of four cases of small AVMs associated with significant bleeding Coenen et аl. used CT-angiographic data for their neuronavigation

Correspondence and reprint request to: Yavor P. Enchev, Clinic of Neurosurgery, “St. I. Rilsky” University Hospital, Medical University, Sofia; E-mail:[email protected] 15 Acad. Iv. Geshov Blv. 1431 Sofia, Bulgaria 40 Received 16 July 2007; Accepted for publication 30 January 2008

Folia Medica, XL, 1/2008 platform.4 They reported that CT-angiography had the advantages of visualizing feeding and draining AVMs vessels simultaneously. What is more, CTangiography gave a nice vessel contrast even when bleeding from the AVMs had already occurred. That is why the authors pointed out that CT-angiography excels MRI as a method of investigation in neuronavigation-assisted localization of AVMs in the immediate proximity of considerable intraparenchymal bleeding. They reported one more advantage of CT-angiography in neuronavigationassisted surgery of AVMs - that technique allowed simultaneous identification of hemodynamic-dependent aneurisms thus helping the identification of the feeding arteries. Aim

The aims of this study were to investigate the effect of neuronavigation - either positive (“+”) or missing (“0”) – on the following parameters: “skin incision”, ”craniotomy”, ”intraoperative anatomical orientation”, “guiding of dissection”, “localization of the pathological formation”, “assessment of the degree of resection” and “duration of surgical procedure” in AVMs; to specify the indications and contraindications for the application of neuronavigation in AVMs; to investigate the early results after application of the system in AVMs; to analyze the different stages of navigated neurosurgical intervention as well as the possible sources of errors and the ways of overcoming them in AVMs. Patients and methods

Clinical material Five patients with AVMs who underwent neuronavigation-assisted surgical treatment between March 2003 and December 2005 were included in the present study. Female to male ratio in the series was 2 : 3 (40% : 60%). The patients’ mean age was 28.2 ± 18.27 (range 10 to 56) years. The study had a prospective character and included all neuronavigation-assisted AVM resections that were realized at the Clinic of Neurosurgery during the above mentioned period of 2 years and 10 months. The patients were examined and followed up in a standardized manner. The following indicators were studied prospectively: effect of neuronavigation at each concrete stage of the surgical intervention (skin incision, craniotomy, intraoperative anatomical orientation, guiding of dissection, localization of the pathological formation, and assessment of the degree of resection), effect of

neuronavigation on the reduction of the operating time and specific peculiarities of cranial neuronavigation in AVMs. Effect

of neuronavigation in the different stages of

AVMs Generally, the effect of neuronavigation concerning any particular stage of surgical intervention may be classified either as a positive one (“+” plus effect) or as a missing one (“0”- zero effect). Plus effect for a concrete stage is accepted in cases in which at least one of the potential effects of neuronavigation has been realized. The neuronavigation was assessed in relation to the following indicators: Skin incision: precise localization; individual design; reduced dimensions compared to the respective classic one; reduced blood loss; better cosmetic effect. Craniotomy: precise localization; individual design; reduced dimensions compared to the classic craniotomy; reduced blood loss; reduced duration compared to the conventional one; better cosmetic effect. Intraoperative anatomical orientation: orientation in dural venous sinuses, bridge veins, cerebral arteries and veins; orientation in cerebral cortex surface area (distances, gyruses, sulcuses and important functional zones); orientation in the direction of the cranial nerves; orientation in the anatomy of the ventricular system (form, dimensions, walls, intraventricular foramens of Monro and aqueduct); orientation in the conditions of pathological or postoperative changes of intracranial anatomy. Guiding of dissection: assessment of the distance to the pathological formation; assessment of the subarachnoidal cisterns – their dimensions, position and content; optimization of the position and dimensions of corticotomy; dissection specifying in relation to the underlying fibers (association, projection and commissural); preservation of the basal ganglia; assessment of the cranial nerves’ tracts and nuclei; maximally short and relatively non traumatic dissection concerning the cerebral vessel structures; dissection with reduced duration under the conditions of improved orientation and increased security. Localization of the pathological formation: precise localization of the pathological formation and/or the target of endoscopy; fast localization on the basis of a better orientation and more secure dissection with reduced duration; eliminated negative traumatic exploration of the surrounding cerebral parenchyma; reduced risk of intraoperative cerebral damage. Assessment of the degree of resection: evaluation of the border between pathological formation surgical intervention in

41

Role of neuronavigation in the resection of intracranial arteriovenous malformations

and cerebral parenchyma; evaluation of the residual tumor size. Duration of procedure: reduction of the surgical intervention duration concerning each particular stage and the entire procedure. Neuronavigation system (Vector Vision 2) Vector Vision 2 (BrainLab Heimstetten, Germany) neuronavigation system was used5,6 (Fig. 1). It is an intraoperative image-guided frameless non-mechanical (without mechanical arms) localizing system which is based on a passive reflection of infrared rays. The system software allowed the following: image fusion of two different techniques; outlining the object (lesion, inlet and outlet, trajectory, anatomical structures which are important for surgical approach); 3-dimensional reconstruction; virtual pointer elongation; mathematical calculations (dimensions, depth and volumes); registration in three different ways, “autopilot” performance; intraoperative documenting, etc. Neuronavigation procedure The neuronavigation procedure included the following steps5: pre-operative image study (most often СТ or МRI), transfer of image data to the work station, pre-operative analysis and planning of the surgical

intervention, fusion of image volumes of the different imaging techniques, transfer of already processed information to the navigation system, recording, surgical planning, intraoperative navigation, possibility of postoperative analysis based on intraoperative neuronavigation data. Results

Arteriovenous malformations of all five patients who underwent neuronavigation-assisted resection were removed totally (Fig. 2). We did not register any case of morbidity or mortality associated with the method. Frameless stereotaxy helped to optimize the localization and the dimensions of the skin incision and craniotomy in 4/5 patients. Neuronavigation was estimated as particularly useful for the intraoperative orientation and, namely, to differentiate feeding arteries from draining veins, to trace their orientation, as well as for guiding, dissection of the nidus (especially concerning its deep periventricular part) in 5/5 patients with AVMs. Intraoperative neuronavigation-assisted evaluation of the degree of resection was found to be necessary in 4/5 patients. We registered high application accuracy of neuronavigation comparing it with anatomical reference points. Reduced duration of neuronavigation-assisted AVM resection and reduced blood loss during surgery (no one of the patients needed blood transfusion) came as result of the cumulative positive effect of frameless stereotactic procedure. The positive effect of neuronavigation in AVMs on various parameters is shown in Table 1. Table 1. Positive effect of cranial neuronavigation in AVMs Indicators of surgical intervention

Figure 1. Neuronavigation system VectorVision 2 (ВrainLab, Неimstetten, Germany).

42

Positive effect (%)

Skin incision

80

Craniotomy

80

Intraoperative orientation

100

Guiding of dissection

100

Localization

100

Evaluation of the degree of resection

80

Reduction of operating time

100

Folia Medica, XL, 1/2008

а - CT scan with contrast medium before surgery.

b - MR-angiography visualizing the feeding arteries, nidus and draining arteries before surgery.

c - Conventional cerebral panangiography before surgery visualizing feeding arteries from the right middle cerebral artery, the nidus and the venous drainage (to sinus sagittalis superior mainly).

d - Intraoperative screenshots. Neuronavigation helps orientation (differentiation of feeding arteries from draining vessels and their direction) and leads guides nidal dissection.

e - Postoperative cerebral panangiography. No malformative cerebral vessels are visualized.

f - Postoperative CT imaging. No data about residual parts. Figure 2 (a,b,c,d,e,f). A patient with right frontoparietal AVM.

43

Role of neuronavigation in the resection of intracranial arteriovenous malformations

DISCUSSION

In contrast to the numerous publications that analyze the application of frameless stereotaxy in the surgical treatment of intracranial neoplasms, there is quite a limited number of reports about clinical investigations on neuronavigation-assisted resections of cerebral arteriovenous malformations (AVMs).1,2,4,7,8 One possible explanation is that conventional cerebral angiography is the gold standard for cerebral AVM image investigation and currently there is no method to introduce it into the neuronavigation platform. What is more, outlining AVM margins in MR images is not as distinct as outlining tumors, although rapid progress of МR technology will probably overcome this problem in the near future. Contrast MR-angiography, however, defines structural peculiarities of AVMs and demonstrates good resolution of the parenchymal anatomical characteristics. In our series (Table 2) we used 3-dimensional reconstructed AVM models, based on the fusion of contrast МR images with МR-angiography, which ensure ample pre-operative information as well as precise intraoperative interactive navigation. The segmented AVM was observed in various directions which facilitated the analysis of its relations to the cerebral parenchyma. Malformation depth and volume were measured. Useful information for the performance of well centered craniotomy and for the selection of the optimal surgical approach was achieved with pre-operative virtual simulation of the trajectory. The 3-dimensional reconstructions demonstrated considerable advantages over the 2-dimentional image slices when specifying the relationship between Table 2. Series of navigated AVMs resections Author and year

Number of navigated AVMs resections

Zamorano, et аl. 19968

4

Nakajima, et аl. 19977

6

Muacevic, et Steiger 19991

22

Russell, et al. 20022

22

Coenen, et аl. 20054

4

Present series

5

44

AVMs and the surrounding anatomical structures during surgery. The technique was recognized as markedly useful for AVMs that were localized near cortical zones of functional importance (in 3/5 cases). That resulted in a less invasive and more selective approach in comparison with the large flaps of the classic method. The main vessels and the nidus of the AVMs were easily localized. The intraoperative interactive visualization ensured constant feed-back which facilitated orientation and increased surgeon’s confidence. It was recognized that the application of neuronavigation-assisted resections of cerebral AVMs optimised skin incisions and craniotomy localization and size in 4/5 which corresponded to literature data.7 Like Muacevic et Steiger1 we used neuronavigation to differentiate and clip the feeding arteries aiming at intranidal decompression before formal nidal resection in 5/5 patients. No concomitant hemodynamicdependent aneurisms were found in the series. Such aneurisms were used by Coenen et аl. to help identification of feeding arteries.4 The ability to define nidal periphery and feeding arteries during AVM resections was qualified as a particular advantage of neuronavigation. It was most useful during the final stage of dissection in the vicinity of deeply situated periventricular margins. As a rule, in such cases the nidus limits the field of vision which may result in unpleasant bleeding. As AVMs were removed en block, cerebral dislocation was not found to be an essential problem. Neuronavigation resulted in shortened duration of procedure and reduced intraoperative blood loss in 5/5 AMV patients (which was in accordance with the results reported by Russell, et аl.2) That was most probably because of the smaller skin incisions and craniotomies, precise nidal margin localization as well as the pre-identification and early ligation of the feeding arteries. Neuronavigation was particularly useful for the resection of only one clinical case with AVM that was deeply situated and had minimal cortical representation. That was in conformity with the results of the most series.2,8 Precise cortical incision and dissection trajectory potentially lead to lower surgical morbidity rates through reduced unnecessary parenchymal exploration. The results of our study did not confirm the statement of Coenen et аl. that in AMVs near significant intraparenchymal bleeding MR-angiography is poorly informative for their neuronavigation-assisted localization.4 In the only case of our study it was evaluated to be effective.

Folia Medica, XL, 1/2008 Conclusions

In conclusion: in AVM resection frameless stereotaxy facilitates the performance of smaller and more accurate skin incisions and craniotomies. Neuronavigation optimizes surgical approach visualizing the interrelations between AVMs, the skull and some critical anatomical structures. Deep vessel components and nidal margins, especially those which are near the ventricles, can be identified precisely. The approach to deeply situated AVMs which are hidden under the cortical surface area can be planned in details. Frameless stereotaxy can improve the early surgical results in cerebral AVMs through reduction of the operating time and blood loss. We believe that greater series of AVM resections assisted by neuronavigation will probably reveal the obvious improvement of the surgical results. References

1. Muacevic A, Steiger HJ. Computer-assisted resection of cerebral arteriovenous malformations. Neurosurgery 1999;45:1164-70. 2. Russell S, Woo H, Joseffer S, et al. Role of frameless stereotaxy in the surgical treatment of cerebral arte-

Место нейронавигации при резекции интракраниальных артериовенозных мальформаций Я. Енчев, Р. Попов, К. Роминский, М. Маринов, В. Бусарский Резюме Цель: Настоящее исследование ставит себе целью изучить эффект нейронавигации на следующие параметры: “кожный разрез”, “краниотомию”, “интраоперативную анатомическую ориентацию”, “ведение диссекции”, локализацию патологической формации”, “оценку степени резекции”, “продолжительность оперативной интервенции” при артериовенозных мальформациях (АВМ) и уточнить индикации применения нейронавигации при АВМ. Методы: За период март 2003 – декабрь 2005 г. в Клинике нейрохирургии при УМБАЛ “Св. Ивана Рилского” осуществлено 5 нейронавигированных резекций интракраниальных АВМ. Соотношение женщины/мужчины -2/3 (40% /60%). Средний возраст больных 28.2 ± 18.27 (от 10 до 56 лет). Проведенное исследование несет проспективный

riovenous malformations: techniques and outcomes in a controlled study of 44 consecutive patients. Neurosurgery 2002;51(5):1108-28. 3. Bussarsky VA, Romansky KV, Marinov MB, et al. Intraoperative neuronavigation in the microsurgical excision of cerebral vascular malformations. Bulg Neurosurg 2004;9(l):10-14 (Bulgarian). 4. Coenen V, Dammert S, Reinges MH, et al. Imageguided microneurosurgical management of small cerebral arteriovenous malformations: the value of navigated computed tomographic angiography. Neuroradiology 2005;47:66-72. 5. Enchev YP. Application of neuronavigation in cranial neurosurgery [dissertation]. Medical University: Sofia; 2007. 6. Gumprecht HK, Widenka DC, Lumenta CB. BrainLab vectorvision neuronavigation system: technology and clinical experience in 131 cases. Neurosurgery 1999;44:97-104. 7. Zamorano L, Planells M, Jiang Z, et al. Vascular malformations of the brain: Surgical management using interactive image guidance. Neurosurg Clin N Am 1996;7:201-14. 8. Nakajima S, Atsumi H, Bhalerao A, et al. Computerassisted surgical planning for cerebrovascular neurosurgery. Neurosurgery 1997;41:403-10. характер. Больных исследовали и прослеживали стандартизованным способом. Результаты: АВМ удалены тотально во всех 5 случаях. Морбидитет и морталитет, связанные с методикой не наблюдались. Нейронавигация позволяет точную локализацию и индивидуальный дизайн кожного разреза и краниотомии. Нейронавигированные интраоперативная анатомическая ориентация, ведение диссекции, локализация формации и оценка степени резекции считаются особо полезными, так как редуцируют оперативную продолжительность по сравнению с конвенциональной нейрохирургией. Заключение: При резекции АВМ нейронавигация оптимизирует хирургический доступ, изображая отношения АВМ к черепу и к критическим анатомическим структурам. Глубокие сосудистые компоненты и нидусные границы, особенно в близости к вентрикулам, могут быть точно идентифицированными. Нейронавигация может улучшить ранние хирургические результаты при АВМ мозга посредством уменьшения оперативной продолжительности и кровопотери.

45

CLINICAL EVALUATION OF RESTORATIONS WITH SELF-ETCH ADHESIVE AND NANOFILLED ...

Dental Investigations CLINICAL EVALUATION OF RESTORATIONS WITH SELF-ETCH ADHESIVE AND NANOFILLED COMPOSITE IN CLASS I AND CLASS II CAVITIES Neshka A. Manchorova, Stoyan B. Vladimirov

Student study group: Zoya K. Donencheva, Ivan S. Drashkovich, Stanislav K. Manolov, Veselina I. Todorova Department of Operative Dentistry and Endodontology, Faculty of Dental Medicine, Medical University, Plovdiv ABSTRACT Introduction: In the era of adhesive restorative dentistry the principles of conventional cavity preparation have acquired a new meaning. The emphasis is shifted from “widening for preventive purposes” and preparation of macro-mechanical retention to “consciously limited” preparation. The clinical and biological durability of restorations depends on the adhesive bond and the quality of the composite material. Objective: The aim of the study was to conduct a clinical evaluation of restorations with a self-etch single-step adhesive and nanofilled composite in class I and class II cavities over a period of 15 months. Patients and Methods: The clinical trial involved 34 patient (volunteers) who received 76 posterior restorations of medium and deep cavities with the self-etch adhesive Adper Prompt L-Pop and nanofilled composite Filtek Supreme (3МESPE Dental products USA). All restorations were evaluated by the modified Ryge and Cvar criteria at 6- and 15-month intervals. Results: Loss of restorations with a self-etch single-step adhesive and nanofilled composite at 6 months and 15 months was 0% and 6.7%, respectively. The evaluation of discolouration in the restorations at 6 months revealed no significant changes (Р > 0.05, u = 1.72). There was certain deterioration in the colour at 15 months (Р < 0.05, u = 2.96). Statistically significant changes in the marginal discolouration were observed at 6 months (P < 0.05, u = 2.79) in comparison with the initial scores. During the 15-month follow-up the discolouration on the margins of the restorations was statistically nonsignificant compared to the 6-month follow-up (Р > 0.05, u = 0.36). There were no С (Charlie) scores recorded during the 15-month follow-up study within the continuing observation. Conclusion: The restorations with nanofilled composite and self-etch single-step adhesive fulfill the requirements for clinical use in posterior restorations. Key words: self-etch adhesive, nanocomposite, clinical evaluation

Introduction

In the era of adhesive restorative dentistry the principles of conventional cavity preparation are given a new meaning. Emphasis has shifted from “widening for preventive purposes” and preparation of macro-mechanical retention to “consciously limited” preparation. The preservation of the healthy dental

tissues while only removing the carious material using non-invasive methods is the philosophy of a new approach known as ‘minimally invasive preparation’. In this sense, the clinical and biological durability of restorations depends on the adhesive bond and the quality of the composite material.1 Conventional adhesive approaches to the dental

Correspondence and reprint request to: N. Manchorova, Department of Operative Dentistry and Endodontology, Faculty of Dental Medicine, Medical University, Plovdiv; E-mail: [email protected] 3 Hristo Botev blvd., Plovdiv 4002, Bulgaria 46 Received 12 January 2007; Accepted for publication 27 June 2007

Folia Medica, XL, 1/2008 tissues require accurate multi-step application with a complex clinical protocol.2 More recent singlestep, self-etch adhesive systems have combined the etching agent, primer and adhesive to form an all-in-one formulation and are applied simultaneously on the smear layer. There is disagreement in literature regarding the strength and durability of the bond which they form with the dental tissues.3 So far there have been few in vivo studies to provide evidence of the effectiveness of this generation of systems. In the past years composites have been increasingly used in the treatment of class I and class II carious defects. As a result of the introduction of nanotechnologies, a novel generation of composite materials has been created. Literature reports about less polymerization shrinkage and improved mechanical properties4 have attracted much attention to their clinical behaviour. We found insufficient information on the evaluation of restorations with nanofilled composites in clinical conditions possibly due to their novelty in dental practice. The inadequate information in literature as well as the absence of similar investigations in this country on clinical evaluation of restorations with self-etch adhesive and nanofilled composite for class I and class II cavities motivated the present study.

The aim of the study was to conduct a clinical evaluation of restorations with a self-etch single-step adhesive and nanofilled composite in class I and class II cavities over a period of 15 months.

molars from both tooth rows • Size of cavity: - the minimum occlusion width of the cavity is bigger or equal to ⅓ of the intercuspal distance; - complex cavity shapes including cuspal restoration; • Occlusion: all restored teeth occlude against an antagonist natural tooth, and restorations make contact (both in centric and functional occlusion); • Approximal contact: all class II restorations are in contact with the adjacent tooth; Cavity preparation for adhesive restoration was performed using rotary tools with water/air cooling. The adhesive system, Adper Prompt L- Pop, was applied according to the manufacturer’s recommendations. The cavities were obturated with FiltekSuprеme, applied in oblique increment coatings of up to 2 mm thick each, and halogen light-cured for 20 sec (Optilux, USA, 450 mW/ cm²). Finishing and polishing procedures were performed with Soft Lex Discs and Brushes - 3MESPE Dental Products. All restorations were evaluated by the modified Ryge and Cvar criteria7 at 6- and 15-month intervals. A colour digital photograph (Canon, A 100) was taken postoperatively, following the preparation and restoration, and at each of the follow-up observations. Data were analysed statistically with SPSS 13.0. Alternative and non-parametric analyses were carried out (Mann-Whitney test) with a significance level of P < 0.05.

patients and Methods

Results

The clinical study included 34 patients who received a total of 76 restorations: class I - 30, class II - 46. * Prior to their participation in the study, all patients signed a form of informed consent. The selection of participants was carried out according to the following criteria in conformity with the requirements of the American Dental Association (ADA)5,6: • Age: 20 - 35 years • Class of carious defect: class I and class II • Diagnosis: Caries media/Caries profunda chronica • Type of tooth: first and second premolars and

The present study evaluated 44 and 48 restorations at 6- and 15-month intervals, respectively. Fig. 1 and Fig. 2 show pre-operative, postpreparation, and 6- and 15-month follow-up clinical cases. The results from the comparative clinical evaluation of the restorations for both periods of observation are given in Table 1. In accordance with the ADA standard, the loss of adhesion in restorations with a self-etch singlestep adhesive and nanofilled composite observed at the 6- and 15-month follow-ups was found to be 0% and 6.7%, respectively. The evaluation of the colour of the restoration material during the 6-month follow-up did not find any significant changes (Р > 0.05, u = 1.72).

Objective

*

The study is part of the project “Student Expertise” supported by 3MESPE Dental products USA.

47

CLINICAL EVALUATION OF RESTORATIONS WITH SELF-ETCH ADHESIVE AND NANOFILLED ...

Figure 1. Clinical case (Class I cavity) observed preoperatively (a), post-preparation (b), and at 6- (c) and 15-month (d) follow-ups.

factory when no more than 5% exceeded a score C (Charlie) in cases when secondary caries was diagnosed. There were none assessed with a score C (Charlie). Secondary caries on the restoration margins was diagnosed in either of the studied periods. According to the ADA standard, clinically satisfactory are class II restorations which retain a tight contact and proper anatomical contour in 95% of the cases. We did not find any significant difference in the contact quality for either of the studied periods. In four restorations there was loss of approximal contact (score Bravo) which was accounted for by a slight rotation of the teeth. According to ADA, complete fractures in restorations are inadmissible. We diagnosed three cases of partial loss of restoration material on the marginal contour in the 15-month period. There was no statistically significant difference from the baseline scores. In all restorations, only enamel surfaces were found, which could be explained with the occlusal stress of the material at contact with the antagonist. Discussion

Figure 2. Clinical case (Class II cavity) observed preoperatively (a), post-preparation (b), and at 6- (c) and 15-month (d) follow-ups.

Certain deterioration in the colour was observed during the 15-month follow-up (Р < 0.05, u = 2.96). All cases that ranked less satisfactory at follow-up examinations were given score B (Bravo). Their number was significantly higher at 15 months (n = 9) than at 6 months (n = 3). The most dynamic change in this study was established in the marginal discoloration. Statistically significant changes were already recorded at 6 months (P < 0.05, u = 2.79) compared to the initial score. The discolouration on the restoration margins observed at 15 months was statistically non-significant in comparison with that of the 6month period (Р > 0.05, u = 0.36). None of the restorations were given score C (Charlie). The anatomical shape of the restorations and their marginal adaptation were evaluated to be clinically satisfactory. No significant difference was observed between the initial scores and the 6- and 15-month parameters. Restorations were considered clinically satis-

48

According to ADA requirements for composite materials used for posterior restorations, 18 months after the restoration score C (Charlie) should not exceed 10% with regard to the following indicators: colour stability, marginal adaptation, colour on the restoration margins. Our study did not record any C (Charlie) scores at 15 months for the period the observation, suggesting a tendency for the studied materials to conform to these standards. We found a high level of restoration retention (93.3%) after 15 months, although some authors hold the view that the strength of the bond with selfetch adhesives is poorer.8,9 Our results significantly differ from those reported by Brackett et al.10 - by 65%. The great difference can be accounted for by the fact that the authors studied the degree of retention in class V cavities, which belong to an area of specific morphology and elastic deformation while mastication. In our study we found that the colour of the restorations after 6 months of use did not reveal a significant change (Р > 0.05, u = 1.72). That is related to the innovative technology and unique structure of Filtek Supreme, which reproduces the optical properties of the hard dental tissues. The spherical particles in the composite structure sized 20 mm contribute to the optimal smoothness of the

Folia Medica, XL, 1/2008 Table 1. Comparative clinical characterization of restorations assigned maximum scores (Alpha) Criteria

Anatomical shape

Colour

Marginal adaptation

Marginal discoloration

Secondary caries

Approximal contact

Loss of restoration

Observation period

Number n*

nA

p ± Sp

Initial scores

76

76

100

After 6 months

44

43

97.7 ± 2.31

After 15 months

48

46

95.8 ± 2.99

Initial scores

76

76

100

After 6 months

44

41

93.1 ± 4.00

After 15 months

48

39

81.2 ± 6.34

Initial scores

76

76

100

After 6 months

44

42

95.4 ± 3.27

After 15 months

48

45

93.7 ± 3.66

Initial scores

76

76

100

After 6 months

44

36

81.8 ± 6.52

After 15 months

48

40

83.3 ± 5.97

Initial scores

76

76

100

After 6 months

44

44

100

After 15 months

48

48

100

Initial scores

46**

46

100

After 6 months

28**

28

100

After 15 months

32**

28

87.5 ± 6.36

Initial scores

76

76

100

After 6 months

44

44

100

After 15 months

48

45

93.75 ± 3.65

n* - Total number of restorations nA - Number of restorations with maximum scores A (Alfa) ** - Evaluation of class II cavities only.

restoration surface and colour stability.4,11 Some researchers12 have established a tendency towards deterioration of the colour of the restorations caused by food. Their view is that the colour stability is directly dependent on the smoothness of the restorations, type of the restoration material and the length of exposure to the effect of the colouring substance. Our observations support these findings. The assessment of the restoration colour after 15 months recorded a statistically significant change in that indicator (Р < 0.05, u = 2.96). Our results about the marginal discoloration of the restorations reveal significant changes after 6 months of use (P < 0.05, u = 2.79). The comparison of the 15-month indicators with the 6-month indicators does not reveal a significant difference

(Р > 0.05, u = 0.36). These changes can be explained with the adhesive bond degradation that occurs in the first months after restoration. The cause of the degradation of the adhesive bond in single-step self-etch systems is accounted for by the specific mechanism of etching, hybridization and morphology of the interdiffusion zone. The demineralizing capacity of Adper Prompt L-Pop is due to the activity of the water-soluble metacrylated phosphoric acid esters, which dissolve away the enamel surface and dentin. Because of their low pH = 0.4, some researchers report that the efficacy of the etching process is commensurate with that of phosphoric acid13, while others have found low etching capacity mainly towards enamel.14,15 We found that the change in marginal

49

CLINICAL EVALUATION OF RESTORATIONS WITH SELF-ETCH ADHESIVE AND NANOFILLED ...

discoloration has only occurred in the enamel of all evaluated restorations, which is a common finding in other clinical studies with Prompt L-Pop.10 During the etching process, the pH of the phosphoric acid esters increases thus self-controlling the demineralization process. The residues – by-products of the process – are incorporated in the hybrid layer or evaporate under a gentle drying airflow. The etching monomers are also responsible for hybridization. The width of the demineralization zone correlates with the permeation depth of the monomer. In addition to the micromechanical bond between the adhesive and dentin, there is probably chemical bonding between the phosphoric acid esters and the calcium ions of apatite15, which should warrant good clinical results. Frankenberger, et al. studied the quality of the hybrid layer with Adper Prompt L-pop and found it to be very fine, hardly visible and non-uniform.16 Miyazaki, et al. supported these observations.17 This could be due to the oxygen inhibition of the polymerization reaction of the adhesive monomers because of the narrow coating application and incomplete polymerization.16-19 Inadequate hybridization can result in leakage within the spaces around the non-impregnated collagen fibrils at molecular level known as nanoleakage. Therefore, some authors regard single-step self-etch adhesives as semi-permeable membranes for diffusion processes.20 The unobstructed passage of ions through the hybrid layer of self-etch adhesives leads to a breakdown of the bond caused by water sorption and dissolution of the incompletely polymerized hydrophilic monomers in the hybrid layer, proven in vivo.20 Water reduces the bonding forces within the polymer chains, a process known as ‘plasticization’.21 The high acidity and hydrophily of the monomers in the single-step self-etch adhesive systems increase the risk of hydrolytic degeneration.22 On the other hand, some enzymes, esterases, produced by microorganisms in vivo catalyze the breakdown of unpolymerized components from the plastic matrix.23 The observed adhesive nonincorporated collagen fibrils can be hydrolyzed by metalloproteinases, which are a product of the oral flora.24 Some researches report that the degradation of the bond in self-etch adhesives occurs within a relatively short period25,26, which is supported by the statistically significant deterioration (P < 0.05, u = 2.79) in the marginal discoloration observed at 6 months in this study.

50

Our results about the change in the anatomical shape, approximal contact and secondary caries do not differ from the results in similar studies.27 The self-etch adhesive system and nanofilled composite possess excellent mechanical properties. Filtek Supreme unites the advantages of microphill and hybrid composites. These characteristics are related to its unique structure and packing density. On the one hand, the small spherical nanomers, sized 20-75 nm, are responsible for the high level of smoothness, gloss and excellent aesthetics. On the other hand, the nanoclusters impart good wear resistance and applicability for posterior restorations.4,11 In this study the marginal adaptation was found to be much better than the results obtained by Bittencourt, et al. in their study of the same adhesive system, but with class V cavities.28 ConclusionS

Considering the constraints of the present clinical study, it can be concluded that restorations with a nanofilled composite material and self-etch singlestep adhesive in class I and class II cavities meet the requirements for clinical application in posterior restorations. The clinical evaluation of restorations over a longer period of observation will be a subject for future research. Acknowledgements

The authors are grateful to 3MESPE Dental Products USA for their technical and material support in carrying out the present study. References

1. Türkün LS. Clinical evaluation of self etching and a one-bottle adhesive system at two years. J Dent 2003;31:527-34. 2. Türkün LS, Aktener BO. Twenty-four-month clinical evaluation of different posterior composite resin materials. JADA 2001;132:196-203. 3. Van Meerbek B, Perdigao J, Lambrechts P, et al. The clinical performance of adhesives. J Dent 1998;26:1‑20. 4. Thomsen KB, Peutzfeldt A. Resin composites: strength of the bond to dentin versus mechanical properties. Clin Oral Invest 2006. (online edition) 5. American Dental Association - Council on Scientific Affairs American Dental Association Program Guidelines: resin based composites for posterior restorations, 2001. 6. American Dental Association - Council on Scientific Affairs American Dental Association Program

Folia Medica, XL, 1/2008 Guidelines: products for dentin and enamel adhesive materials, 2001. 7. Ryge G, Cvar JF. Criteria for the clinical evaluation of dental restorative materials. US Dental Health Center, San Francisco: US Goverment Printing Office Publication; 1971. Publication № 7902244. 8. Rosin M, et al. Polymerization shrinkage – stain and microleakage in dentin borders cavities of chemically and light-cured restorative materials. Dent Mater 2002;18:521-8. 9. Spohr AM, Conceicao EN, Pacheco JF. Tensile bond strength of four adhesive systems to dentin. Am J Dent 2001;14:247-51. 10. Brackett WW, Covey DA, St- Germain HA Jr. Oneyear clinical performance of self-etching adhesive in class V resin composites cured by two methods. Oper Dent 2002;27:218-22. 11. Mitra SB, Wu D, Holmes BN. An application of nanotechnology in advanced dental materials. J Am Dent Assoc 2003;134(10):1382-90. 12. Lu H, Roeder LB, Lei L, Powers JM. Effect of surface roughness on stain resistance of dental resin composites. J Esthet Restor Dent 2005;17(2):102-8. 13. Perdigao J, et al. Effects of self-etching primer on enamel shear bond strength and SEM morphology. Am J Dent 1997;10:141-6. 14. K anemura N, Sano H, Tagami J. Tensile bond strength to and SEM evaluation of ground and intact enamel surfaces. J Dent 1999;27:523-30. 15. Perdigao J, Geralelli S. Bonding characteristics of self-etching adhesives to intact versus prepared enamel. J Esthet Restor Dent 2003;15:32- 41. 16. Frankenberger R, et al. “No- bottle” vs “multi- bottle” dentin adhesives: a microtensile bond strength and morphological study. Dent Mater 2001;17:373-80. 17. Mijasaki M, Iwasaki K, Onose H. Adhesion of single application bonding systems to bovine enamel and

dentin. Oper Dent 2002;27:88-94. 18. Pashley EL, et al. Effects of one versus two applications of unfilled, all-in-one adhesive on dentin bonding. J Dent 2002;30:83-90. 19. King NM, et al. Conversion of one-step self etch adhesives for improved efficacy and extended application. Am J Dent 2004;13:456-78. 20. De Munk J, et al. A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 2005;84(2):118-32. 21. Santerre JP, Shajii L, Leung BW. Relation of dental composite formulations to their degradation and the release of hydrolyzed polymeric-resin-derived products. Crit Rev Oral Biol Med 2001;12:136-51. 22. Tay FR, Pashley DH. Aggressiveness of contemporary self etching systems. Part I: Depth of penetration beyond dentin smear layers. Dent Mater 2001;17:296-308. 23. Finer Y, Santerre. Salivary esterase activity and its association with biodegradation of dental composites. J Dent Res 2004;83;22-6. 24. P ashley DH, et al. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83:216‑21. 25. Giannini M, Seixas CAM, Reis AF, Pimenta LAF. Six-month storage-time evaluation of one-bottle adhesive systems to dentin. J Esthet Rest Dent 2003;15:43-9. 26. Meiers JC, Young D. Two-year composite/dentin bond stability. Am J Dent 2001;14:141-44. 27. Swift EJ, et al. Clinical evaluation of two one-bottle dentin adhesives at three years. J AmDent Assoc 2001;130:1117-123. 28. Bittencourt DD, et al. An 18-month evaluation of selfetch and etch-and-rinse adhesive in non-carious cervical lesions. Acta Odont Scand 2005;63:173‑8.

Клиническая оценка обтураций из self-etch adhesive и нанофильного композита при І и ІІ классах кавитетов

от качеств адгезивной системы и от материала композита. Ц ель : Настоящее исследование ставит себе целью сделать клиническую оценку обтураций из self-etch adhesive и нанофильного композита при І и ІІ классах кавитетов за период 15 мес. Пациенты и мetodы: Клиническое исследование поставлено на 34 пациентах (добровольцы). Поставлены обтурации на 76 жевательных зубах со средним и глубоким кариесом. Использован адгезив Adper Prompt L- Pop и нанофильный композит Filtek Supreme (3 MESPE Dental products USA). Все обтурации оценены посредством модифицированных критериев Ryge и Cvar на 6-ой и 15-ый месяц. Результаты: Потеря обтураций из self-etch adhesive и нанофильного композита на 6-ой и 15-ый

Н. Манчорова, С. Владимиров, З. Доненчева, И. Драшкович, С. Манолов, В. Тодорова

Резюме Введение: Во время адгезивного восстановительного зуболечения принципы классической препарации кавитетов переосмысливаются. Акцент смещается с “расширения с целью предохранения” и препарирования макромеханической ретенции к “сознательно ограниченной” препарации. Клиническая и биологическая долговечность обтураций зависит

51

CLINICAL EVALUATION OF RESTORATIONS WITH SELF-ETCH ADHESIVE AND NANOFILLED ...

мес соответственно 0% и 6.7%. При оценке цвета обтураций на 6-ой мес существенные изменения не наблюдаются (Р > 0.05, u = 1.72). Некоторое ухудшение цвета наблюдается на 15-ый мес (Р < 0.05, u = 2.96). Статистически значимые изменения в маргинальном окрашивании наблюдались еще на 6-ой мес (Р < 0.05, u = 2.79) по сравнению с начальной оценкой. При контроле на 15-ый мес изменение в цвете по границам обтураций статистически несущественно по сравнению с оценкой на 6-ой мес (Р < 0.05, u = 0.36).

52

Анатомическая форма обтураций и их маргинальная адаптация оцениваются как клинически приемлемы; статистически значимое различие между начальными оценками и параметрами на 6-ой и 15-ый мес не установлено. При контрольном осмотре на 15-ый мес в течение наблюдения не регистрирована ни одна оценка С (Charli). Заключение: Обтурации из вышеуказанных материалов соответствуют требованиям к клиническому применению на жевательных зубах.

Folia Medica, XL, 1/2008

Experimental Investigations Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line Milena N. Draganova-Filipova, Milena G. Georgieva1, Ekaterina N. Peycheva1, George A. Miloshev1, Victoria S. Sarafian, Lyudmil P. Peychev2

Department of Biology, Medical University, Plovdiv, 1Laboratory of Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Science, Sofia, 2Department of Pharmacology and Drug Toxicology, Medical University, Plovdiv, Bulgaria Abstract The mechanisms of action of propolis can be studied in detail by comparing the effects of propolis and the effects of its constituent components. Aim: To clarify and compare the effects of Bulgarian propolis and caffeic acid phenethyl ester (CAPE, a chemically synthesized component of propolis) - by using a set of cellular, molecular-biological and immunological techniques. Material and methods: The McCoy-Plovdiv cell line was treated with propolis and САРЕ in increasing concentrations (0.01, 0.1, 1.0, 10 mg/L, and 2.5, 4, 8, 16 mg/L, respectively). The expression of the proliferating cell nuclear antigen (PCNA) and the tumour-suppressor protein p53 was studied immunocytochemically. Apoptosis was measured using a highly sensitive microgel electrophoresis technique (comet assay). Results: The results of the study showed corresponding changes in the expression of the examined proliferative antigens. PCNA was detected in all examined concentrations of the tested substances the expression being dose-dependent. Molecule localization changed from the nucleus to the cytoplasm. Treatment with САРЕ brought about gradual attenuation of PCNA expression. High propolis concentrations induced increased synthesis of p53. No p53 expression was found when cells were treated with САРЕ. The studied substances in their highest concentrations (10 mg/L propolis and 16 mg/L САРЕ) had a cytotoxic effect. The comet assay showed DNA degradation kinetics characteristic for apoptosis. Conclusions: The present study demonstrates that high concentrations of propolis and САРЕ cause apoptosis-induced cell death in McCoy-Plovdiv cells. Key words: propolis, САРЕ, apoptosis, McCoy-Plovdiv, comet assay Introduction

The life-activity products of the honey bee Apis melifera have unique nutritional and healing properties. Honey, beeswax, pollen, propolis and bee venom are widely used. Royal jelly or propolis is composed of more than 300 chemical compounds with biological properties of importance for human health.1-3 It has been proved that propolis and several of its components have an inhibiting effect on the reproduction and the realization of genetic information and induce apoptosis in different types of tumour cells.4 Propolis provokes cell death through activating caspases 8

and 9.5,6 It thus participates in the induction stages of apoptosis, triggered by intrinsic and extrinsic signals. Propolis treatment changes the expression of some adhesion molecules (β-catenins), which disrupts normal intercellular contacts and lowers the proliferative potential.7 The mechanisms by which propolis induces programmed cell death in neoplastic cells have not been fully clarified yet. The phenethyl ester of caffeic acid is one of the many flavonoids found in propolis. It is known that it is a carrier of the antiproliferative activity and the antitumour effect of propolis.8,9 In vitro САРЕ induces apoptosis through p53-dependent

Correspondence and reprint request to: M. Draganova-Filipova, Department of Biology, Medical University, Plovdiv, Bulgaria 15A Vassil Aprilov St., 4002 Plovdiv, Bulgaria Received 11 December 2007; Accepted for publication 30 January 2008

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Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line

or independent pathways, which makes it a potent antiproliferative agent.10 It is well known that the content of different chemical components in propolis varies according to the geographical region it is obtained from. Gardjeva P, et al. have provided evidence that the quantity of САРЕ in Bulgarian propolis equals that of Brazilian and Argentinian propolis.11 Information about the mechanisms of the processes of cell proliferation and cell death can be obtained by tracing out the changes in the expression of basic regulatory molecules. PCNA is a key factor, participating in the replication and the reparation of nuclear DNA. It is found in the S and G2 phases of the cell cycle as part of the DNA-synthetase complex.12 The regulation of PCNA expression is accomplished at transcriptional and post-translational level. The basic transcriptional factor of the PCNA-gene is the p53 protein. The latter takes part in arresting the cell cycle in G1 phase and allows the cell to repair the lesions in DNA. If repair is impossible, p53 induces apoptosis.13 Despite the advances in synthetic chemistry which keeps creating new drugs to treat different diseases, the interest in using natural products for treatment has increased in the recent years. The rich chemical composition of propolis is responsible for its application as a therapeutic and prophylactic means in infectious, tumour and immunodeficient conditions. Progressive tumour growth is often the result of suppressed apoptosis. In this respect, it is of great importance to elucidate the molecular and cellular mechanisms via which propolis itself and its components influence cell proliferation and cell death. The lack of comparative studies on Bulgarian propolis and some of its isolated components in vitro makes it necessary to clarify and compare their mechanisms of action. The aim of this study was to investigate the effects of Bulgarian propolis and to compare them with those of САРЕ. Material and methods

Materials We used propolis produced in the Eastern Rhodope mountains, extracted in 96% ethanol in final concentrations of 0.001; 0.01; 0.1; 1.0; 10 mg/L; САРЕ (Sigma, Cat. N:С8221-1G) – in concentrations of 2.5; 4; 8; 16 mg/L. The amount of ethanol in the final working concentrations did not exceed 0.1%.

54

Methods Cell cultivation. The McCoy-Plovdiv cell line is a fibroblast synovial cell line, obtained from McCoy cell line and adapted for cultivation in serum-free conditions.14 It was cultivated in Ham’s 12/DMEM culture medium (Sigma-Aldrich Cat. N:4388) in 1:1 ratio. 100 IU/ml penicillin-G and 100 µg/ml streptomycin were added to the medium. The cells were incubated at 37°С, 5% СО2 and high humidity. After formation of a confluent monolayer the culture medium was removed and a fresh stock, containing different concentrations of the examined substances was added. The cells were treated for 24 and 48 hours with propolis and for 24 hours with САРЕ. They were fixed in ice-cold acetone. Cells cultivated in propolis and САРЕ-free medium were used as controls. Immunocytochemistry. The expressions of the proliferative antigen PCNA and of the tumour-suppressor protein p53 was examined using the biotinstreptavidin peroxidase method with the universal DАКО kit (Cat.N:K0672). Monoclonal antibodies against PCNA (Cat. N:1529) and p53 (Cat. N: 1581) were used as primary antibodies. Visualization of the reaction was performed by aminoethylcarbasol, while hematoxylin was used to contrast the nuclei. The expression was assessed by the presence of red-brown granular staining. The reaction intensity was measured according to the following semi-quantitative scale: «+» – slight staining in single cells, «++» – presence of expression in 50% of the 100 counted cells, «+++» – highly intensive homogenous expression in 100% of the cells. Comet assay. The comet assay or gel electrophoresis of single cells is a sensitive method for assessing DNA fragmentation. The method was used according to the protocol of Matassov et al.15 Propolis- and САРЕ-treated cells were included in low melting agarosis and were spread as microgel on coverslips. Electrophoresis was performed after cell lysis and DNA denaturation. The DNA-lesions typical for apoptosis were observed under a fluorescent microscope. The DNA-binding dye SYBR Green 1 was used (Molecular Probes, Cat. N: S-7567). Statistical methods. The analysis of data was performed with Statistica 4.5 (StatSoft, Inc. Microsoft). Differences were considered statistically significant at P < 0.05. Results were presented as mean ± SE. The reliability of the comparison was

Folia Medica, XL, 1/2008

Figure 1. Immunocytochemical detection of PCNA and p53 in McCoy-Plovdiv cells treated with propolis and САРЕ. A. PCNA expression in untreated cells at 48 hours. Homogeneous high intensity cytoplasmic expression - «+++». × 40. B. PCNA expression in cells treated with propolis (0.01 mg/L) at 48 hours. The marker is positive on the membranes of cells with high intensity of expression. - «+++». × 40. C. p53 expression in untreated cells at 48 hours. The marker is not expressed. × 40. D. p53 expression in cells treated with propolis (0.01 mg/L) at 48 hours. The antigen is expressed in the cytoplasm with «++» intensity. × 40. E. PCNA expression in cells treated with CAPE (2.5 mg/L) at 24 hours. Nuclear localization in single cells with «+» intensity. × 40. F. PCNA expression in cells treated with CAPE (4 mg/L) at 24 hours. Homogeneous expression in the nuclei and in the cytoplasm of all cells with «+++» intensity. × 40.

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Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line

determined by the Student’s t-test with separate variance estimates and by one-way analysis of variance (ANOVA). Results

Immunocytochemistry PCNA was found in the cytoplasm one hour after the start of cell cultivation. The intensity of the reaction increased thereafter reaching its maximum at 72 hours when the nuclei and the perinuclear zone became positive (Fig. 1A). After that the PCNA expression began to attenuate. The marker was not detected after the formation of a confluent cell monolayer. In the propolis-treated cells the marker expression was found in concentrations from 0.001 to 1.0 mg/L and the localization changed from the nucleus to the cytoplasm. The intensity of the reaction increased with the elevation of propolis concentration. After a 48-hour propolis treatment a clear staining of the plasma membrane was observed. (Fig. 1B). After 48 hours of propolis treatment (1 mg/L) the characteristic red-brown staining was seen in fewer cells most of them being rounded. PCNA outlined the cell membrane and showed lower intensity compared with smaller concentrations. The p53 marker was not detected in the control cells (Fig. 1C). After treatment with 0.01 mg/L of propolis a positive reaction with homogenous cytoplasmic localization was established. The number of positive cells increased in the subsequent concentrations (Fig. 1D). The most intensive reaction was observed 48 hours after treating the cells with 1 mg/L of propolis. The level of expression and the localization of PCNA and p53 markers changed depending on the concentration and treatment duration. In prolonged exposures the intensity of the expression increased. There was a correlation between the two markers. Intense proliferative activity was observed in the presence of PCNA and in the absence of p53. Enhanced expression of p53 was accompanied by a slight expression of PCNA. CAPE treatment upregulated the PCNA expression and the localization changed from the nucleus towards the cytoplasm. At 2.5 mg/L the antigen was detected in the cell nuclei (Fig. 1E). At 4 mg/L the reaction was the most intensive and homogeneously distributed in the cytoplasm (Fig. 1F). In the subsequent concentrations the marker gradually decreased its expression and at the highest concentration (16 mg/L) was not expressed at all. The lowest proliferation level was also observed 56

at this point. There was no expression of p53 in any of the studied concentrations. Comet assay The comet assay showed that DNA was significantly damaged when the McCoy-Plovdiv cells were treated with propolis and САРЕ. The DNA lesions were typical of apoptosis. As the method is highly sensitive, comets were seen even at the lowest concentrations of the examined substances. With the increase of concentration there was a natural rise in the percentage of apoptotic cells. The lowest propolis concentration (0.01 mg/L) induced apoptosis in 22% of the cells, while in the highest concentration (10 mg/L) the apoptotic cells were as high as 46% of all cells (Fig. 2A). In treatment with 2.5 mg/L САРЕ apoptosis was detected in 30% of the cells reaching 72% in the highest concentration – 16 mg/L (Fig. 2B). A lower percentage of comets was observed in propolis-treated cells than in САРЕ treated cells. The chemically pure propolis component exhibits higher cytotoxicity than the product as a whole. The data were obtained by calculating the percentage of apoptotic cells in counting 1500 cells (nuclei and comets) for each concentration of the examined substances. Discussion

In a number of studies on tumour and normal cells propolis and some of its components have been demonstrated to affect proliferation and lower the number of surviving cells by inducing apoptosis.16 The natural honey-bee product affects cell homeostasis because it participates in the apoptosis-inducing process via two mechanisms – by external and internal signals. The extrinsic apoptotic pathway is initiated by specific propolis compounds which bind with cell membrane receptors and activate the apoptotic process.4,5 The intrinsic pathway is activated at mitochondrial level.5,9 In case of non-repairable DNA damage the p53 protein changes the expression of genes from the Bcl-2 family. The disrupted balance between the pro- and antiapoptotic members of this family induces a change in mitochondrial membrane permeability, release of cytochrome C from the mitochondria into the cytosol and cascade activation of caspases. Propolis from different geographic regions has different components depending on the flora, the bee species, the season of pollen collection and the method of extraction. Because of insufficient data

Folia Medica, XL, 1/2008 60 *

comets, %

50 40

*

30

*

20 10

A

0 control

0.01

1,0

10

comets, %

concentration of propolis, mg/L

80 70 60 50 40 30 20 10 0

**

**

** *

B

control

2,5

4

8

16

concentration of CAPE, mg/L Figure 2. Results from the comet assay. A. Comet assay in propolis treatment; B. Comet assay in CAPE treatment. The data are significantly different from the controls at *p < 0.05, n = 9; **p < 0.01, n = 9

from in vitro studies on cell cultures of Bulgarian propolis, the choice of treatment time and the applied concentrations were based on other authors’ research. The latter vary according to the type of the cell lines used and the research design.16,17 McCoy-Plovdiv is a highly sensitive test-system for the detection of biologically active substances.14,18 In our study the highest concentrations (10 mg/L propolis and 16 mg/L САРЕ) had a pronounced cytotoxic effect. When compared with the controls there were significant differences (р < 0.05, р < 0.01 when n = 9) (Fig. 2). The comet assay showed the DNA degradation kinetics characteristic of apoptosis. Apoptosis in McCoy-Plovdiv cells is induced by both the whole compound and САРЕ, but the pathways of activating cell death differ. Most probably this effect is due to the presence of specific components in propolis whose individual actions either complement or neutralize each other. The changes that occur in the expressions of PCNA and p53 are an indication of subtle alterations

in the complex mechanisms of cell proliferation. Propolis upregulates the p53 expression which is evidence of initial DNA damage, but the simultaneous detection of PCNA suggests that the mechanisms controlling the DNA repair are also activated. This is supported by the comet assay which showed fewer apoptotic cells. The fact that p53 is expressed in propolis-treated cells implicates this protein in the induction of cell death. Propolis is composed of many compounds which synergetically use different mechanisms of action from those of its ingredient compounds acting independently. Propolis induces apoptosis in the McCoy-Plovdiv cells activating a p53-dependent pathway, while САРЕ’s action is most probably due to activation of external apoptosis triggering mechanisms. There is no correlation between the two markers when cells are treated with САРЕ. Only PCNA is positively expressed and the reaction intensity changes in a proportional dose-effect dependent manner. The fact that there was no p53 expression

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Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line

in any of the studied concentrations suggests that САРЕ induces apoptosis in McCoy-Plovdiv cells via p53-independent pathways. Nomura M, et al. use murine epidermal JB6 Cl 41 cells and prove that in vitro САРЕ triggers apoptosis through p53-dependent and independent pathways. This fact determines this protein as a potent antitumour agent.10 Our results suggest that САРЕ interacts with surface death receptors and activates cell death through an extrinsic pathway. ConclusionS

The present study reports the first ever data about the proliferative action of Bulgarian propolis and its compounds in vitro. On this basis the effects of propolis and CAPE on normal and tumour cell lines could be further compared showing the potential of Bulgarian propolis to be used independently or in combination with chemotherapeutic drugs to manage carcinogenic conditions. Acknowledgments

The authors wish to extend their gratitude to Assoc. Professor M. Draganov for placing the McCoy-Plovdiv cell line at their disposal and for his invaluable methodological help. The study was funded under project no. MU-L-1408 from the Ministry of Education and Science and project no. 01/2005; 06/2003 of the Medical University, Plovdiv. References

1. Almeida EC de, Menezes H. Anti-inflammatory activity of propolis extracts: a review. J Venom Anim Toxins 2002; 8:191-212. 2. Peychev L, Sarafian V, Murdjeva M. Therapeutic use of Bulgarian propolis: a review. Union of Scientists research, Plovdiv, series General Medicine, Pharmacy and Dentistry, Medicine and Stomatology 2006; vol.VІІ:52-8. 3. Borrelli F, Izzo AA, Di Carlo G, et al. Effect of propolis extract and caffeic acid phenethyl ester on formation of aberrant crypt foci and tumor in the rat colon. Fitoterapia 2002; 73:38-43. 4. Kimoto T, Arai S, Kohguchi M, et al. Apoptosis and suppression of tumor growth by Artepillin C extracted from Brazilian propolis. Cancer Detect Prev 1998;22:506-15. 5. Chen CN, Wu CL, Lin JK. Propolin C from propolis induces apoptosis through activating caspases, Bid

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and cytochrome C release in human melanoma cells. Biochem Pharmacol 2004;67:53-66. 6. Lee YJ, Kuo HC, Chu CY, et al. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Biochem Pharmacol 2003;66:2281-9. 7. Wang D, Xiang DB, He YJ, et al. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J Gastroenterol 2005;11:4008-12. 8. Orsolic N, Terzic S, Mihaljevic Z, et al. Effects of local administration of propolis and its polyphenolic compounds on tumor formation and growth. Basic Biol Pharm Bull 2005;28:1928-33. 9. Woo KJ, Jeong YJ, Park JW, et al. Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells. Biochem Biophys Res Commun 2004;325:1215-22. 10. Nomura M, Kaji A, Ma W, et al. Suppression of cell transformation and induction of apoptosis by caffeic acid phenethyl ester. Mol Carcinogen 2001;31:83‑9. 11. Gardjeva P, Alexandrov A, Dimitrova S, et al. A study on chemical composition and antifungal activity of Bulgarian propolis. Union of Scientist research, Plovdiv, series General Medicine, Pharmacy and Dentistry, Medicine and Stomatology 2006;7:201‑5. 12. Ivanova V, Karaivanov M, Marinov E. Assessment of cell proliferation in clinical pathology. Clin Аpplic Immunol 2002;1:28-33. 13. Macdonald F, Ford CHJ. Molecular biology of cancer. BIOS Scientific Publishers;1997:53. 14. Kamberov E, Draganov M, Murdjeva M. Development of a new serum-free cell culture system, McCoy-Plovdiv. In Vitro Cell&Dev Biol - Animal 2000;36:284-6. 15. Matassov D, Kagan T, Leblanc J, et al. Measurement of apoptosis by DNA fragmentation. Methods Mol Biol 2004;282:1-17. 16. C hen MF, Wu CT, Chen YJ, et al. Cell killing and radiosensitization by caffeic acid phenethyl ester (CAPE) in lung cancer cells. J Radiat Res 2004;45:253-60. 17. S cifo C, Cardile V, Russo A, et al. Resveratrol and propolis as necrosis or apoptosis inducers in human prostate carcinoma cells. Oncol Res 2004;14:415‑26. 18. Ganchevska P, Draganov M, Sarafian V. Characterization of the proliferative activity of the serumfree cell line McCoy-Plovdiv. Trakia J Sci 2004; 2:12‑5.

Folia Medica, XL, 1/2008 Эффекты прополиса и САРЕ на пролиферацию и апоптоз клеточной линии МсСоу-Plovdiv М. Драганова-Филипова, М. Георгиева, Е. Пейчева, Г. Милошев, В. Сарафян, Л. Пейчев

Резюме Сравнительное рассматривание эффектов прополиса и его отдельных составных частей позволяет детально изучить механизмы его действия. Цель: Применить набор клеточных, молекулярнобиологических и иммунологических техник в целях выяснения и сравнения эффектов болгарского прополиса и химически синтезированного прополисного компонента САРЕ. Материалы и mетоды: Клеточная линия МсСоуPlovdiv третирована нарастающими концентрациями прополиса (0.01; 0.1; 1.0; 10 mg/L) и САРЕ (2.5; 4; 8; 16 mg/L). Экспрессия пролиферативного антигена PCNA и опухолево-супрессорного белка р53 исследована иммуноцитохимично. Для детекции

апоптоза использован высокочувствительный метод – микрогель электрофорез (Кометный тест). Результаты: Результаты исследования показывают закономерное изменение в экспрессии исследуемых пролиферативных антигенов. PCNA устанавливается при всех исследованных концентрациях тестированных веществ, при чем экспрессия является доза-зависимой. Локализация молекулы изменяется от ядра к цитоплазме. При третировании САРЕ проявляется тенденция к постепенному затиханию экспрессии PCNA. Высокие концентрации прополиса индуцируют повышенный синтез р53. При третировании САРЕ отсутствует экспрессия р53. Самые высокие применяемые концентрации (10 mg/L прополиса и 16 mg/L САРЕ) имеют цитотоксический эффект. При Кометном тесте наблюдается характерная для апоптоза кинетика расщепления ДНК. Выводы: Исследование доказывает, что высокие концентрации прополиса и САРЕ приводят к клеточной смерти вследствие апоптоза в клетках МсСоуPlovdiv.

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SUICIDE BY UNUSUAL MANNER OF HANGING

CASE REPORTS SUICIDE BY UNUSUAL MANNER OF HANGING. A CASE REPORT Ivan D. Doichinov, Yonka А. Doichinova, Svetlozar S. Spasov, Nikolai D. Marinov1

Department of Forensic Medicine, Medical University, Plovdiv, 1Ward of Forensic Medicine, Regional Hospital, Kirdjali, Bulgaria ABSTRACT We present a case of a 35-year-old man who committed a suicide by unusual manner of hanging. The man positioned his neck between two branches of a cherry tree which were V-shaped. The dead body hung on the tree with the feet 50 cm off the ground. The external examination showed strangulation grooves on both sides of the neck with skin abrasion caused by the compression of the neck between the two tree branches as well as submucosal hemorrhages of both eyelids. Autopsy revealed some of the common signs of asphyxia (hemorrhages under the epicardium and the visceral pleura, acute blood stagnation of the internal organs, liquid blood); hemorrhages in the sternocleidomastoid muscles at their attachment to the sternum; horizontal and parallel ruptures of the intima of the left common carotid artery (Amussat’s sign); ruptures of the ligaments and the disc between cervical vertebras 6 and 7 and hemorrhages (Simon’s sign). Toxicological blood studies, which were additionally performed, found no evidence of drugs or alcohol. Key words: forensic medicine, mechanical asphyxia, suicide, hanging, unusual manner

INTRODUCTION

Hanging is the most common type of mechanical asphyxia in forensic medical practice and it is the most popular method used to commit a suicide.1,2 Hanging is best defined as compression of neck by a ligature tightened by the weight of the body that causes instant loss of consciousness and results in a very rapid death.1,2 The major mechanism causing death in hanging is reduced blood flow and disturbed brain circulation caused by pressure on the neck blood vessels - veins and arteries. Experimental studies have measured the force needed to obstruct neck blood vessels and disturb brain circulation and have shown it to be in the range of 2.5 to 30 kg.3 In forensic medicine hanging is defined either as typical or atypical depending on the position of the noose around the neck of the victim and the position of the body itself. Atypical or unusual cases are also cases in which various objects (not ligature) are used for neck constriction by hanging, the way

these objects are used to compress the neck, the position of the body in such cases, etc. Atypical, unusual hanging provides grounds to suspect other people/peoples’ intervention in committing a murder. We present a case of committing suicide by an unusual manner of hanging. CASE DESCRIPTION

Scene of the accidents The dead body of a 35-year-old man was found in the woods, 1.5 km from a mountain village. The neck was positioned between two branches of a cherry tree which were V-shaped. The dead body hung on the tree and the feet were 50 cm off the ground (Fig. 1). There were fresh marks of damage of the tree bark (arrows, Fig. 1). Bark particles were found stuck to the shoes of the victim. The neck was compressed tightly between the two branches (Fig. 2). Examination of the dead body The neck is deformed and flattened laterally. One

Correspondence and reprint request to:Iv. Doichinov, Department of Forensic Medicine, Medical University, Plovdiv 15A Vassil Aprilov Blv., 4002, Plovdiv, Bulgaria Received 19 March 2007; Accepted for publication 27 June 2007 60

Folia Medica, XL, 1/2008

Figure 1. Position of the dead body on the tree.

Figure 2. Position of the head and the neck between the two branches.

abrasion is found оn each side of the neck, 8х4 сm on the right side and 7х4 сm on the left side. The abrasions are of brownish colour, compact and rough – imprints of the rough uneven surface of the tree bark. There are dark-red pinpoint submucosal hemorrhages of the eyelids. Hemorrhages (2х2х1 cm) are found at the sites of attachment of the two sternocleidomastoid muscles to sternum. There are 4 ruptures of the intima of the left common carotid artery which are horizontal and parallel to each other and are 0.2-0.3 cm in length (Amussat’s sign). The intervertebral ligaments and the disc between C6 and C7 are lacerated and have hemorrhages (Simon’s sign). There are pinpoint hemorrhages of darkred colour under the epicardium and the visceral pleura. Acute blood stagnation is revealed in the internal organs. The blood in the heart cavities and the large blood vessels is liquid. Toxicological studies found no evidence of drugs or alcohol in the victim. The man had no history of mental disorders.

DISCUSSION

There are isolated reports in forensic literature of cases of hanging by neck compression by various objects (not nooses): compression of neck between the door and the side column of a car2; compression of neck by the two wings of an automatic door4; compression of neck by the lift door5; compression of neck with the collar of a sweater and hanging from a motorcycle6; accidents with elderly people with neck compression between the side bars of а bed under the weight of their bodies.7,8 In the reported cases the characteristics of the atypical neck compression arouse suspicion whether they concern hanging or some other type of mechanical asphyxia - strangulation by ligature for example. Some of the authors of these reports4 accept that there is a double form of mechanical asphyxia – atypical strangulation and atypical hanging, an opinion which we do not share.2 In the present case there are data from the scene of the accident which show fresh damage of the tree bark and bark particles stuck on the shoes

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SUICIDE BY UNUSUAL MANNER OF HANGING

of the victim. On this basis we conclude that the man climbed the tree unaided. Examination of the corpse reveals data about rapid death setting in as well as the presence of some general signs of asphyxia – submucosal hemorrhages of the eyelids, subepicardial and subpleural hemorrhages, liquid blood and acute blood stagnation in the parenchymal organs. Specific signs of hanging are also evident – a strangulation groove, (imprint of compression of the tree branches), hemorrhages in the neck muscles, ruptures of the intima of the left common carotid artery (Аmussat’s sign), certain damages between C6 and C7 (Simon’s sign). Death is caused by disturbed blood circulation of brain as a result of the compression of neck blood vessels by the tree branches. The weight of the body of the man is big enough to lead to constriction of the neck blood vessels. After thorough evaluation of the data in the scene of accident and the examination of the dead body we came to the conclusion that the case we present is a suicide committed through unusual manner of hanging - positioning the neck between two branches of a tree. We consider it a unique case in the forensic medical practice. Самоубийство через повешение необычайным способом. Сообщение об одном случае И. Дойчинов, Й. Дойчинова, Св. Спасов, Н. Маринов

Резюме Авторы сообщают об уникальном случае самоубийства 35-илетнего мужчины через повешение необычайным способом. Шея мужчины вклинена между двумя ветками черешни (ветки с V-образной формой). Труп человека висел на дереве, при чем стопы находились в 50 см от земли. При внешнем осмотре с обеих сторон шеи обнаружены странгуляционная борозда от протирания кожи

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REFERENCES

1. Medicine Legale. Ed. L. Derobert, Paris, Flammarion, 1974. 2. Radanov S, Doichinov I, Lissaev P, Stanchev N, Hristov S. Forensic Medicine and Medical Deontology. Sofia; Sieala; 2006. 3. Khokhlov VD. Pressure on the neck calculated for any point along the ligature. Forensic Sci Int 2001;123:178-81. 4. Hejna P, Hottmar P. Unusual form of accidental mechanical asphyxia. Arch Med Sadowej Kryminol 2006;56:61-3. 5. Verma SK, Agarwal BB. Accidental hanging with delayed death in a lift. Med Sci Law 1999;39:342‑4. 6. Nurhantari Y, Asano M, Nushida H, et al. Accidental hanging by a sweater: an unusual case. Am J Forensic Med Pathol 2002;23:199-201. 7. Osculati A, Fassina G. Two cases of accidental asphyxia by neck compression between bed bars. Am J Forensic Med Pathol 2000;21:217-9. 8. Di Nunno N, Vacca M, Costantinides F, Di Nunno C. Death following atypical compression of the neck. Am J Forensic Med Pathol 2003;24:364-8.

вследствие прижатия шеи между ветками дерева, кровоизлияния под слизистой век обоих глаз. Вскрытие установило признаки асфиксии (кровоизлияния под эпикардом сердца и под плеврой легкого, острый застой крови во внутренних органах, жидкая кровь), кровоподтеки в стерноклейдомастоидных мышцах в месте их прилегания к грудной кости, горизонтальные, параллельные разрывы интимы левой сонной артерии (признак Амюса), разрыв связок и диска между 6 и 7 шейными позвонками с кровоподтеком (признак Симона). Дополнительное токсикологическое исследование не устанавливает наличие наркотиков и алкоголя в крови. В литературе не имеется сообщение о самоубийстве через повешение таким необычайным способом.

Folia Medica, XL 1/2008

Breast reconstruction in Poland anomaly with endoscopicallyassisted latissimus dorsi muscle flap and autologous fat tissue transfer: a case report and review of the literature Ali Mojallal, Christo Shipkov1, Fabienne Braye

Department of Burns and Plastic, Reconstructive and Esthetic Surgery, Hospital Eduard Herriot, Lyon, France, 1Division of Plastic and Craniofacial Surgery, “St. George” University Hospital, Plovdiv, Bulgaria Abstract Objective: The breast deformity in females with Poland anomaly (Poland syndrome) can be particularly embarrassing from esthetic and social point of view. Various methods of breast reconstruction were reported comprising breast implants, latissimus dorsi (LD) flap or the combination of these two. The aim of this study was to present a breast reconstruction in Poland anomaly with endoscopically-assisted LD muscle flap in association with autologous fat tissue transfer and to analyse the results in the light of the literature data. Patients and methods: An 18-year-old woman presented with severe left breast hypoplasia (in the context of a Poland anomaly) with heavy social and psychological impact on both private and professional life. No thoracic deformity was clinically and radiologically detectable. The preoperative imaging studies of the breast (mammography and ultrasound) did not show any pathological findings. A three-stage autologous breast reconstruction was performed. An endoscopically assisted LD muscle flap was carried out during the first stage. This was followed by two stages of autologous fat tissue transfer and reduction mammaplasty of the opposite breast. Results: No postoperative complications were observed. The postoperative pain was less intensive than in the classic open technique. The LD muscle harvesting was carried out through a smaller incision which left a smaller back scar. The final result was breast symmetry of excellent quality. Conclusions: The breast reconstruction in Poland anomaly can be performed with entirely autologous techniques. Endoscopic techniques in LD harvesting leave smaller scars and cause less postoperative pain. The autologous fat tissue transfer can be an useful adjunct to the classic flap techniques provided that safety requirements in technique and follow up are strictly observed. Key words: Poland’s syndrome, latissimus dorsi, endoscopic harvest, lipofilling Introduction

Poland anomaly, described by Alfred Poland1 in 1841, is characterized by congenital unilateral absence or hypoplasia of pectoralis major muscle, often accompanied with ipsilateral breast agenesis or hypoplasia in women.2 Chest wall deformity and hand anomalies may be encountered as well.3 Breast reconstruction in female patients with severe breast deformity is frequently demanded because of the significant esthetic, social and psychological impact. The basic surgical reconstructive options include breast implants, latissimus dorsi (LD) muscle flap, the combination of these two techniques, as well as free tissue transfer.4 The fat tissue transfer, as

described by Coleman5, was reported as an useful adjunct to these treatment options and can be even used as a single treatment in this pathology.6 This latter technique is currently under evaluation at our institution following strict criteria of surveillance. Partial or total correction of the breast volume in Poland syndrome in single or multiples stages can be achieved by fat tissue transfer even in the most severe cases. This is particularly efficient in the restoration of anterior axillary fold even in association with a breast implant.6 Advances in endoscopy (minimally invasive surgery, MIS) in the last 15 years have gained popularity in reconstructive surgery as well. Using

Correspondence and reprint request to: Ali Mojallal, Department of Plastic Surgery and Burns, Eduard Herriot Hospital, 5 Place d’Arsonval, 69437 Lyon, France; E-mail: [email protected] Received 4 September 2007; Accepted for publication 30 January 2008

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Breast reconstruction in Poland anomaly...

MIS techniques and sutable instruments certain procedures can be safely performed through smaller incisions, placed at more cosmetically suitable locations with less postoperative pain. 7 This is particularly true for the harvest of muscle flaps as the TRAM and latissimus dorsi flap where conventional open techniques necessitate long incisions with heavier postoperative period and eventual wound healing problems. In this respect the ednoscopically assisted LD muscle flap represents an atractive option which provides a smaller scar and less painfull postoperative period.7 Case report

An 18-year-old woman presented with severe left breast hypoplasia (in the context of a Poland anomaly) with heavy social and psychological impact on both private and professional life (Figs 1A,B). No thoracic deformity was clinically and radiologically detectable. No upper limb malformations were present. The preoperative imaging studies (mammography and ultrasound) did not show any pathological findings. A three-stage autologous breast reconstruction was performed. An LD muscle flap without prosthesis was carried out at the first stage. This was followed by two stages of autologous fat tissue transfer. First stage A standard endoscopic surgery equipment was used to harvest the LD flap with a 10 mm camera of

zero and 30 degrees. The operation was performed under general anesthesia, with the patient in semiprone lateral decubitus position. The procedure was started by identifying the lateral border of the LD muscle through a small 5 cm long laterothoracic incision. This incision was carefully placed at the middle axillary line so that it would be hidden by the brassiere. The initial dissection of the muscle from the thorax and subcutaneous tissue was comfortably achieved under direct vision through this incision. Once the limits of this dissection were reached, a 10 mm port was introduced and secured to the axillary incision. The 10-mm camera was introduced through the port. Another two 5 mm ports were inserted and secured in the middle axillary line and in the lumber region. They were used to pass the manipulating instruments for the subcutaneous dissection and muscle-from-thorax dissection. The skin was externally retracted by means of transcutaneous sutures which served to tract on the skin by the assistant. After the area around the axillary incision was dissected under direct vision the dissection proceeded distally by means of the endoscopic instruments. Initially, the subcutaneous dissection was performed, ligaclipping all the encountered musculo-cutaneous perforators. Afterwards the elevation of the muscle from thorax was carried out. Once the entire muscle was dissected, it was divided distally with endoscopic ultracision. The optic field was provided

A

Figure 1. Preoperative view. A: Frontal view; B: Left oblique view.

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B

Folia Medica, XL 1/2008 by means of internal and external retraction. No balloon retraction was used. The vascular pedicle dissection was accomplished under direct vision. Once the entire flap was elevated, it was rotated and passed through a subcutaneous tunnel in a retromammary position. The flap was fixed with transcutaneous stitches. The inframammary crease symmetrization was performed by means of an abdominal advancement flap.8 Two suction drains were inserted – one under the flap and the other one in the donor site. They were removed when drain output reached less than 30 ml per 24h. No postoperative complications were observed, including seroma or back pain, and the postoperative period was uneventful (Figs 2A,B). Second stage The second stage was carried out six months later. A symmetrization of the contralateral breast was preformed by means of a superior pedicle mastopexy with a “J” scar. This procedure was combined with autologous fat tissue transfer in the reconstructed breast. The aim was to increase the volume of the reconstructed breast, to recreate the anterior axillary fold and to augment the volume of the supero-medial quadrant which remained undercorrected after the LD flap. At this stage 350 cc were harvested from the abdomen and 285 cc were injected. The fat tissue harvest was performed as described by Coleman SR.5 A 3 mm

aspiration cannula, 15 cm long, with a rounded tip and double lateral openings was used to harvest the fat tissue. The cannula was attached to a 10 ml Luer Lock syringe. The vacuum in the syringe was created manually and progressively to avoid any excessive depression on the adipocytes. The fat tissue harvested was centrifuged thereafter. The centrifugation was carried out for 3 minutes at 3000 turns per minute. The injection of fat tissue was performed by means of 2 mm cannulas with various form and length. It was of paramount importance to inject the fat in the muscle flap. Any fat injection in the breast parenchyma was avoided. Ten ml syringes were used to inject the purified fat and 1 ml syringes for more precise injecting. Third stage The 3rd stage (Figs 3A,B) was carried out 6 months later with the harvest of 300 cc and the injection of 250 cc. The same technique of fat tissue transfer was used as described above. The final result one year after the third stage is shown in Figure 4 (Figs 4A,B). Discussion

The breast reconstruction in Poland anomaly is a challenging problem which needs a thorough preoperative analysis. The choice of the technique

A

B

Figure 2. Result 6 months after the endoscopic latissimus dorsi muscle flap. A: Frontal view; B: Left oblique view.

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Breast reconstruction in Poland anomaly...

A

B

Figure 3. Preoperative markings before the second stage of fat tissue transfer. A: Frontal view; B: Left oblique view.

A

B

Figure 4. Final result at one year. A: Frontal view; B: Left oblique view (note the small scar at the donor site).

depends on a number of parameters among which the volume of the affected gland, the quality of the skin envelope and the association of thoracic wall deformity. The reconstruction with a breast implant is feasible when a reliable coverage of the prosthesis can be achieved. Since the pectoralis major muscle is generally hypoplastic or even absent the prosthesis is inserted in a subcutaneous pocket or in a

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subglandular manner depending on the presence and volume of the breast parenchyma. This technique was reported to have a big percentage of implant exposure and high incidence of capsular contracture due to the unreliable coverage.4,9 Previous tissue expansion was recommended in cases where skin envelope is of poor quality.10 Longaker M, et al. advocated the microvascular

Folia Medica, XL 1/2008 free tissue transfer which requires special equipment and training, and is associated with elevated morbidity of the donor and recipient site.11 Free tissue transfer was also reported for simultaneous breast and chest wall reconstruction by Liao HT et al.12 The reconstruction with a LD muscle flap was initially proposed by Hester and Bostwick.13 It offers well-vascularised, autologous tissue, but the muscle flap usually undergoes some hypotrophy with possible reduction of the final volume of the reconstructed breast. However, this method is largely used and can be combined with a breast implant. In this case the muscle flap provides an excellent coverage for the prosthesis. Nevertheless, the possible complications from the implant (infection, capsular contraction, rupture, etc.) should be born in mind.13 In most of our cases we favour the autologous breast reconstruction with LD muscle flap. Harvesting an LD muscle flap for breast reconstruction is a well-established procedure.8 However, it requires a long incision with significant scar and post-operative pain. The endoscopic harvest offers a remarkable opportunity to decrease these two disadvantages of the open technique as reported by Lin CH, et al.14 Their results did not show any statistically significant differences in the amount of intraoperative bleeding, the incidence of postoperative hematoma and seroma, and the incidence of donor-site wound infection between the open and endoscopic technique. Endoscopically assisted harvest of the LD muscle was associated with less pain and allowed earlier and better movement of the upper extremity of the donor site. The patients’ feelings about the scar and overall satisfaction were higher in the endoscopic group, with statistically significant difference.14 Our impression from this case is that the patient felt significantly less pain and the postoperative pain was very easily controlled. In addition, she was highly satisfied with the smaller scar on her thorax. One of the greatest disadvantages of the endoscopic technique are the difficulties in the exposure when performing the endoscopic dissection. To overcome this several solutions were reported. The most commonly used techniques are the external skin retraction and internal skin and/or muscle lifting. 7 We also used external traction on the skin in association with internal lifting with an endoscopic liver retractor. The external retraction was performed by transcutaneous sutures to tract

on the skin. The use of CO2 gas for creation of virtual cavity by gas distention was reported by Pomel et al.15 We do not have any experience with this method which is difficult to realise because of the lack of sufficient skin laxity. Another option for the creation of an optical work space is the use of a sterile balloon filled with saline as reported by Karp NS et al.16 We do not have any experience with this technique but it seems that it provides good exposure for endoscopic dissection.16 The average axillary incision length is reported to be 5.6 cm.16 In our case a 5-cm incision was used which seems sufficient for the initial dissection. The number of access ports vary between 3 and 4, with the four-port technique reported as more reliable.16 We feel that whether to use 3 or 4 ports is more a matter of experience and personal choice. In our case the operative time was longer than in the open technique due to our small experience with the endoscopic harvesting of LD muscle flap. The technique of injecting autogenous fat for breast augmentation has been considered highly questionable for the last 20 years. As far back as the 1980s, there have been multiple published reports of fat necrosis and soft tissue calcification associated with this procedure.17,18 The concern that fat injections could hamper the diagnosis of breast cancer rendered this technique contraindicated for most surgeons. However, there is increasing data that fat tissue transfer is probably a safe and reliable technique in both postoncologic breast reconstruction and aesthetic indications.19,20 As recently reported by Spear et al. and Coleman et al. the detailed clinical examination in association with imaging diagnosis are sufficient in detecting breast cancer after fat tissue transfer in the breast.19,20 Furthermore, the injecting of fat itself does not seem to decrease the detection rate of breast cancer. This is supported by the data of Pierrefeu-Lagrange AC, et al. who demonstrated safety and reliability of radiologic surveillance of the reconstructed breast with autologous fat tissue transfer.21 However, it should be pointed out that it is of primary importance to inject the autologous fat in the underlying muscles (pectoralis major muscle, latissimus dorsi muscle flap) and not in the breast parenchyma. This is a detail which is particularly important, as reported by Coleman20, in order to avoid the formation of any intraparenchymatous post-injectional lesions. We tried to follow strictly

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Breast reconstruction in Poland anomaly...

these recommendations for fat injection. The volumetric recreation of anterior axillary fold in Poland anomaly was reported as one of the most difficult problems to treat.9 The satisfactory restoration of this element can be hardly achieved either with the LD flap or with breast implants.9 We feel that the fat tissue transfer represents an useful adjunct in achieving satisfactory volumetric restoration of the anterior axillary fold and the supero-medial quadrant of the breast. The patient presented in this report was followed up twice a year. The result demonstrated on figure 4 is stable over time. No pathological findings were detected at the follow-up visits. We used the technique of fat tissue transfer in association with autologous endoscopically-assisted LD muscle flap to perform an entirely autologous reconstruction, with the patient extremely satisfied with the final result. Conclusions

The breast reconstruction with a LD muscle flap remains an important option in Poland syndrome. The endoscopic harvest of the flap might provide a shorter scar, decreased postoperative pain and elevated overall satisfaction. The endoscopic approach can be used in all ages but should be particularly considered in young patients. The autologous fat tissue transfer represents a complementary procedure of choice to treat the regions that cannot be repaired by the flap. It seems that the autologous techniques for breast reconstruction are gaining increasing importance. Provided the fat tissue transfer in normal breasts would be accepted as a safe and reliable procedure, it will considerably ameliorate the possibilities of breast reconstruction in Poland anomaly. References

1. Poland A. Deficiency of the pectoral muscles. Gyus Hosp Rep 1841;6:191. 2. Shipkov CD, Anastassov Y. Bilateral Poland anomaly: Does it exist? Am J Med Genet 2003;118A:101. 3. Da Silva Freitas R, Dall’oglio Tollazi AR, Martins VD, et al. Poland’s syndrome: different clinical presentations and surgical reconstructions in 18 cases. Aesthet Plast Surg 2006;8:78-82. 4. Fourcas L, Grolleau-Raoux JL, Chavoin JP. Poland’s syndrome: clinic series and thoraco-mammary reconstruction. Report of 27 cases. Ann Chir Plast Esthet 2003;2:54-66.

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5. Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg 1997;2:347-67. 6. Mojallal A, Foyatier JL. Historical review of the use of adipose tissue transfer in plastic and reconstructive surgery. Ann Chir Plast Esthet 2004;50:419‑45. 7. Miller MJ, Robb GL. Endoscopic techniques for free flap harvesting. Clin Plast Surg 1995;4:755-73. 8. Delay E, Jorquera F, Pasi P, et al. Autologous latissimus breast reconstruction in association with the abdominal advancement flap: a new refinement in breast reconstruction. Ann Plast Surg 1999;1: 67-75. 9. Shipkov HD, Anastassov Y, Mikhaylov T. Surgical reconstruction options in Poland’s syndrome. Khirurgiia 2000;2:26-9 (Bulgarian). 10. Versaci AD, Balkovich ME, Goldstein SA. Breast reconstruction by tissue expansion for congenital and burn deformities. Ann Plast Surg 1986;1:20-31. 11. Longaker MT, Glat PM, Colen LB, et al. Reconstruction of breast asymmetry in Poland’s chest wall deformity using microvascular free flaps. Plast Reconstr Surg 1997;2:429-36. 12. Liao HT, Cheng MH, Ulusal BG, et al. Deep inferior epigastric perforator flap for successful simultaneous breast and chest wall reconstruction in a Poland anomaly patient. Ann Plast Surg 2005;4:422-6. 13. Hester TR, Bostwick J. Poland’s syndrome: correction with a latissimus muscle transposition. Plast Reconstr Surg 1982;69:226-32. 14. Lin CH, Wei FC, Levin LS, et al. Donor-site morbidity comparison between endoscopically assisted and traditional harvest of free latissimus dorsi muscle flap. Plast Reconstr Surg 1999;4:1070-7. 15. Pomel C, Missana MC, Atallah D, et al. Endoscopic muscular latissimus dorsi flap harvesting for immediate breast reconstruction after skin sparing mastectomy. Eur J Surg Oncol 2003;2:127-31. 16. Karp NS, Bass LS, Kasabaian AK, et al. Balloon assisted endoscopic harvest of the latissimus dorsi muscle. Plast Reconstr Surg 1997;5:1161-7. 17. Pulagam SR, Poulton T, Mamunas EP. Long-term clinical and radiologic results with autologous fat transplantation for breast augmentation: Case reports and review of the literature.  Breast J 2006; 12:63‑5. 18. Kwak JY, Lee SH, Park HL, et al. Sonographic findings in complications of cosmetic breast augmentation with autologous fat obtained by liposuction. J Clin Ultrasound 2004;32:299-301.   19. Spear SL, Wilson HB, Lockwood MD. Fat injection to correct contour deformities in the reconstructed breast. Plast Recosntr Surg 2005;116:1300-5.

Folia Medica, XL 1/2008 20. Coleman SR, Saboeiro AP. Fat grafting to the breast revisited: safety and efficacy. Plast Reconstr Surg 2007;119:775-85.

21. Pierrefeu-Lagrange AC, Delay E, Guerin N, et al. Radiological evaluation of breasts reconstructed with lipomodeling. Ann Chir Plast Esthet 2006;1:18‑28.

Р е конс т ру к ц и я г рудно й жел е зы при синдроме Poland с помощью эндоскопически ассистированного мышечного лоскута (ламбо) от latissimus dorsi и аутотрансплантации жировой ткани – клинический случай и обзор литературы

сильное социальное и психологическое воздействие на ее личную и профессиональную жизнь. Клинические и рентгенологические данные о сопутствующей деформации грудной стенки не выявлены. Дооперативные исследования груди (маммография и эхография) не показывают патологические изменения. Проведена аутореконструкция груди в трех этапах. Первый этап - эндоскопически ассистированный мышечный лоскут от LD. На следующих двух этапах проведена симметризация контралатеральной груди редукционной маммопластикой и аутотрансплантацией жировой ткани. Результаты: Постоперативные осложнения не имеются. Отмечена более слабая постоперативная боль по сравнению с той при классической “открытой” технике отпрепарирования ламбо от LD, которое проводится через меньший разрез и с меньшим цикатриксом на спине. Достигнутая симметрия грудных желез отлична. В ыводы : Реконструкция груди при синдроме Poland можно осуществить целиком аутоложными средствами. Эндоскопические техники отпрепарирования ламбо от LD приводят к меньшим цикатриксам и к меньшей постоперативной боли. Аутотрансплантация жировой ткани может оказаться полезным дополнением к классическим техникам с применением ламбо при условии выполнения требований к обеспеченности и про-ведения регулярного постоперативного контроля. (1/2008)

A. Mojallal, Хр. Шипков, F. Braye

Резюме Введение: Поражение грудной железы у женщин с аномалией Polandу (синдром Poland) представляет собой проблему с эстетической и социальной точки зрения. В целях реконструкции груди в таких случаях предлагаются различные методы – протезы, ламбо от latissimus dorsi (LD) или комбинация этих двух методов. Цель: Настоящее исследование ставит себе целью представить реконструкцию груди при синдроме Poland с помощью эндоскопически ассистированного мышечного лоскута (ламбо) от latissimus dorsi в сочетании с аутотрансплантацией жировой ткани и анализировать полученные результаты в соответствии с данными литературы. Пациенты и методы: Представляется женщина в возрасте 18 лет с тяжелой гипоплазией левой груди (в контексте синдрома Poland), что оказывало

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In memoriam

PROFESSOR ATANAS DJURDJEV 1941 - 2007 We are all deeply saddened by the death of Professor Atanas Djurdjev. He was Rector and Dean of the Plovdiv Medical University for two terms, Head of First Department of Internal Medicine, and Editor-in-Chief of the University Medical Journal Folia Medica. This grievous loss deeply saddens us all. Professor Djurdjev was a great spirit, a man of unflagging kindness whose goodness inspired all who knew him. He combined consummate professionalism with an abiding moral and ethical sense. He combined profound and encyclopaedic knowledge with open-minded generosity. His erudition was a constant credit to his University. He was a venerated teacher and colleague. There are no words to describe the unforgettable, inspirational man that he was. An administrator of unsurpassed abilities, he could find solutions and options for even the most vexing day-to-day problems. He was the driving force behind the construction and equipping of the Cardiology and Cardiosurgery Units, and it was thanks to his initiative and foresight that the university’s New Dental and Pharmaceutical Departments were founded. Everything in this world is transient. But we who knew him will continue to honour the memory of our

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beloved teacher, colleague and friend. We honour the intelligent and humane way he did his work and fulfilled his vocation. He cared deeply about people, he loved life, and he lived his life in a most worthy way. While we were honoured to be numbered among his friends, for us he was and will always be Professor Djurdjev. That title of respect and authority accurately reflects his many academic and administrative accomplishments, and it is one that he fully earned and most richly deserved. Professor Djurdjev was a consummate physician and clinician who successfully treated literally thousands of patients. The key to his success as a doctor was his ability to both treat his patients’ illnesses and at the same time comfort and console them. Patients from all over the country sought him out as the highest authority in his field, fully confident in his professional abilities, and he never rejected anyone who wanted to consult him. He was always willing to share with his colleagues his wealth of clinical experience. He was always available, always did his utmost to put people at their ease, was always ready with a kind word and a helping hand. Professor Djurdjev was long one of Bulgaria’s most distinguished scholars. He had to his credit more than 200 publications in Bulgarian and international journals, was author and co-author of two monographs, and contributed articles to more than ten medical textbooks. Under his supervision, more than fifteen doctoral dissertations were successfully defended and four major scientific projects developed. He was always apprised of the latest developments in his field, thanks to his active participation for more than twenty years in scholarly conferences related to cardiology. While his expertise was firmly grounded in both general medicine and cardiology, it extended to interdisciplinary issues related to the diagnosis, assessment, and treatment of disease. He was a long-standing Chairman of the Plovdiv Society of Cardiologists, and served a term as a national consultant in cardiology. For many years, he was a member and vice chairman of the Bulgarian Cardiology Accreditation Commission. Professor Djurdjev was named Physician of the Year in 2000, awarded the City of Plovdiv’s Medal of Honour for his distinguished service, and made an honorary citizen of the Chepelare municipality and the village of Pavelsko. In 2002, he won the annual Professor Konstantin Chilov prize for distinguished service to the field of medicine. His reputation as a physician and cardiologist far exceeded Bulgaria’s national boundaries. He was named Doctor Honoris Causa by the Polish Academy of Sciences, awarded the gold medal by the Albert Schweitzer World Academy of Medicine, and honoured with the Nagasaki Order of Peace. When engaged in scholarly discussion and debate, his prodigious memory, insatiable scientific curios-

Folia Medica, XL, 1/2008 ity, and profound insight were a constant source of encouragement and amazement to his colleagues. We will never forget his brilliant, innovative approach to problems in the medical profession. What’s more, he never underestimated the contribution of his colleagues, constantly encouraged us to apply a rigorously scientific approach when introducing new technology and techniques into Bulgaria. Professor Djurdjev could express even the most difficult ideas clearly and simply, and that skill made him a highly respected teacher for generations of students. He had the rare ability to communicate his enthusiasm to his audience along with the complexities and subtleties of his discipline. Some of his former students now hold key positions in Bulgaria, while others working abroad serve as his ambassadors, maintaining the high standards of scholarship and service that he both taught and embodied. Professor Djurdjev was not only a highly skilled professional, he was also committed to the cause of justice and human dignity both within and beyond the bounds of his profession. He always displayed the highest respect for his colleagues’ efforts and achievements. As an administrator, he was always industrious

and energetic, and through his extraordinary sense of humour and generosity of spirit could motivate his staff to successfully implement his far-reaching plans. At home, Dr. Djurdjev was a loving and devoted husband, a caring father and grandfather, an avid supporter of his favourite football team. He could never get enough of his favourite music, which he was always eager to share with his friends. His inexhaustible wit, vitality, and powerful intellect were a constant delight, and made working with him a joy. Scores of his friends and colleagues all over the world join us in mourning his passing. Professor Djurdjev was living proof that those who find and follow their vocation can be productive, respected, and contented human beings. We who had the privilege of knowing him – as colleague, teacher, scholar, physician, administrator, and friend – will be forever grateful for all that he gave us. He led us by example, and he bequeathed to us a lasting legacy of selfless dedication to the medical profession. The loss of this great man is a heavy personal blow to all of us who knew and admired him, and to Bulgarian cardiology. May our service and achievements in his chosen profession continue to honour his memory.

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FOLIA MEDICA, which began publication in 1959, is the peer-reviewed official journal of the Medical University in Plovdiv, Bulgaria. It is published quarterly in English giving high priority to original contributions of the medical staff of the Medical University. Original contributions of authors from other medical establishments in Bulgaria and abroad can also be considered for publication. The materials are submitted to the Editorial Office in Bulgarian with the signatures of all authors on the last page of the manuscri pt. A cover letter must be enclosed separately giving the full name of the corresponding author and providing this author’s complete address,telephone and fax numbers, and e-mail address. The latter should coordinate the suggested review corrections with the co-authors. Each author is required to attest in written in a Submission Form that the manuscri pt is not under simultaneous consideration by another publication at the time of its submission, and that it has not been published elsewhere either in print or electronic format. The submission of the manuscri pt by the authors means that the authors automatically agree to assign exclusive copyright to FOLIA MEDICA if and when the manuscri pt is accepted for publication. The materials and the procedures used in the study should be in conformity with the established ethical criteria concerning experimental studies involving human beings or animals. No patient should be referred to by a name, initials or photographs by which identification of the person could be done. Authors are responsible for all statements, opinions, conclusions, and methods of presenting their data in the submitted materials. All submissions are assigned to a review process by expert referees to determine the originality, validity, and importance to the field of their content and conclusions and then accepted for publication upon approval by the Editorial Board. Editorial Office: Library & Information Center FOLIA MEDICA Medical University 15A Vassil Aprilov Street Plovdiv 4002 tel.:+359 32/602 224 E-mail: [email protected]

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These instructions have been revised in accordance with the “Uniform Requirements for Manuscri pts Submitted to Biomedical Journals,” (N Engl J Med 1997; 336: 309-315.) Submissions in the following categories will be deemed appropriate for consideration by the Editors and Editorial Board of FOLIA MEDICA: Review articles and special articles these should not exceed 12 standard-sized doublespaced pages, including tables, figures and text appended to them. The review articles should include Introduction, the remaining sections being given appropriate headings by the authors. The Abstract and References (no more than 25 references) sections are submitted separately. Original articles - up to 10 standard-sized double-spaced pages, including tables, figures, and accompanying text. The Abstract and References (no more than 20 references) sections are submitted separately. The sections in the articles are: Introduction; Materials and Methods; Results; Discussion and Conclusions. Short Communications - these should be no more than 4 standard-sized double-spaced pages including tables, figures and accompanying text. The Abstract and References (no more than 20 references) sections are submitted separately. The format is identical to that of a full-length article with only the following sections bearing a heading: Introduction (with the aim of the communication clearly stated); Materials and Methods; Results and Discussion; Conclusions. Case Reports - these are limited to 3 standard-sized double-spaced pages including tables, figures and illustrations. The Abstract and References (no more than 8 references) sections are submitted separately. Letters to the Editor - these include comments arising from recent articles published in the FOLIA MEDICA, or discussions on topical problems. They should be short, up to 2 standard-sized double-spaced pages including references (no more than 5 references). The authors are required to e-mail or submit by post two copies of the original manuscri pt, the tables and figures accompanying the text and a 3.5”-disk containing the manuscri pt. The abstract is submitted in two copies separately from the manuscri pt.

 TECHNICAL REQUIREMENTS AND

PREPARATION OF THE MANUSCRIPT

1. All manuscri pts must be either typewritten or printed on one side of white bond paper (A4, 212 x 297 mm). Allow margins of at least 25 mm on all sides of the typed pages. Papers should be double-spaced throughout in 30 lines and 60 characters per line. Number manuscri pt pages consecutively throughout the paper beginning with the title page. If the hard copy is printer generated, use only 12-point font size of the letters. The copies should contain no smudges or pencil or pen marks. Manuscri pts written in block type font will be rejected. 2. The first (title) page of the paper should carry the following obligatory sections: - Title, no abbreviations - Authors’ names - full first and family name, and middle initial - Names of the Department(s) and Institution(s) with which the authors are currently affiliated. Indicate by an asterisk the institutions other than these if the research was performed in them. 3. Acknowledgements - Individuals with direct involvement in the study but not included in authorshi p, collaboration or preparation, financial or material support may be acknowledged. Authors are responsible for obtaining written permission from persons acknowledged by name. ABSTRACT. Authors submitting manuscri pts should prepare an abstract of no more than 250 words under the following headings: Objective, Methods, Results, and Conclusions. They should briefly describe the problem being addressed in the study, how the study was performed, the salient results, and what the authors conclude from the results. The abstract should be on a separate page. Do not include the institutional affiliation of authors. Abbreviations should be avoided in the abstract. Include up to 5 key words or phrases for subject indexing (use terms from the Medical Subject Headings from Index Medicus). FIGURES. Figures should be professionally designed and photographed; freehand or typewritten lettering is unacceptable. Send sharp, glossy, black-and-white photographic prints usually 90x120 mm or 127x178 mm (5x7 in.). Figures should be numbered consecutively according to the order in which they have been first cited in the text and the numbers should be given in the left-hand margin (e.g., Fig. 1) when first cited. Letters, numbers, and symbols should be clear and even throughout and of sufficient size

that when reduced for publication each item will still be legible. The back of each figure should include the sequence number and the proper orientation (e.g., top). Each figure should be enclosed in an envelope with the author’s name and the title. When symbols, arrows, numbers, or letters are used to identify parts of the illustrations, they should be marked on a transparency, attached to one side of the photograph. Titled and detailed explanations belong in the legends for illustrations,not on the illustrations themselves and these should be stated on a separate sheet at the end of the manuscri pt, entitled “Legends for Illustrations”. TABLES. Type or print out each table with double spacing on a separate sheet of paper. Number tables consecutively in the order of their first citation in the text and give the number of the table in the left-hand margin. Supply a brief title for each. Give each column a short or abbreviated heading. Place explanatory matter in footnotes, not in the heading. Explain in footnotes all non-standard abbreviations that are used in each table. STATISTICS. All data must be analyzed statistically. Detailed statistical methodology must be reported. Describe randomization procedures. Describe the specific tests used to examine each part of the results. Care should be taken with respect to a) parametric vs. non-parametric data, b) corrections for multi ple comparisons, and c) rounding errors (summary statistics should not contain more significant digits than the original data). Variability should be expressed either as median and range (or percentiles) for non-parametric data, or mean ± standard error, standard deviation (or confidence intervals) for normally distributed data. Traditionally FOLIA MEDICA uses the following symbols: x ± S x (for mean and standard error) and Sx (for standard deviation). UNITS OF MEASUREMENT. All measurements should be reported in accordance with the International System of Units (SI). ABBREVIATIONS. Use only standard abbreviations. Avoid abbreviations in title and abstract. The full term for which an abbreviation stands should precede its first use in the text unless it is a standard unit of measurement. REFERENCES. References should be numbered consecutively in the order in which they are first mentioned in the text (not alphabetically). Identify references in the text, tables, and legends by Arabic numerals in parentheses.

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ARTICLES IN JOURNALS. List all authors when they are three or fewer; when there are more than three, list the first three, followed by “et al”. The names of all authors are cited in inverted order - first the family name in full followed by the author’s initials without dots between them. The journal names are given in the abbreviated form in the style used in Index Medicus, followed by the year of publication, volume number, the month and issue number and inclusive page numbers. Example: Parkin DM, Clayton D, Black RJ, Masuyer E, Friedl HP, Ivanov E, et al. Childhoodleukaemia in Europe after Chernobyl: 5 year follow-up. Br J Cancer 1996; 73: 1006-12. BOOKS AND OTHER MONOGRAPHS. List the name of the author(s), title, place of publication, publisher and date.

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Example: Kassirer JP, Kopelman RI. Learning clinical reasoning. Baltimor (MD): Williams & Wilkins;1991. CHAPTERS IN A BOOK. List the name of the author, title, followed by In:, name of editor(s), place of publication, publisher, date and pages. Example: Philli ps SJ, Whisnant JP. Hypertension and stroke. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis, and management. 2nd ed. New York: Raven Press; 1995: 465-78. DISSERTATION. List the name of author, title of dissertation, specified in square brackets [dissertation], the locale where the dissertation was written, the name of institution and year of writing. Example: Kaplan SJ. Post-hospital home health care: the elderly’s access and utilization [dissertation]. St. Louis (MO): Washington Univ.; 1995.

Back cover: One of the most beautiful reliefs of the healing cults in the Greco-Roman pantheon is the unique “Frieze of the healing family” exhibited in the Archeology Museum in Plovdiv. It was excavated in the foundations of an old ruined Turkish mosque in 1921. The correct identification of all figures was performed by Professor Zapryanov* in 1964 - Department of Social Medicine. The frieze, according to him, used to adorn a Roman valetudinaria - a military hospital - off the walls of the east entrance of the ancient city which was called Trimontium by the Romans in the late III century. It weighs about 3000 kg and is 2.80 m long and 1.08 m high. The figures on it are framed in a wide rim; it bears the personified images of the Moon (on the left) and the Sun (on the right). Presented on the frieze are (from left to right): Jaso and Panacea - Asclepios’ daughters, Telesphor - the fortunate genius of the healing process, Asclepios - the god of healing art, Hygeia - his daughter, Epione - Asclepios’ wife, Machaon and Podaleirios - his sons worshi pped as military physicians. All figures, except Panacea, are entirely in full face which is very rare in a general composition picture. The frieze’s sculptor depicted in great detail the figures’ anatomic features, clothes and peculiar attributes. All deities in the composition are on a par with the only association seen between Panacea and Asclepios (Panacea touches a bundle of herbs next to Telesphor’s cowl with her left hand, while pouring the cure all (panacea) in Asclepios’ bowl).

_____________________________ * Folia Medica 1964; 6(3): 152 - 156