Effects of immunosuppression on regrowth of adult rat retinal ganglion cell axons into peripheral nerve allografts

Analysis of the effectiveness of allografts and immunosuppression in the repair of nerve defects in the adult peripheral nervous system (PNS) has a long experimental and clinical history. There is little information, however, on the use of allografts in peripheral nerve (PN) transplantation into the injured central nervous system (CNS). We assessed the ability of PN allografts (from Dark-Agouti rats) to support regeneration of adult rat retinal ganglion cell (RGC) axons in immunosuppressed host Lewis rats. PN allografts were sutured onto intraorbitally transected optic nerves. Three weeks after grafting, regenerating RGC axon numbers were determined using retrograde fluorescent labelling, and total axons within PN grafts were assessed using pan-neurofilament immunohistochemistry. In the absence of immunosuppression, PN allografts contained few axons and there were very few labelled RGC. These degenerate grafts contained many T cells and macrophages. Systemic (intraperitoneal) application of the immunosuppressants cyclosporin-A or FK506 reduced cellular infiltration into allografts and resulted in extensive axonal regrowth from surviving RGCs. The average number of RGCs regenerating axons into immunosuppressed allografts was not significantly different from that seen in PN autografts in rats sham-injected with saline. Many pan-neurofilament-positive axons, a proportion of which were myelinated, were seen in immunosuppressed allografts, particularly in proximal regions of the grafts toward the optic nerve-PN interface. This study demonstrates that PN allografts can support axonal regrowth in immunosuppressed adult hosts, and points to possible clinical use in CNS repair.     

Matching of motor-sensory modality in the rodent femoral nerve model shows no enhanced effect on peripheral nerve regeneration

The treatment of peripheral nerve injuries with nerve gaps largely consists of autologous nerve grafting utilizing sensory nerve donors. Underlying this clinical practice is the assumption that sensory autografts provide a suitable substrate for motoneuron regeneration, thereby facilitating motor endplate reinnervation and functional recovery.This study examined the role of nerve graft modality on axonal regeneration, comparing motor nerve regeneration through motor, sensory, and mixed nerve isografts in the Lewis rat. A total of 100 rats underwent grafting of the motor or sensory branch of the femoral nerve with histomorphometric analysis performed after 5, 6, or 7 weeks. Analysis demonstrated similar nerve regeneration in motor, sensory, and mixed nerve grafts at all three time points. These data indicate that matching of motor-sensory modality in the rat femoral nerve does not confer improved axonal regeneration through nerve isografts.     

End-Organ reinnervation does not prevent axonal degeneration in nerve allografts following immunosuppression withdrawal

Previous work indicated that appropriate end-organ reinnervation fails to influence axonal degeneration in nerve allografts following immunosuppression withdrawal. In the present study, we examined if differences existed in axonal degeneration when axons regenerated across nerve allografts are allowed or completely denied end-organ reinnervation. Two ACI rat nerve allografts (3 cm long) were sutured into gaps created in both peroneal nerves in Lewis rats. In the right leg, the distal end of the graft was connected to the distal host nerve stump to allow end-organ reinnervation. In the left leg, the distal end was turned back and double ligated (unconnected) to prevent end-organ reinnervation. Rats received Cyclosporin A daily for 12 weeks to allow for regeneration and were sacrificed at 16 (n = 5) or 18 (n = 5) weeks following engraftment to assess axonal degeneration following immunosuppression withdrawal. Five Lewis rats receiving autografts served as control and were sacrificed at 12 weeks. Morphometric analysis was performed. In the control group (autografts) the cross-sectional area of and the number of myelinated fibres in the unconnected grafts was double that of the connected grafts, suggesting a sprouting effect. There was a tenfold reduction in the mean number of fibres at weeks 16 and 18 in the allografts compared to controls, without any significant differences in the connected versus unconnected sides. End-organ reinnervation decreases sprouting of axons within the graft but does not protect axons from degeneration following immunosuppression withdrawal.     

Indefinite survival of peripheral nerve allografts after temporary Cyclosporine A immunosuppression

It is hypothesized that unlike solid organ transplants immunosuppression of peripheral nerve allografts is needed only for the finite time period required for regeneration of proximal host nerve axons through the allograft and subsequent re-establishment of host end-organ connections. The aim of this study was to explore the consequences of temporary and continuous systemic Cyclosporine A (CsA) immunosuppression upon peripheral nerve allograft survival. Buffalo rats received Lewis nerve allografts under CsA immunosuppression (5 mg/kg/day) either continuously for 20 weeks, or for only 10 weeks followed by abrupt withdrawal. At 20 weeks, the nerve segments from both groups were regrafted into na?ve Buffalo or Lewis recipients without further immunosuppression. These grafts were compared with isografts, unimmunosuppressed allografts and allografts immunosuppressed for 10 weeks in situ. By eight weeks following regrafting, the secondary Lewis recipients had rejected the temporarily immunosuppressed allografts and accepted the continuously immunosuppressed allograft, while the secondary Buffalo recipients accepted both the temporarily and continuously immunosuppressed allografts as assessed by histology and morphometry. Functional recovery was earlier in secondary recipient strain animals that received temporarily immunosuppressed allografts in comparison to those that received continuously immunosuppressed allografts. Analysis of secondary recipients of temporarily immunosuppressed allografts demonstrated greater in vitro MLR and LDA reactivity than did those receiving continuously immunosuppressed allografts. These findings support the hypothesis that donor alloantigens are lost or replaced by the recipient after immunosuppression withdrawal. Moreover, the change to recipient antigenicity in the nerve allograft is retarded and incomplete under continuous CsA immunosuppression, resulting in acceptance by both secondary donor and recipient strains upon regraftment.     

Schwann cell involvement in the neurological lesion of the dystonic mutant mouse: A nerve grafting study

Schwann cell function in the dystonic mutant mouse was studied by grafting peripheral nerve from normal into affected littermates of a C57/BL (Fa.) dt dystonic mouse colony and vice versa. In a control experiment, only unaffected animals of the colony were used, and nerve isografts were found to be ultrastructurally indistinguishable from normal nerve autografts. In addition, the isografts showed no features of the lymphocytic inflammatory rejection reaction observed in normal nerve allografts, and there was evidence that donor Schwann cells remained viable and were active in all the isografts examined. When nerve isografts from affected dystonic mutants were implanted into normal littermate nerves, the normal host axons regenerating through the grafted region acquired degenerative changes characteristic of naturally occurring dystonic peripheral nerve. These changes were not seen in the host axons regenerating either outside the dystonic graft regions, or more distally in the host nerve stumps. When normal nerve isografts were implanted into affected dystonic mutant nerves, the dystonic axons regenerated through the normal graft region and became normally myelinated. It is concluded that an underlying Schwann cell defect may be responsible for the abnormalities of the dystonic mouse peripheral neuropathy.     

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Abstract

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently,the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Basement membrane component changes in nerve allografts and isografts

This study describes immunocytochemical changes in laminin, which is an integral basement membrane (BM) component, during axonal regeneration through antigenic nerve allografts and nonantigenic nerve isografts. In normal rat nerve, laminin was localized in the BM of Schwann cells and the perineurium. During nerve allograft rejection, the perineurium and Schwann cells disappeared. However, the Schwann cell BMs persisted and became distorted and collapsed. In isografted nerves, the perineurium and Schwann cells were present, and only a few Schwann cell BMs appeared to be distorted; however, the staining for laminin was faint, indicating a possible BM breakdown. A new BM appeared as small rings around the Schwann cells after they had become associated with regenerated axons. Because only a limited axonal regeneration occurred in allografts as compared to isografts, it is concluded that the viable Schwann cells, and their BM architecture, are essential for regeneration through long nerve grafts.     

Immunomodulation by costimulation blockade inhibits rejection of nerve allografts

The aim of this study was to investigate if costimulation blockade could be used to modulate the immune response, to prevent rejection, and to stimulate regeneration into nerve allografts. Nerve allografts from Balb/C mice, and isogenic nerve grafts (isografts) from C57/BL6 mice, were used to bridge a 7-mm gap of the sciatic nerve in C57/BL6 mice. Allograft recipients were treated with either a triple treatment with anti-lymphocyte function antigen-1 (anti-LFA), anti-CD40 ligand (anti-CD40L), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (anti-CTLA4Ig) or isotype antibodies (placebo) at postoperative days 0, 2, 4, and 6 (intraperitoneal). After 5 or 9 days, the nerve grafts, together with the proximal and the distal nerve segments, were evaluated by histology and immunocytochemistry for inflammatory cells [CD4-positive (CD4+) and CD8-positive (CD8+) staining cells] and axonal outgrowth (neurofilaments). The immune response was inhibited by costimulation blockade with less extensive inflammation and a lower number of CD4+ staining cells in triple-treated allografts at 9 days. The regeneration rate was significantly faster in isografts (0.75 mm/day) compared with allografts with placebo treatment (0.39 mm/day), but not when compared with triple-treated allografts (0.49 mm/day). At 9 days, the axons were significantly longer in nerve isografts than in nerve allografts, irrespective of treatment. Hence, costimulation blockade neither increased the regeneration rate nor the outgrowth length in triple-treated allografts. We conclude that costimulation blockade inhibits the immune response in nerve allografts without deterring early axonal outgrowth.     

Treatment modality affects allograft-derived Schwann cell phenotype and myelinating capacity

We used peripheral nerve allografts, already employed clinically to reconstruct devastating peripheral nerve injuries, to study Schwann cell (SC) plasticity in adult mice. By modulating the allograft treatment modality we were able to study migratory, denervated, rejecting, and reinnervated phenotypes in transgenic mice whose SCs expressed GFP under regulatory elements of either the S100b (S100-GFP) or nestin (Nestin-GFP) promoters. Well-differentiated SCs strongly expressed S100-GFP, while Nestin-GFP expression was stimulated by denervation, and in some cases, axons were constitutively labeled with CFP to enable in vivo imaging. Serial imaging of these mice demonstrated that untreated allografts were rejected within 20 days. Cold preserved (CP) allografts required an initial phase of SC migration that preceded axonal regeneration thus delaying myelination and maturation of the SC phenotype. Mice immunosuppressed with FK506 demonstrated mild subacute rejection, but the most robust regeneration of myelinated and unmyelinated axons and motor endplate reinnervation. While characterized by fewer regenerating axons, mice treated with the co-stimulatory blockade (CSB) agents anti-CD40L mAb and CTLAIg-4 demonstrated virtually no graft rejection during the 28 day experiment, and had significant increases in myelination, connexin-32 expression, and Akt phosphorylation compared with any other group. These results indicate that even with SC rejection, nerve regeneration can occur to some degree, particularly with FK506 treatment. However, we found that co-stimulatory blockade facilitate optimal myelin formation and maturation of SCs as indicated by protein expression of myelin basic protein (MBP), connexin-32 and phospho-Akt.     

Acellular nerve allografts in peripheral nerve regeneration: a comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011     

Chondroitinase treatment increases the effective length of acellular nerve grafts

Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.     

Vascularization is delayed in long nerve constructs compared with nerve grafts

Introduction: Nerve regeneration across nerve constructs, such as acellular nerve allografts (ANAs), is inferior to nerve auto/isografts especially in the case of long defect lengths. Vascularization may contribute to poor regeneration. The time course of vascular perfusion within long grafts and constructs was tracked to determine vascularization. Methods: Male Lewis rat sciatic nerves were transected and repaired with 6 cm isografts or ANAs. At variable days following grafting, animals were perfused with Evans Blue albumin, and grafts were evaluated for vascular perfusion by a blinded observer. Results: Vascularization at mid‐graft was re‐established within 3–4 days in 6 cm isografts, while it was established after 10 days in 6 cm ANAs. Conclusions: Vascular perfusion is reestablished over a shorter time course in long isografts when compared with long ANAs. The differences in vascularization of long ANAs compared with auto/isografts suggest regenerative outcomes across ANAs could be affected by vascularization rates. Muscle Nerve 54 : 319–321, 2016     

In vitro pre-degenerated nerve autografts support CNS axonal regeneration

In the present study we compared, in adult rats, the axonal regeneration of central respiratory neurons within autologous fresh (f-; grafted immediately after removal) and pre-degenerated (pd-; grafted after being stored during 3 days in saline at + 8°C) peripheral nerve grafts (PNGs) implanted within the C2 cervical spinal cord. The proximal end of the left peroneal nerve was implanted in the site of projection of medullary respiratory neurons (ventro-lateral quadrant) and the distal part of each nerve graft was left unconnected (blind-ended graft). PNGs were examined 2 to 4 months after grafting. Central neurons regenerating axons within the PNGs were studied by recording spontaneous unit activity from small strands teased from the grafts. In control f-PNGs (n = 9), 248 filaments had spontaneous activities, 58 of these were respiratory-related, i.e. had discharge patterns identical to those of normal respiratory (inspiratory and expiratory) neurons. The presence of regenerated nerve fibers with spontaneous unitary impulse traffic (n = 216) was found in all pd-PNGs (n = 5). Thirty-four had respiratory patterns identical to those found within f-PNGs and corresponded to efferent activity. No statistically significant differences in axonal regrowth were found between f- and pd-PNGs. In conclusion, f- and pd-PNGs were equally capable of promoting axonal regeneration of central neurons. The neural components (Schwann cells and others) required for axonal regeneration of adult central neurons are still effective following 3 days of in vitro peripheral nerve degeneration without special storage conditions (oxygenation, medium inducing ATP synthesis). These results have clinical implications for nerve graft surgery when time is required for typing the tissues of both donor and recipient (post-mortem allografts) or transportation of graft material.     

Nerve allograft regeneration and immunological tolerance of rats with sciatic nerve transplantation after intragastric pretreatment with ultraviolet B-irradiated donor splenocytes★

BACKGROUND: Nerve allograft rejection is an unavoidable problem for nerve allografts. Traumatic peripheral nerve injuries are commonly reconstructed using autogenous nerve grafts. However, this form of reconstruction is limited by insufficient autologous nerves for large gap repairs and by morbidity at the nerve donor site. OBJECTIVE: To examine sciatic nerve regeneration and immune tolerance reaction after intragastric administration of ultraviolet B-irradiated (UVB) donor splenocytes. DESIGN, TIME AND SETTING: A complete randomized grouping design and controlled experiment. The experiments were conducted in the Department of Orthopedics, the First Affiliated Hospital to Shanxi Medical University, China, between March and October 2007. MATERIALS: Fourteen adult male SD rats and fourteen male Wistar rats, weighing 250–300 g, were randomly matched as donors and acceptors. A further seven male SD rats (weight 250–300 g, age 12–16 weeks) were used for nerve isografts. Immune preparations and the Epics XL flow cytometer were purchased from B-D Company, USA. A computer-assisted electromyograph machine was provided by Keypoint P, Dantel Company, Denmark. METHODS: Splenocytes from Wistar rats were isolated, purified, and cultured, and then irradiated with ultraviolet B. In the first control group (Group 1), the SD rats received a syngeneic SD nerve isograft. In the second control group (Group 2), the SD rats received a nerve allograft from Wistar rats without pretreatment. In the oral-tolerance treated group (Group 3), the SD recipient rats were inoculated with 2.5×107 Lewis UVB-irradiated donor splenocyte cells by intestinal tract administration at seven days before transplantation. MAIN OUTCOME MEASURES: (1) The recent end and the middle and distal end of the transplanted nerve were cut at 8 and 12 weeks after operation. Recovery of nerve regeneration was measured with HE staining using the total number, density, and diameter of the nerve fibers. (2) The level of CD25+T lymphocytes in peripheral blood was detected with the Epics XL flow cytometer at one week after operation. (3) The bilateral sciatic nerves were exposed from the sciatic notch up to 0.5 mm beyond the distal graft site at eight weeks post-engraftment. Bipolar platinum stimulating electrodes were placed under the sciatic nerve proximally and the mean conduction velocity was recorded with recording electrodes on the posterior tibial nerve at 0.3 cm distal to the nerve graft. RESULTS: Eight weeks after operation, total axon number and fiber density in Group 3 were higher than that in Group 1 (P < 0.05), neural regeneration in Group 3 was lower than that in Group 1 (P < 0.05) , The level of CD25+T lymphocytes in peripheral blood of Group 3 was significantly lower than that of Group 2 (P < 0.05). There was no significant difference between Group 3 and Group 1 (P > 0.05). At eight weeks post-engraftment the mean conduction velocity of Group 3 approximated that of Group 1. The untreated allografts in Group 2 demonstrated no measurable recovery of conduction velocity. CONCLUSION: Pretreatment with a single intragastric dose of UVB-modified donor antigen specifically induced tolerance to peripheral nerve allografts in rats. Key Words: sciatic nerve; transplantation, homologous; immune tolerance     

Processed allografts and type I collagen conduits for repair of peripheral nerve gaps

Autografting is the gold standard in the repair of peripheral nerve injuries that are not amenable to end‐to‐end coaptation. However, because autografts result in donor‐site defects and are a limited resource, an effective substitute would be valuable. In a rat model, we compared isografts with Integra NeuraGen® (NG) nerve guides, which are a commercially available type I collagen conduit, with processed rat allografts comparable to AxoGen's Avance® human decellularized allograft product. In a 14‐mm sciatic nerve gap model, isograft was superior to processed allograft, which was in turn superior to NG conduit at 6 weeks postoperatively (P < 0.05 for number of myelinated fibers both at midgraft and distal to the graft). At 12 weeks, these differences were no longer apparent. In a 28‐mm graft model, isografts again performed better than processed allografts at both 6 and 22 weeks; regeneration through the NG conduit was often insufficient for analysis in this long graft model. Functional tests confirmed the superiority of isografts, although processed allografts permitted successful reinnervation of distal targets not seen in the NG conduit groups. Processed allografts were inherently non‐immunogenic and maintained some internal laminin structure. We conclude that, particularly in a long gap model, nerve graft alternatives fail to confer the regenerative advantages of an isograft. However, AxoGen processed allografts are superior to a currently available conduit‐style nerve guide, the Integra NeuraGen®. They provide an alternative for reconstruction of short nerve gaps where a conduit might otherwise be used. Muscle Nerve, 2008     

Long term evaluation of experimental median nerve repair by frozen and fresh nerve autografts,allografts and allografts repopulated by autologous Schwann cells

The authors used different kinds of peripheral nerve grafts to reconstruct a terminal branch of the brachial plexus (the median nerve) gap of adult Sprague-Dawley rats, including fresh or frozen autografts and allografts from Norway rats. They also performed acellular allograft repopulation by autogenous Schwann cells, to improve the environment for nerve regeneration. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. Initially, the functional recovery of frozen grafts was lower than fresh graft recovery, but twelve months after surgery it was similar for both types of graft.     

An ultrastructural comparison of peripheral nerve allografts and autografts

Summary There is a marked difference in the cellular response of the host to peripheral nerve allografts and autografts. The response elicited by allografts is characterised by invasion of tissue with lymphocytes, plasma cells and activated macrophages. These cells disrupt the nerve architecture, and cause rupture and consequent compression of the neurolemmal tubes which are the essential conduit element of a nerve graft.By contrast, in the autograft, regeneration follows the initial process of Wallerian degeneration without the complication of immune reaction. The Schwann cells and macrophages rapidly remove the myelin and axons from the neurolemmal tubes of the donor nerve; new axonal sprouts are then able to traverse these channels and reach the periphery in large numbers. If this process is delayed, the neurolemmal tubes in the distal nerve segment are compressed by surrounding collagen which limits the size of regenerating axons.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Costimulation blockade in transplantation of nerve allografts: long-term effects

Costimulation blockade can prevent rejection of nerve allografts in short-term studies. We tested if costimulation blockade also prevented rejection of nerve allografts in long-term experiments, thereby improving functional recovery. A 7-mm sciatic nerve defect in C57/BL6 mice was bridged either by nerve allografts from Balb/C mice or by isogenic nerve grafts (isografts) from C57/BL6 mice. Costimulation blockade in the form of a triple treatment with anti-LFA-1, anti-CD40L, and CTLA4Ig was given at post-operative days 0, 2, 4, and 6 (intraperitoneal). Control mice (placebo; allografts) with nerve grafts were treated with isotype antibodies during the same time period. After 49 days, tetanic muscle force, wet weight of gastrocnemius muscle, histology, and morphometry in the tibial nerve were evaluated. Costimulation blockade diminished rejection of the nerve allografts. Axons bridged the graft. Treatment increased wet weight of the gastrocnemius muscle and resulted in a higher mean myelin area/nerve fiber in the tibial nerve distal to the nerve grafts. Tetanic muscle force and number of axons in tibial nerve showed no differences between groups. We conclude that rejection is suppressed by costimulation blockade. Treatment improves recovery of target muscle and myelination after nerve allografting.