GROWTH HORMONE TREATMENT ENHANCES THE FUNCTIONAL RECOVERY OF SCIATIC NERVES AFTER TRANSECTION AND REPAIR PABLO DEVESA, PhD,1 MIGUEL GELABERT, MD, PhD,2 TAMARA GONZ´LEZ-MOSQUERA, BSc,1 ´ LUIS RELOVA, MD, PhD,1 JESU ´ S DEVESA, MD, PhD,1 and VI´CTOR M. ARCE, MD, PhD1 ROSALI´A GALLEGO, MD, PhD,3 JOSE 1
Department of Physiology, School of Medicine, University of Santiago de Compostela, San Francisco 1, 15782 Santiago de Compostela, Spain 2 Department of Surgery, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Accepted 7 September 2011 ABSTRACT: Introduction: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration. Methods: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance. Results: GH-treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH. Conclusion: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair. Muscle Nerve 45: 385–392, 2012
Despite progress in understanding the physiopathology of peripheral nervous system injury and regeneration, repair of peripheral nerve injuries continues to be a major challenge. Injuries to peripheral nerves cause partial or total loss of motor, sensory, and autonomic function due to axon discontinuity and degeneration, which finally result in significant functional loss and decreased quality of life.1,2 However, although unassisted axonal regeneration is, at best, rare in the central nervous system, in the peripheral nervous system the nerves can spontaneously regenerate without any treatment if nerve continuity is maintained.3,4 This is the case in crush injuries in which there is a complete axotomy with intact endoneurium or in transected nerves repaired by epineurial suture.5–8 In both cases, the endoneurial sheath provides the proximal axonal ends with neurotrophic support together with a physical guide to their original targets.6 Although regeneration of peripheral nerves may compensate for functional deficits caused by Abbreviations: BDNF, brain-derived neurotrophic factor; CMAP, compound muscle action potential; GDNF, glial-cell-line–derived neurotrophic factor; GH, growth hormone; IGF-1, insulin-like growth factor-1; NF-L, neurofilament-L; NGF, nerve growth factor; NT, neurotrophin-3 Key words: growth hormone, nerve injury, nerve regeneration, peripheral nervous system, sciatic nerve Correspondence to: V. M. Arce; e-mail:
[email protected] C 2011 Wiley Periodicals, Inc. V
Published online in Wiley DOI 10.1002/mus.22303
Online
GH and Sciatic Nerve Recovery
Library
(wileyonlinelibrary.com).
nerve injuries, clinical and experimental evidence shows that the results are often disappointing, especially after severe injuries. There is thus a need for more effective strategies to improve nerve regeneration.2,4 Until now, several approaches have been proposed to have beneficial effects on peripheral nerve regeneration. These include administration of neurotrophic factors, blockade of axonal regeneration inhibitory molecules, transplantation of stem cells, or the use of biomaterials.4 Other therapeutic approaches, such as brief electrical stimulation or treadmill exercise in the immediate postoperative period after transection and surgical repair, have also been proven to exert positive effects on axonal repair.9–12 Despite these developments, none have been able to achieve completely satisfactory results, and the development of techniques and approaches to accelerate axon regeneration after peripheral nerve injury continues to be an area of intense research. In this study we investigated the ability of growth hormone (GH) treatment to promote axonal regeneration after surgical section and repair of the sciatic nerve in rats. Although not classically considered to be a neurotrophic factor, accumulating evidence indicates that GH plays an important role in the regeneration of neural cells.13–15 Our results indicate that GH treatment is also useful in promoting sciatic nerve regeneration, thus supporting the possibility of using the hormone, alone or in combination with other therapeutic approaches, for treatment of peripheral nerve injuries. METHODS Experimental Design. All studies used adult male Sprague-Dawley rats (9–10 weeks old). Animals were housed at a constant temperature (22 –24 C) and exposed to a 14:10-h light–dark cycle. Standard laboratory rodent chow (Panlab, Barcelona, Spain) and tap water were available ad libitum. All animal procedures were conducted according to the principles approved by the institutional animal care committee of the Universidad de Santiago de Compostela. All rats were subjected to complete transection of the left sciatic nerve and immediate repair by epineurial suture. The animals were then MUSCLE & NERVE
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FIGURE 1. GH treatment promoting axonal repair. Sciatic nerve samples were obtained from both saline- and GH-treated rats 8 weeks after nerve transection and epineural repair, then fixed and stained with hematoxylin and eosin. Traces show nerve organization at the site of the lesion in saline-treated (a, c) and GH-treated (b, d) rats. The boxed areas in (a) and (b) correspond to magnified images (c) and (d), respectively. Note the lack of nerve organization and the presence of abundant eosinophilic material that can be seen in the saline-treated rats but not in the GH-treated rats. The GH-treated animals also show increased cellularity compared with saline-treated rats. Results are representative of 5 animals. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
randomly assigned to one of two treatment groups (10 animals per group): GH-treated (0.15 mg/kg/ day subcutaneously); or vehicle-treated controls given saline solution (once per day subcutaneously). Treatments started the day after surgery and lasted for 8 weeks. At the end of the treatment period, 5 animals from each experimental group were used for the electromyographic studies, and 5 animals underwent rotarod and treadmill performance tests. Sciatic nerve samples were collected from the animals used for the performance tests. Nerve Lesion and Repair. Surgical procedures were performed using a surgical microscope (OPMI; Carl Zeiss MicroImaging GmbH, Go¨ttingen Germany). Rats were anesthetized (ketamine 80 mg/kg and xylazine 10 mg/kg intraperitoneally), and a small incision was made in the upper left thigh. Muscles were then separated to expose the sciatic nerves, which were sharply transected at a constant point and immediately repaired. Nerve transection was performed with straight microsurgical scissors, and nerve repair was done using epineurial sutures (8-0 Vicryl; Ethicon, Inc., Cornelia, Georgia). To prevent misalignment of regenerating axons, care was taken to preserve the original alignment of tibial, sural, and fibular fascicles. Muscle and skin were then sutured separately, the skin was scrubbed with antiseptic solution, and the animals were left to recuperate in individual cages. This surgical procedure caused complete paralysis of the left hindlimb below the knee. 386
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Histological Evaluation of Nerve Regeneration. Sciatic nerve samples were fixed by immersion in 10% buffered formalin for 24 h, then dehydrated and embedded in paraffin using a standard procedure. Serial 4.5-lm sections were consecutively cut with a microtome (Leica Microsystems GmbH, Wetzlar, Germany) and transferred to adhesive-coated slides. Immunohistochemical detection of single antigen labeling was performed on these sections using an autostainer (TechMate TM50; Dako, Glostrup, Denmark). To enhance antigen retrieval, slides were submerged in TE buffer (pH 9.2) and preheated in a microwave oven (H2800 Microwave Processor; EBSciences, East Granby, Connecticut). Endogenous peroxidase activity was blocked by incubating the slides in peroxidase-blocking solution (Dako Diagno´sticos, S.A., Barcelona, Spain) for 5 min. The EnVision Detection Kit (Dako) was used as the staining detection system. Antibodies against the S100 protein or the 70-kDa neurofilament (NF)-L protein were purchased from Millipore (Billerica, Massachusetts) and used at dilutions of 1:100 and 1:200, respectively. For negative controls, nerve sections were incubated with normal rabbit serum instead of the primary antibody (data not shown). Semithin sections (1 lm thick) were stained with toluidine blue. The number of myelinated axons was counted from at least six randomly selected fields under 600 magnification. Image processing and analysis software (ImageJ; National Institutes of Health, Bethesda, Maryland) was used MUSCLE & NERVE
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FIGURE 2. GH treatment increases the presence of NF-L immunoreactivity. Sciatic nerve samples were obtained from both saline- and GH-treated rats 8 weeks after nerve transection and epineural repair and immunostained with antibodies against NF-L. The longitudinal sections show nerve organization at the site of the lesion in saline-treated (a, c) or GH-treated (b, d) rats. The boxed areas correspond to the magnified images. Transverse sections were obtained 4 mm distally to the suture in saline-treated (e) or GH-treated (f) animals. Sciatic nerves from intact rats were used as controls (g). Note the increased NF-L immunoreactivity in GH-treated compared with saline-treated rats. Results are representative of 5 animals. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to evaluate the myelinated axon diameter and the thickness of the myelin sheath. Electromyography. Muscle reinnervation was assessed by nerve conduction tests. Eight weeks after surgery, rats were anesthetized with ketamine/ xylazine as indicated earlier, and placed on a smooth table. The sciatic nerve and tibialis anterior muscle were exposed, and a pair of stimulating electrodes was gently placed on the proximal side of the nerve, 2 mm from the sutured site. Compound muscle action potentials (CMAPs) were obtained by stimulating the sciatic nerve. Stimulation was performed with a dual-output square-pulse stimulator (Grass S-88; Grass Technologies, West Warwick, Rhode Island) coupled to a photoelectric isolation unit (PSIU-6; Grass Technologies), with a current intensity ranging from 0.01 to 1.5 mA. For CMAP recording, a pair of needle electrodes with an impedance of
