Neurochemical Research, Vol. 30, No. 8, August 2005 ( 2005), pp. 1021–1026 DOI: 10.1007/s11064-005-6786-7
Effect of General Anesthetics on Amyloid Precursor Protein and mRNA Levels in the Rat Brain Miklo´s Palota´s,1,2 Andra´s Palota´s,1,3,4 Annama´ria Bjelik,1 Magdolna Pa´ka´ski,1 Marietta Hugyecz,1 Zolta´n Janka,1 and Ja´nos Ka´lma´n1 (Accepted June 22, 2005)
The incidence of Alzheimer’s disease is elevated after exposure to surgical interventions. Since amyloid precursor protein (APP) and its neurotoxic derivatives play key roles in the development of Alzheimer dementia, the role of general anesthesia is controversial in the development of cognitive decline. As such, the effect of anesthetics on APP protein and mRNA levels was assessed utilizing semiquantitative Western-immunoblot and reverse transcription polymerase chain reaction (RT-PCR) in brains of rats following intraperitoneal treatment with propofol and thiopental. The anesthetics did not change cortical APP protein and mRNA concentration considerably. These results indicate that both propofol and thiopental are considered to be relatively safe with respect to APP metabolism. KEY WORDS: Alzheimer’s disease; amyloid precursor protein; cognitive dysfunction; general anesthetic; general surgery; propofol; thiopental.
after non-cardiac surgery. Cognitive dysfunction is most notable immediately after the operation, but it can persist. It leads to longer hospitalization and increased acuity of institutionalization or discharge care especially in the elderly. Further, there is great personal loss to patients who might not return to their baseline level of cognitive function, despite a successful surgical procedure. The incidence of cognitive dysfunction has been reported to be between 1% and 60%, depending on the type of operation (2). Up to 83% of patients exhibit cognitive dysfunction in the immediate postoperative period, which persists in 20% to 30% of patients. Apart from cognitive decline, the incidence of Alzheimer’s disease (AD) is also markedly increased after cardiac surgical interventions (3–6). We have recently reported that the level of cognitive biomarkers in the cerebrospinal fluid of patients that had undergone offpump coronary artery bypass grafting reflect severe neuronal injury and mimics those seen in AD patients (7), and hypothesized that anesthetics might play a
INTRODUCTION Cardiac operations frequently are complicated by postoperative cognitive decline (POCD) (1). Less common are postoperative psychiatric symptoms 1
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Department of Psychiatry, Albert Szent-Gyo¨rgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged, H-6721, Szeged, Semmelweis u. 6, Hungary. Department of Anesthesiology and Intensive Therapy, Albert Szent-Gyo¨rgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged, H-6721, Szeged, Semmelweis u. 6, Hungary. Division of Cardiac Surgery, Center for Cardiology, Albert Szent-Gyo¨rgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged, H-6720, Szeged, Pe´csi u. 4, Hungary. Address reprint requests to: Andra´s Palota´s, Division of Cardiac Surgery, Center for Cardiology, Albert Szent-Gyo¨rgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged, H-6721, Szeged, Pe´csi u. 4, Hungary. Tel.: +36-30255-6225; Fax: +36-62-545-701; E-mail:
[email protected]. u-szeged.hu
1021 0364-3190/05/0800–1021/0 2005 Springer Science+Business Media, Inc.
1022 key role in initiating the process that may lead to the development of dementia. The neurotoxic (-amyloid peptide ((AP) is thought to play a crucial role in the development of AD, which is a proteolytic derivative of the amyloid precursor protein (APP) (8). Even though increased APP level is a precondition to excessive (AP formation, one of the inducible neuroactive (trophic) factors at site of neural injury is the APP that may participate in cell-to-cell recognition, re-establishment of synaptic contacts, alleviation of glutamate-induced membrane depolarization and over-excitation, and stabilization of the intracellular Ca2+ homeostasis (9,10). Therefore, it is assumed that APP is an important protein for tissue repair and that its expression is up-regulated following nerve damage (11), as might be expected to occur following surgery. The purpose of this study was to evaluate whether the two most frequently used anesthetics: thiopental (Thiopental, Thio) and propofol (Diprivan, Dipr) might interfere with APP metabolism. As such, we utilized semiquantitative Westernimmunoblot to detect APP protein levels and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) to assess the abundance of APP mRNA (mRNAAPP).
MATERIALS AND METHODS Animals. Male, Sprague–Dawley rats weighing 200–250 g were maintained under standard laboratory conditions with a 12-h light/dark cycle; they were given rodent chow and water ad libitum. With respect to thiopenthal (Thiopental, Sandoz Ltd., Hungary), groups of six rats were injected intraperitoneally (ip.) on a daily basis with therapeutic (5 mg/kg) or toxic (20 mg/kg) doses dissolved in saline. In case of propofol (Diprivan, Astra Zeneca Ltd., Hungary), therapeutic (4 mg/kg) or toxic (20 mg/kg) doses, dissolved in saline, were administered. Control rats received the vehicle alone. No other animals were housed in the room or allowed contact with the study animals. Experiments were performed in accordance with a protocol approved by the university ethics committee on laboratory animals. Preparation of Rat Cortical Samples. After acute (6, 12, 24 and 96 h) and chronic (1, 2, 3 and 4 weeks) administration of the anesthetics in both therapeutic and toxic doses, rats were decapitated under ether anesthesia. The cerebellum was removed and the fronto-temporo-parietal cortices were dissected. In order to eliminate post-mortem decay, preparation was performed on dry ice. Cortical samples were homogenized in 50 mM of Tris buffer (pH 7.5) containing 0.15 M NaCl, 2 mM phenyl-methyl-sulfonylfluoride, 2 mM EDTA, 2 (g/ml leupeptin, 1 (g/ml pepstatin, 1% Nonidet-P-40 and 0.1% sodium deoxycholate by using a glass-teflon potter (1500 rpm, 1 min). The homogenates were centrifuged at 10,000 g for 30 min at 4C. The supernatants were used for the
Palota´s et al. detection of total APP. Protein concentrations of samples were determined by the method of Folin as modified by Hess et al. (12). Western Immunoblot. Proteins (30 (g/lane) were separated on a 9% of sodium-dodecyl-sulfate (SDS)-polyacrylamide gel and electroblotted onto nitrocellulose membranes by using the BioRad Mini-Protean II system. After protein quenching with 5% non-fat dry milk in 50 mM of Tris-buffered saline (TBS, pH 7.5) containing 0.2% of Tween-20, the blotted samples were incubated overnight at room temperature in monoclonal 22C11 antibody (5 lg/ml, against residues 68–81 of APP). After being washed (5(), the membranes were incubated with horse-radish-peroxidase (HRP)-conjugated anti-mouse-IgG (1:500, Sigma-Aldrich, USA). For the detection of blots, the Renaissance Western Blot Chemiluminescence Reagent (Pierce, USA) was employed, followed by exposure to an autoradiographic film. Optical densities of immunoreactive bands were detected and quantified by means of the NIH-Image Analyser Program (NIH, USA). The levels of APP in the control group were taken as 100%, and changes were calculated with respect to this value (data are given in relative units). RT-PCR. Total cellular RNA was extracted by the methods of acid guanidium-thyocianate phenol/chloroform extraction (13). In brief, 5 lg of total mRNA was transcribed into cDNA with oligo(dt)18 primers using RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). cDNA were amplified by PCR with oligonucleotides for (-actin and APP. The oligonucleotide primers for (-actin were as follows (14). Sense: 5¢GGCTGTGTTGTCCCTGTAT-3¢ (2203–2221 bp), and antisense: 5¢-CCGCTCATTGCCGATAGTG-3¢ (2554–2536 bp; Genebank access number J00691). The numbers of nucleotide bases were according to the rat APP genetic sequence numeration from Genebank (access number X074648). The APP oligonucleotides were as follows. Sense, 5¢-GGATGCGGAGTTCGGACATG-3¢ (788–1807 bp), and antisense, 5¢-GTTCTGCATCTGCTCAAAG3¢ (2085–2067 bp). PCR was performed in a final volume of 25 ll, containing 2.5 ll of 10 PCR puffer, 2 ll of 25 mM MgCl2, 1 ll of 5 mM dNTP, 6 pmol of b-actin, 25 pmol of APP, 2 ll of the cDNA described above and 0.25 ll Taq DNA polymerase (5 units/ ll) (Fermentas, St. Leon-Rot, Germany). Each cycle consisted of 1 min 94C, followed by 94C for 30 s, 54 C for 30 s and 72C for 1 min. After 30 cycles, extension was performed at 72C for 5 min. PCR products were separated by 1.3% agarose gel electrophoresis. The ratios of APP695/b-actin mRNA and APP770/b-actin mRNA were calculated. Data Analysis. Results presented are ‘‘arithmetic means ± standard deviation’’ of experiments performed in triplicate. Student’s t-test was used to determine the significance of observed differences. Level of significance was set at 5% (P < 0.05).
RESULTS AND DISCUSSION This is the first published report on the effect of Thio and Dipr on APP metabolism in vivo. Using monoclonal 22C11 antibodies against residues 68–81 of APP, and by utilizing RT-PCR we were able to detect APP at 110–120 kDa, and mRNAAPP at 290– 300 bp (Figs. 1–3). Results were normalized to b-actin: a house-keeping gene whose expression is thought to be constant irrelevant of any drug treatment.
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Fig. 1. APP/mRNAAPP abundance. Quantification of gel electrophoresis delineates that neither acute, nor chronic administration of Thio or Dipr, neither in therapeutic (TH) nor toxic (TX) doses, changed rat cortical APP (a) protein and (b) mRNA levels when compared to that seen in controls (c). Concentrations were measured at 6, 12, 24, 96 h, 1 week, 2–3–4 weeks, however given an insignificant effect of the anesthetics, only 12 h and 4 weeks are depicted in the figure.
cognitive dysfunction does occur, especially in the elderly (15,16). Conversely, no specific complications have been reported of anesthesia for patients with AD. Given the substantial number of people affected with this disease, it is likely that anesthesiologists will encounter many patients with AD. Questions as to potential problems including informed consent, drug interactions, and preoperative progression of the disease may arise in this growing population. Even with large scale, prospective, randomized studies of contemporary anesthetic practice in the elderly, there appears to be no
Interestingly, neither Thio, nor Dipr did change APP and mRNAAPP levels considerably. There was neither time-, nor concentration-dependence with respect to the abundance of APP protein and mRNA. Development of cognitive dysfunction and AD after cardiac surgeries is a well-recognized and intensively studied phenomenon. Cognitive decline after major non-cardiac operations is not well characterized. Numerically, more non-cardiac interventions are performed each year, which could lead to a greater absolute number of patients who have cognitive impairment. Several reports indicated that
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Fig. 2. Semi-quantitative evaluation of rat cortical APP-Western-blot after intraperitoneal Thio and Dipr administration. Rat cortical APPlevels were approximated utilizing Western-blotting, and chemiluminescent visualization of bands were quantified by densitometry. Anesthetic-treatment revealed insignificant alterations in both therapeutic (TH) and toxic (TX) doses with regards to APP when compared to that seen with control (C) animals.
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Fig. 3. Time-course of anesthetics on rat cortical mRNAAPP. Ip. treatment of rats with Thio and Dipr do not significantly alter mRNAAPP in vivo as detected by RT-PCR (C: control; TH, TX: therapeutic and toxic anesthetic treatment, respectively).
clinical or statistical significant advantage to regional vs. general anesthesia. Similarly, there is no evidence to support the preferential selection of a specific general anesthetic agent in these patients (17). Treatment with anesthetics resulted in stable APP levels in our experiments, which leads to constant (AP production in the brain. As such, both Thio and Dipr are considered to be safe with respect to APP metabolism and they do not facilitate (AP production leading to POCD or AD. Moreover, experimental evidence supports a neuroprotective role for several commonly used anesthetics through anti-oxidative (e.g. Dipr), anti-apoptotic (e.g. sevoflurane) and other mechanisms. One of the difficulties in human research in this area is the fact that anesthesia is hardly ever administered as a sole procedure but is almost invariably given to facilitate surgery. Consequently, theories of surgical stress-induced POCD also exist. It is therefore reasonable to believe that POCD is not attributable to the anesthesia per se. Broadly in line with this hypothesis, there appeared to be a trend towards an increase in APP with time in our study irrespective of treatment. This might be attributable to the prolonged stress caused by daily ip. injections, forasmuch as APP is a marker of stress and early neuronal injury (18,19), even though increased APP level is a precondition to excessive neurotoxic (AP formation. These neuroactive (trophic) factors and functions of APP participate in cell-to-cell recognition, re-establishment of synaptic contacts, alleviation of glutamate-induced membrane depolarization
and over-excitation, and stabilization of the intracellular Ca2+ homeostasis (20,21). Importantly, the mechanism through which (AP aggregation leads to apoptotic neuronal death is associated with increased oxidative stress and disruption of cellular Ca2+ homeostasis. It is hypothesized that AD may result from a chronic low-grade inflammatory response in the brain, as supported by the increased incidence of AD in patients who have suffered head injury, and by the protective effects of non-steroidal anti-inflammatory drugs (22). It is therefore likely that, instead of the use of various anesthetics, the major inflammatory response associated with surgery would induce POCD and AD. Thio, being a barbiturate derivative, is known to potentiate c-amino-butyric acid (GABA)-induced chloride-current through binding to the GABAAreceptor. On the other hand, Dipr, a representative of steroid-based anesthetics, is a GABAA- and glycineagonist, inhibitor of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, and it also modulates the a2-adrenoreceptor system (23,24). Dipr also increases dopamine levels and decreases serotonin concentrations, probably through its action on GABA receptors, in the area postrema to yield antiemetic actions (25). Even though both drugs have a broad range of cellular effect in the brain, neither of them had an impact on APP metabolism in our study. This observation suggests that anesthetics with different pharmacological characteristics share similarities at the proteomics and genetic level with respect to certain proteins.
Impact of General Anesthetics on Amyloid Precursor Protein Despite major advances in surgical techniques and anesthesia management, central nervous system complications remain a common problem after surgical procedures. The etiology of neuropsychologic dysfunction after operations remains unresolved and is probably multifactorial. Demographic predictors of POCD and AD include age and years of education. Perioperative factors including number of cerebral emboli, temperature, mean arterial pressure, and jugular bulb oxygen saturation have varying predictive power. Apparently, the use of anesthetics does not facilitate cognitive dysfunction at the cellular level. Thus, other unknown factors influence this decline in cognition, some of which might not be unique to the nature of operations, including surgical stress. Furthermore, it might also be possible that people who have (subclinical) AD might for various reasons be more likely to undergo surgery, rather than the other way around. Surgical stress, in turn, may accelerate development of clinical signs and symptoms of AD. Despite successful surgical intervention, a patient might not be able to return to their baseline status and could require more intensive care by the family or by a long-term care facility due to a devastating cognitive dysfunction, which can therefore create an enormous cost to society in suffering and consumption of resources. By identifying the incidence, severity, and predictors of cognitive dysfunction, we might prevent these adverse neuropsychologic outcomes.
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ACKNOWLEDGMENTS This study was sponsored by the following grants: NKTH 8/ 2004 and OTKA T043418 (2003).
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