Vascular endothelial growth factor gene therapy improves nerve ...

Vascular endothelial growth factor gene therapy improves nerve regeneration in a model of obstetric brachial plexus palsy Matthias Hillenbrand1, Thomas Holzbach2, Kaspar Matiasek3, Ju¨rgen Schlegel4, Riccardo E. Giunta2 1

Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Germany, 2Department of Hand Surgery, Plastic Surgery and Aesthetic Surgery, University Hospital of the Ludwig-Maximilians-University, Munich, Germany, 3Institute of Veterinary Pathology, LudwigMaximilians-University, Munich, Germany, 4Division of Neuropathology, Institute of Pathology, Technical University of Munich, Germany The treatment of obstetric brachial plexus palsy has been limited to conservative therapies and surgical reconstruction of peripheral nerves. In addition to the damage of the brachial plexus itself, it also leads to a loss of the corresponding motoneurons in the spinal cord, which raises the need for supportive strategies that take the participation of the central nervous system into account. Based on the protective and regenerative effects of VEGF on neural tissue, our aim was to analyse the effect on nerve regeneration by adenoviral gene transfer of vascular endothelial growth factor (VEGF) in postpartum nerve injury of the brachial plexus in rats. In the present study, we induced a selective crush injury to the left spinal roots C5 and C6 in 18 rats within 24 hours after birth and examined the effect of VEGF-gene therapy on nerve regeneration. For gene transduction an adenoviral vector encoding for VEGF165 (AdCMV.VEGF165) was used. In a period of 11 weeks, starting 3 weeks post-operatively, functional regeneration was assessed weekly by behavioural analysis and force measurement of the upper limb. Morphometric evaluation was carried out 8 months post-operatively and consisted of a histological examination of the deltoid muscle and the brachial plexus according to defined criteria of degeneration. In addition, atrophy of the deltoid muscle was evaluated by weight determination comparing the left with the right side. VEGF expression in the brachial plexus was quantified by an enzyme-linked immunosorbent assay (ELISA). Furthermore the motoneurons of the spinal cord segment C5 were counted comparing the left with the right side. On the functional level, VEGF-treated animals showed faster nerve regeneration. It was found less degeneration and smaller mass reduction of the deltoid muscle in VEGF-treated animals. We observed significantly less degeneration of the brachial plexus and a greater number of surviving motoneurons (P , 0.05) in the VEGF group. The results of this study confirmed the positive effect of VEGF-gene therapy on regeneration and survival of nerve cells. We could demonstrate a significant improvement on the motorfunctional as well as on the histomorphological level. However, increased vascularization of the nerve tissue caused by VEGF does not seem to be the major reason for these effects. The clinical use of adenoviral VEGF-gene therapy in the newborn cannot be justified so far. Keywords: Nerve regeneration, Obstetric brachial plexus palsy, Vascular endothelial growth factor, Gene therapy

Introduction Obstetric brachial plexus palsy occurs depending on birth weights with an incidence of 0.9 to 2.6 per 1000 births.1 This complication is caused by injury to the brachial plexus during complicated child delivery and occurs mostly in macrosomic children of diabetic mothers.2,3 In many cases of obstetric brachial plexus

Correspondence to: M. Hillenbrand, Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Krankenhausstr. 12, 91054 Erlangen, Germany. Email: [email protected]

ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1743132814Y.0000000441

palsy, the continuity of the peripheral nerves remains preserved, so it usually comes to complete spontaneous regression of symptoms.4 Although many children recover spontaneously, in 20–25%, a permanent impairment of the affected limb remains.5 The treatment of peripheral nerve injuries has been limited to surgical reconstruction of neural continuity. Despite better understanding of the pathophysiology of nerve injuries and advanced microsurgical techniques, the surgical treatment remains a challenge. In addition to the damage of the brachial plexus itself it also leads to a loss of motoneurons in

Neurological Research

2015

VOL .

37

NO .

3

197

Hillenbrand et al.

VEGF-gene therapy improves nerve regeneration

the spinal cord-associated segment.6 The death of maturing motoneurons after peripheral nerve injuries is due to the lack of interaction with their target structures, which is essential for motoneuron survival during the early postpartum period.7 An interruption of the interaction in this critical period ultimately leads to the death of the corresponding motoneurons in the spinal cord.6,8 Current concepts of treatment have focused on the injury of the peripheral nerve without taking the participation of the central nervous system into account. In addition to the surgical treatment of peripheral nerve injuries, the application of biologically active messengers with neurotrophic effects on the peripheral nervous system could represent a complementary method for improved nerve regeneration.9,10 The vascular endothelial growth factor (VEGF) has an essential function during embryogenesis and in disease by stimulating the formation of blood vessels.11 Its biological effects are mediated via two receptor tyrosine kinases (VEGFR-1/Flt-1 and VEGFR-2/Flk-1), which are also expressed in human nerve grafts.12 Recent discoveries have been shown a direct effect of VEGF on neuronal cells.13 VEGF has been identified as a promoter of neuronal recovery and axonal growth, which improves survival of neurons.14 Reduced VEGF concentrations lead to degeneration of motoneurons by limited tissue perfusion and VEGF-dependent neuroprotection.15 The neuroprotective effect of VEGF is also mediated by reduced sensitivity of motoneurons against ischemia.16 These effects are caused by the binding of specific VEGF isoforms to neuropilin receptors (NRP-1 and NRP-2), which leads to an inducement of several signalling cascades in neuronal cell types.17 VEGF-mediated mechanisms of neuroprotection are not fully understood, but the effects seem to result from a combination of direct stimulation of neural cells and increased vascularization.18 In the present study, we examined the effect of VEGF-gene therapy on nerve regeneration after a selective crush injury of the left spinal roots C5 and C6 within 24 hours after birth. For gene transduction, an adenoviral vector encoding for VEGF165 (AdCMV.VEGF165) was used.

Materials and Methods A total of 18 newborn Sprague–Dawley rats with an average weight of 7 g were used in this study. All experiments were performed in accordance with institutional guidelines and were approved by the government of Oberbayern and the local animal care committee (AZ 55.2-1-54-2531-66-06). The animals were kept in standard Makrolon cages located in the animal facility of the Center of Preclinical Research of the Technical University of Munich/Klinikum

198


2015

VOL .

37

NO .

3

rechts der Isar (Munich, Germany). In all surgical procedures, anaesthesia was induced and maintained by spontaneous inhalation of isoflurane and oxygen.

Gene transduction The animals were randomly assigned to the VEGF (n 5 9) or the control group (n 5 9). Application was performed by a blinded surgeon with either 20 ml of viral solution containing 107 pfU AdCMV.VEGF165 for the VEGF group or 0.9% NaCl solution for the control group. The adenoviral vectors encoding for VEGF165 under the cytomegalovirus (CMV) promoter were constructed at the Institute of Virology and Immunology at the Technical University of Munich/ Klinikum Rechts der Isar, Munich. AdCMV.VEGF165 is a recombinant, replication deficient adenovirus, which carries the cDNA of the human VEGF isoform 165 as a transgene. We used a CMV immediate-early promoter to achieve high transgene delivery.19 In vitro studies have been shown a maximum production of VEGF (1473.37 ng/ml) 48 hours after transduction, followed by a consecutively decrease (208.41 ng/ml after 10 days).

Crush injury Within 24 hours after birth, an identical crush injury of the left spinal roots C5 and C6 was performed in each animal. The surgical area was carefully shaved, cleaned and disinfected. The incision was set caudal to the left clavicle. Pectoralis major and minor muscles were split longitudinally. Subsequently, blunt preparation was performed into the depth up to the brachial plexus. The individual trunks of the left brachial plexus were identified under microscopic magnification (AxiovertH 25; Fa. Zeiss, Jena, Germany). Then spinal roots C5 and C6 were crushed for 4 seconds using microforceps. Either virus or 0.9% NaCl solution was then injected around the nerve injury. After wound closure, animals were returned to their mothers.

Behavioural analysis In a period of 11 weeks, starting 3 weeks postoperatively, functional regeneration was assessed weekly by behavioural analysis and force measurement of the upper limb. In all experiments, motorfunctional recovery of the upper limb could be precisely evaluated. Each test was conducted at an interval of 2 weeks.

Grid walk test To detect latent deficits of balance and in particular the motor function of the upper limb, the movement pattern of the animals regarding accurate limb placement was examined using the grid walk paradigm.20 Without postoperative training, animals had to cross an 80 cm long ladder raised 1 m horizontally above the ground with rung spacing of 2 cm. Animals had to cross the ladder a minimum of three times.

Hillenbrand et al.

The number of missteps or searching movements of the left and right forepaws was counted and a mean error of the three tests calculated.

Grasping test This objective behavioural test provides a simple quantitative method for assessing functional recovery of the upper limb.21 Animals were gently lifted by the tail and held above a measuring device equipped with a handle that enables recording of tensile force (in gram). The free groping forepaws automatically grasped the handle while the animal is raised slowly until it lost its footing. The measuring device recorded the highest measured value before releasing, which gave information about the maximum force of the grasping movement. This test was performed alternately with the left and right forepaw a minimum of three times. Subsequently, the mean value of the three tests was calculated.

Grooming test Motor-functional behaviour of the upper limb can also be assessed using the grooming test.22 The application of sweet syrup onto the animals’ snout causes bilateral grooming response, which allows exact analysis of elbow flexion and abduction. Movement of the left and right upper limb was evaluated and graded using a non-parametric score: grade 1 (no movement or mouth), grade 2 (region below the eye), grade 3 (eye), grade 4 (front of ears), and grade 5 (behind the ears).

Morphometric and biochemical analysis Morphometric evaluation was carried out 8 months post-operatively and consisted of a histological examination of the deltoid muscle and the brachial plexus according to defined criteria of degeneration. In addition, atrophy of the deltoid muscle was evaluated by weight determination comparing the left with the right side. VEGF expression in the brachial plexus was quantified by an enzyme-linked immunosorbent assay (ELISA). Furthermore, the motoneurons of the spinal cord segment C5 were counted comparing the left with the right side. For euthanasia isoflurane inhalation (AttaneTM Isoflurane; Provet AG, Lyssach, Switzerland) followed by intramuscular injection of 100 mg/ kg body weight ketamine (NarketanH; Chassot GmbH, Ravensburg, Germany) and 10 mg/kg body weight xylazine (RompunH; Bayer, Leverkusen, Germany) was used. The aorta was exposed and the fixation-solvent was injected. Histomorphological examinations on the microscope (Axio Scope; Zeiss, Jena, Germany) were taken at 610 magnification.

Weight and degeneration of the deltoid muscle The deltoid muscles were harvested and measured 8 months post-operatively. The muscle weight of the left operated side was compared with that of the contralateral healthy side. The difference between


the left and right side gave information about the extent of atrophy resulting from the neural injury. Histological examination of the deltoid muscle was performed according to defined criteria of degeneration, allowing an assessment of muscle fibre density, homogeneity of the muscle fibre size, formation of microfascicles, perimysial fibrosis, formation of adipose tissue, and inflammatory reactions in different degrees. Sections were stained with haematoxylin and eosin (H&E).

VEGF expression and degeneration of the brachial plexus For quantification of VEGF expression in the brachial plexus an ELISA was used to demonstrate a successful gene therapy and furthermore to allow a comparison of VEGF concentrations in the examined preparations. Each preparation was examined according to the concentration of VEGF pg/g tissue, VEGF pg/mg protein, and mg protein/g tissue. Analysis was performed in a total of six preparations from four different animals of the VEGF and the control group. Histological examination of the brachial plexus was also carried out according to defined criteria of degeneration, allowing an assessment of nerve fibre density, homogeneity of the nerve fibre size, vessel density, fibre degeneration, epineural fibrosis, and inflammatory reactions in different degrees. Sections were also stained with H&E.

Motoneuron counting In all animals, motoneuron counting was carried out in the corresponding pool of the C5 segment in 5 mm cross-sections. Motoneurons were incubated with anticalcitonin gene-related peptide (CGRP) polyclonal antibodies (100 mg in 1 mg/ml; Fa. Abcam, Cambridge, UK) and a secondary anti-rabbit antibody, which is conjugated with a peroxidase enzyme (ImmPRESSTM Peroxidase Polymer Detection Reagent Kit; Fa. Vector, Burlingame, CA, USA). Binding of diaminobenzidine (DAB) as a substrate produced an observable brown colour for motoneuron identification (Fig. 1). The absolute number of motoneurons of the left operated side was compared with that of the contralateral healthy side. The difference displays the extent of motoneuron survival after peripheral nerve injury in the VEGF and the control group.

Statistical analysis The non-parametric Mann–Whitney test was used to compare the results of all examinations between the VEGF and the control group. The level of significance was set to P , 0.05.

Results Behavioural analysis Grid walk Test Results of the grid walk test revealed obvious deficits of the movement pattern regarding accurate limb


2015

VOL .

37

NO .

3

199

Hillenbrand et al.


Figure 1 Staining of motoneurons in the corresponding pool of the C5 segment in 5 mm cross-sections. Motoneurons were incubated with anti-calcitonin gene-related peptide (CGRP) polyclonal antibodies and a secondary anti-rabbit antibody, which is conjugated with a peroxidase enzyme. Binding of diaminobenzidine (DAB) produced an observable brown colour. Scale bar: 50 mm.

placement. During a period of 11 weeks, the average of missteps or searching movements showed a continuous decline in both groups. Throughout the experiment it was seen a less number of missteps in the VEGF group. The grid walk test did not show a significant difference in the number of missteps between the VEGF and the control group in any week. Grasping test During a period of 11 weeks, a continuous increase of the maximum force of the grasping movement was observed in both groups. Results of the operated left side showed a stronger increase in force development in the VEGF group. Comparison between the operated left side of the VEGF and the control group revealed only in week 1, a significant difference in the maximum force (P , 0.05). Grooming Test The average range of movement of the operated left side during a period of 11 weeks showed an increase in abduction and elbow flexion in both groups. Throughout the experiment the range of movement on the operated left side was superior in the VEGF group, which returned back to normal from week 9 on. In week 11, no complete range of movement with the operated left side could be carried out in the control group. The grooming test did not show a significant difference in abduction and elbow flexion between the VEGF and the control group in any week.

Morphometric and biochemical analysis Weight and degeneration of the deltoid muscle There was a distinct reduction in muscle mass of the operated left side compared to the healthy right side

200


2015

VOL .

37

NO .

3

Figure 2 Difference of muscle weight (g) between the VEGF and the control group. *P 5 0.1.

in both groups. It was found a reduction of 47.2% in the control group and 34.2% in the VEGF group. Based on these results, it revealed a less reduction in muscle mass of 27.5% in the VEGF group (Fig. 2). There was no statistically significant difference in muscle weight between the two groups. Histological examination of the deltoid muscle according to defined criteria of degeneration did not show a significant difference of the operated left side between the two groups. VEGF expression and degeneration of the brachial plexus VEGF concentration in the VEGF-treated brachial plexus was almost 90% higher compared to the 0.9% NaCl-treated brachial plexus. Histological examination of the operated left brachial plexus according to defined criteria of degeneration revealed significantly less degeneration of neural tissue in the VEGF group (P , 0.05). It was shown that inflammatory reactions in the operated left plexus were marginally higher in the control group. Motoneuron counting It was found a difference in the number of motoneurons in the corresponding pool between the operated left and non-operated right side in both groups with a higher number of motoneurons on the right side in each animal (Fig. 3). In the VEGF group, there was a total difference of 32 motoneurons with a standard deviation of 12.4%. In the control group, there was a total difference of 108 motoneurons with a standard deviation of 31.3%. In conclusion, it revealed 60.4% more surviving motoneurons in the VEGF group. The motoneuron counting showed a significant difference in the number of surviving motoneurons between the two groups (P , 0.05) (Fig. 4).

Hillenbrand et al.


Figure 3 Motoneuron counting in the corresponding pool of the C5 segment. The difference between the right (a) and the left (b) side was compared. Scale bars: 100 mm.

Discussion The results of the behavioural analysis revealed a positive effect of VEGF gene therapy on nerve regeneration in each performed test. The grid walk paradigm allowed an evaluation of complex movement patterns such as balance and accurate placement of the upper limb.20 The number of missteps in the course showed a faster decline in the VEGF group. In addition to functional limitation by paresis, psychological factors might have also influenced the results of this test regarding fear of the animals when crossing a ladder 1 m above the ground. Functional recovery regarding force development of the upper limb was examined using the grasping test.21 The results of the operated left side showed a greater increase of force development in the VEGF group. The grasping test allowed an evaluation of the maximum force of the grasping movement that might not necessarily be limited by an injury of the spinal roots C5 and C6. It has been observed that during the measurement

Figure 4 Motoneuron counting showed a significant difference in the number of surviving motoneurons between the two groups. *P , 0.05.

animals used the entire musculoskeletal system of the upper limb in order not to lose the grip. The results of the grasping test can therefore be regarded as representative for the entire upper limb. Motorfunctional recovery of the upper limb was also examined using the grooming test.22 At week 9, each animal in the VEGF group could reach all anatomical landmarks with the operated left side during grooming movements, whereas in the control group, a motion deficit remained. Throughout all behavioural analysis, a distinct faster regeneration on the motor-functional level was seen in the VEGF group. Reduction in muscle mass of the operated left deltoid muscle indicates tissue atrophy due to a lack of innervation. In the VEGF group, it was observed 27.5% less reduction in muscle mass compared to the control group, which might be an indication of the indirect effect on muscle tissue as a result of enhanced reinnervation. Histological examination of the deltoid muscle confirmed less degeneration in the VEGF group, which was especially seen by higher density of muscle fibre and less perimysial fibrosis (Fig. 5). Comparison of the VEGF concentration in the operated left brachial plexus showed a distinct difference between the treatment with AdCMV.VEGF165 and 0.9% NaCl. The concentration in the VEGF-treated group was almost 90% higher compared to the 0.9% NaCl-treated control group. This result was indicative of an induced increase of VEGF expression by AdCMV.VEGF165 in the treated tissue and demonstrated a successful gene therapy. Histological examination of the brachial plexus allowed a direct assessment of the neural tissue based on defined criteria of degeneration. In addition to the direct effect of VEGF on nerve regeneration, its influence on vascularization in neural tissue has to be taken into account.23 A sufficient blood supply to the injured nerve is crucial for the survival and functional integrity of axons.24 It was only found a slight difference in vessel density between both groups with a higher number of vascular structures in the VEGF-treated


2015

VOL .

37

NO .

3

201

Hillenbrand et al.


Figure 5 Histological examination of the deltoid muscle revealed higher density of muscle fibre and less perimysial fibrosis in the VEGF (a) compared to the control group (b). H&E staining. Scale bars: 100 mm.

plexus. There was significantly less neural degeneration found in the VEGF group mainly represented by higher nerve fibre density and homogeneity of the nerve fibre size at little epineural fibrosis (P , 0.05) (Fig. 6). The higher number of vascular structures found in the VEGF treated plexus was not proportional to the overall extent of degeneration found in the 0.9% NaCltreated group. Hence, the effect of VEGF on neural regeneration only mediated by enhanced vascularization does not seem possible. The difference of motoneurons in the corresponding pool of the C5 segment between the left and right anterior horn represents the number of lost cells after peripheral nerve injury. The counting revealed a significant difference in the number of motoneurons with 60.4% more surviving motoneurons in the VEGF group (P , 0.05). Owing to the fact that motoneurons were not directly treated with AdCMV.VEGF165, maintaining the interaction with their target structures by enhanced peripheral nerve regeneration after injury might be the reason for the higher survival rate. Hence, results confirmed the importance of interaction during the early postpartum period.7 After peripheral nerve

injury, the speed of axonal growth, which is a crucial factor of peripheral nerve recovery, determines a possible survival of motoneurons in the newborn.27 These findings were also shown in other experiments with newborn rats that demonstrated a high plasticity of the premature central nervous system.25,26 In conclusion, we could demonstrate that peripheral nerve regeneration after crush injury was significantly improved by adenoviral gene therapy with VEGF165. The results gave no information in which proportion the angiogenic and neurotrophic effects of VEGF contributed to improved nerve regeneration. VEGF has a supportive effect on the growth of regenerating nerve fibres, which is most likely mediated by a combination of angiogenic, neurotrophic, and neuroprotective effects. The use of recombinant adenoviral vectors should be viewed critically since associated with increased inflammation.28 Thus new methods using non-viral gene therapy have been investigated.29,30 Owing to the high rate of spontaneous and complete regressions of symptoms in patients with obstetric brachial plexus palsy, a mandatory implementation of a high-risk

Figure 6 Histological examination of the brachial plexus revealed higher nerve fibre density and homogeneity of the nerve fibre size in the VEGF (a) compared to the control group (b). H&E staining. Scale bars: 100 mm.

202


2015

VOL .

37

NO .

3

Hillenbrand et al.

therapy with AdVEGF cannot be justified at the moment. The risk–benefit assessment represents an important prerequisite for such therapy. The known risks and disadvantages of the therapy must be in reasonable proportion against the anticipated benefit for the person concerned. To date, there are no results of an appropriate therapy in the newborn, in which the use of viral vectors with respect to the barely developed immune system appears questionable anyway.

Disclaimer Statements Contributors Matthias Hillenbrand is the corresponding author who performed all experiments and wrote the presented article. All other contributors gave instructions that were necessary to realise the experiments. Funding None. Conflicts of interest The authors have no conflict of interest with regard to this publication. Ethics approval All experiments were performed in accordance with institutional guidelines and were approved by the government of Oberbayern and the local animal care committee (AZ 55.2-1-54-2531-6606).

References 1 Rouse DJ, Owen J, Goldenberg RL, Cliver SP. The effectiveness and costs of elective cesarean delivery for fetal macrosomia diagnosed by ultrasound. JAMA. 1996;276:1480–6. 2 Shenaq SM, Berzin E, Lee R, Laurent SP, Nath R, Nelson MR. Brachial plexus birth injuries and current management. Clin Plast Surg. 1998;25:527–36. 3 Bahm J. [Obstetric brachial plexus palsy — clinics, pathophysiology and surgical treatment]. Handchir Mikrochir Plast Chir. 2003;35:83–97. 4 Bager B. Perinatally acquired brachial plexus palsy — a persisting challenge. Acta Paediatr. 1997;86:1214–9. 5 Gilbert A. Long-term evaluation of brachial plexus surgery in obstetrical palsy. Hand Clin. 1995;11:583–95. 6 Schmalbruch H. Motoneuron death after sciatic nerve section in newborn rats. J Comp Neurol. 1984;224:252–8. 7 Lowrie MB, Vrbova G. Dependence of postnatal motoneurones on their targets: review and hypothesis. Trends Neurosci. 1992;15:80–4. 8 Oppenheim RW. Cell death during development of the nervous system. Annu Rev Neurosci. 1991;14:453–501. 9 Zacchigna S, Giacca M. Chapter 20: Gene therapy perspectives for nerve repair. Int Rev Neurobiol. 2009;87:381–92. 10 Tannemaat MR, Boer GJ, Eggers R, Malessy MJ, Verhaagen J. From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve. Prog Brain Res. 2009;175:173–86.


11 Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9(6):669–76. 12 Yla-Kotola TM, Kauhanen SC, Leivo IV, Haglund C, Tukiainen E. Vascular endothelial growth factor and its receptors are expressed in human nerve grafts. J Reconstr Microsurg. 2011;27(3):173–8. 13 Pereira Lopes FR, Lisboa BC, Frattini F, Almeida FM, Tomaz MA, Matsumoto PK, et al. Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy. Neuropathol Appl Neurobiol. 2011;37(6):600–12. 14 Sondell M, Sundler F, Kanje M. Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur J Neurosci. 2000;12:4243–54. 15 Storkebaum E, Lambrechts D, Carmeliet P. VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays. 2004;26:943–54. 16 Lambrechts D, Storkebaum E, Morimoto M, Del-Favero J, Desmet F, Marklund SL, et al. VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet. 2003;34:383– 94. 17 Zachary I. Neuroprotective role of vascular endothelial growth factor: signalling mechanisms, biological function, and therapeutic potential. Neuro-Signals. 2005;14(5):207–21. 18 Storkebaum E, Carmeliet P. VEGF: a critical player in neurodegeneration. J Clin Invest. 2004;113(1):14–8. 19 Muhlhauser J, Merrill MJ, Pili R, Maeda H, Bacic M, Bewig B, et al. VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res. 1995;77:1077–86. 20 Metz GA, Merkler D, Dietz V, Schwab ME, Fouad K. Efficient testing of motor function in spinal cord injured rats. Brain Res. 2000;883:165–77. 21 Bertelli JA, Mira JC. The grasping test: a simple behavioral method for objective quantitative assessment of peripheral nerve regeneration in the rat. J Neurosci Methods. 1995;58:151– 5. 22 Bertelli JA, Mira JC. Behavioral evaluating methods in the objective clinical assessment of motor function after experimental brachial plexus reconstruction in the rat. J Neurosci Methods. 1993;46:203–8. 23 Wongtrakul S, Bishop AT, Friedrich PF. Vascular endothelial growth factor promotion of neoangiogenesis in conventional nerve grafts. J Hand Surg Am. 2002;27:277–85. 24 Sunderland S. The anatomy and physiology of nerve injury. Muscle Nerve. 1990;13:771–84. 25 Korak KJ, Tam SL, Gordon T, Frey M, Aszmann OC. Changes in spinal cord architecture after brachial plexus injury in the newborn. Brain. 2004;127(Pt 7):1488–95. 26 Aszmann OC, Winkler T, Korak K, Lassmann H, Frey M. The influence of GDNF on the timecourse and extent of motoneuron loss in the cervical spinal cord after brachial plexus injury in the neonate. Neurol Res. 2004;26(2):211–7. 27 Tannemaat MR, Boer GJ, Eggers R, Malessy MJ, Verhaagen J: From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve. Prog Brain Res. 2009;175:173–86. 28 Cichon G, Boeckh-Herwig S, Schmidt HH, Wehnes E, Mu¨ller T, Pring-Akerblom P, et al. Complement activation by recombinant adenoviruses. Gene Ther. 2001;8:1794–800. 29 Mykhaylyk O, Sanchez-Antequera Y, Vlaskou D, Hammerschmid E, Anton M, Zelphati O, et al. Liposomal magnetofection. Methods Mol Biol. 2010;605:487–525. 30 Vlaskou D, Plank C, Mykhaylyk O. Magnetic and acoustically active microbubbles loaded with nucleic acids for gene delivery. Methods Mol Biol. 2013;948:205–41.


2015

VOL .

37

NO .

3

203