Chondroitinase ABC Enhances Axonal Regeneration After Peripheral Nerve Autograft Repair

The proliferating Schwann cells following peripheral nerveinjury provide a favorable environment for axonal regeneration.They can act as the substratum for axonal growth, secrete a varietyof neurotrophic factors, express cell adhesion molecule andintegrin on their surface, and produce extracellular matrixmolecules including laminin, fibronectin, collagen, and variousproteoglycans. They can also secret a variety of monocytechemotactic factors to stimulate the recruitment of macrophagesinto the degenerating nerves. The recruiting macrophages can removethe debris of the degraded axons and myelin sheaths, and removemyelin-associated glycoprotein (MAG), which would otherwiseinhibit axonal growth. The endoneurial basal laminae that arecomposed of various extracellular matrix molecules areindispensable substratum for peripheral nerve regeneration. Afterperipheral nerve injury, the basal laminae distal to the injury canremain intact, which is prerequisite for nerve regeneration. Theextracellular matrix components are composed of axongrowth-promoting molecules, such as laminin, fibronectin andⅣtype collagen, as well as growth-inhibiting molecules, such aschondroitin sulfate proteoglycans (CSPGs). The latter also ispresent in central nervous system, and is one of the major inhibitingfactors for axonal regeneration after central nervous injury. Theremodeling of the extracellular matrix, e. g. enhancing the axongrowth-promoting molecules and removing the inhibiting molecules,is very important for nerve regeneration. It has been known thattreatment with chondroitinase ABC which can degrade the GAG sidechains of CSPGs specifically can enhance nerve regeneration and functional recovery after central nervous system injury. Thestudies about the role of chondroitinase ABC on peripheral nerveregeneration are quite rare. Whether the treatment withchondroitinase ABC has effects on Schwann cells, other componentsof basal laminae, and the structural integrity of the basal laminaeremain unresolved.In this study, we established the autograft model of rat sciaticnerve, and treated the nerve grafts with chondroitinase ABC beforethey were interposed into the nerve gaps to increase the degradationof CSPGs. Axonal regeneration into the grafts was assessed byelectrophysiological test and morphologic study. The effects oftreatment with chondroitinase ABC on the Schwann cells and basallamina laminin of the nerve grafts, which are the major axongrowth-promoting factors, were assessed by immuno-histochemicaltechnique.Methods:1. Effect of treatment with chondroitinase ABC on axonalregeneration in nerve graftsThirty rats were randomised into three groups containing 10 ratseach. Group A, controls group (no treat); Group B, vehicle treatgroup; Group C, chondroitinase ABC treat group.Under anesthesia with intraperitoneal ketamine hydrochloride,the sciatic nerves on both sides were approached through a glutealmuscle-splitting incision and isolated free of surrounding fascia.A 5mm segment of the right sciatic nerve was excised distally fromthe point that was 8 mm distal to the inferior border of thepiriformis. This made a 10 mm gap defect after retraction of thenerve ends. A 10 mm segment of the left sciatic nerve was excisedto act as the graft. In group A, the 10 mm segment of the left sciaticnerve was interposed into the gap defect of the right sciatic nerveimmediately. In group B, the 10 mm segment of the left sciatic nervewas incubated in 100μ1 of PBS (vehicle) for 2 h before grafting into the defect of the right sciatic nerve. In group C, the 10 mmsegment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml)for 2h before grafting.At postoperative 8 weeks, the MCV was measured for each nervegraft. After electrophysiological test,, a 5 mm nerve segment wasexcised from the midway trunk of the graft. One nerve segment wasselected randomly from each group and was performed transmissionelectron microscopic investigation. The other segments of eachgroup were made into transverse sections and stained with Palmgrenmethod for light microscopic investigations. The numbers of theregenerating axons in the grafts were counted and compared betweenthree groups.2. Effect of treatment with chondroitinase ABC on laminin/basallamina of nerve graftsSixteen rats were randomized into two groups containing 8 ratseach. Group A, controls group (no treat); Group B, chondroitinasetreat group.Surgical technique was the same as above-mentioned. In group A,the 10 mm segment of the left sciatic nerve was interposed into thegap defect of the right sciatic nerve immediately. In group B, the10 mm segment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml)for 2h before it was grafted.At postoperative 5 days, a5 mm nerve segment was excised fromthe proximal trunk of the graft and made into transverse sections.Polyclonal anti-laminin antibody was used to label basal laminae.3. Effect of treatment with chondroitinase ABC on Schwann cellsin nerve graftsSixteen rats were randomized into two groups containing 8 ratseach. Group A, controls group (no treat); Group B, chondroitinasetreat group.Surgical technique was the same as above-mentioned. In group A,the 10mm segment of the left sciatic nerve was interposed into the gap defect of the right sciatic nerve immediately. In group B, the10 mm segment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml) for 2h before it was grafted.At postoperative 14 days, a5mm nerve segment was excised fromthe proximal trunk of the graft and made into transverse sections.Polyclonal anti-S-100 antibody was used to label Schwann cells. Thenumbers of Schwann cells were counted and compared between twogroups.Results:1. Effect of treatment with chondroitinase ABC on axonalregeneration in nerve graftsThere was no significant difference in MCV between the threegroups at postoperative 8 weeks (P=0.381). The difference in numberof regenerative axons between group A and B was insignificant (P=0.607). Group C had more regenerating axons than group A and B,and the difference was significant (P＜0.05).2. Effect of treatment with chondroitinase ABC on laminin/basallamina of nerve graftsAt 5 days, both group A and B revealed intense laminin labelingin grafts, and the two groups were similar in structural integrityof basal laminae of grafts.3. Effect of treatment with chondroitinase ABC on Schwann cellsin nerve graftsAt 14 days, both group A and B revealed positive S-100 labelingin grafts. No significant difference was found between group A andB in number of Schwann cells in grafts (P=0.945).The above-mentioned results suggest:1. Treating nerve autografts before they are grafted withchondroitinase ABC can enhance axonal regeneration into the grafts.2. Treating nerve autografts before they are grafted withchondroitinase ABC does not influence the number of Schwann cellsin the grafts, and does not impair the laminin and the structural integrity of basal laminae of the grafts, i.e. does not impair themajor axon growth-promoting factors.In conclusion, treatment with chondroitinase ABC is a logicalstrategy to enhance peripheral nerve regeneration. The furtherresearch in this field may improve functional recoveryI afterperipheral nerve injury.