Use of cold-preserved allografts seeded with autologous Schwann cells in the treatment of a long-gap peripheral nerve injury.

BACKGROUND: Limitations in autogenous tissue have inspired the study of alternative materials for repair of complex peripheral nerve injuries. Cadaveric allografts are one potential reconstructive material, but their use requires systemic immunosuppression. Cold preservation (> or =7 weeks) renders allografts devoid of antigens, but these acellular substrates generally fail in supporting regeneration beyond 3 cm. In this study, the authors evaluated the reconstruction of extensive nonhuman primate peripheral nerve defects using 7-week cold-preserved allografts repopulated with cultured autologous Schwann cells. METHODS: Ten outbred Macaca fascicularis primates were paired based on maximal genetic disparity as measured by similarity index assay. A total of 14 ulnar nerve defects measuring 6 cm were successfully reconstructed using autografts (n = 5), fresh allografts (n = 2), cold-preserved allografts (n = 3), or cold-preserved allografts seeded with autogenous Schwann cells (n = 4). Recipient immunoreactivity was evaluated by means of enzyme-linked immunosorbent spot assay, and nerves were harvested at 6 months for histologic and histomorphometric analysis. RESULTS: Cytokine production in response to cold-preserved allografts and cold-preserved allografts seeded with autologous Schwann cells was similar to that observed for autografts. Schwann cell-repopulated cold-preserved grafts demonstrated significantly enhanced fiber counts, nerve density, and percentage nerve (p < 0.05) compared with unseeded cold-preserved grafts at 6 months after reconstruction. CONCLUSIONS: Cold-preserved allografts seeded with autologous Schwann cells were well-tolerated in unrelated recipients and supported significant regeneration across 6-cm peripheral nerve defects. Use of cold-preserved allogeneic nerve tissue supplemented with autogenous Schwann cells poses a potentially safe and effective alternative to the use of autologous tissue in the reconstruction of extensive nerve injuries.

Neuroregenerative effects of preinjury FK-506 administration.

BACKGROUND: FK-506 is used in organ transplantation because it promotes neurite outgrowth in vitro and enhances neuroregeneration in peripheral nerve injury transection models. Immunosuppressive mechanisms of FK-506 are well defined, with demonstration of decreased neuroregenerative effects with delayed administration. The purpose of this study was to describe the effects of preinjury administration of FK-506 in rats with tibial nerve transection injury. METHODS: Eight inbred male Lewis rats per group in three separate groups underwent tibial nerve transection with primary repair. Group I received placebo, group II received FK-506 treatment at 1 day before surgery, and group III received FK-506 preloading 3 days before surgery. RESULTS: Histologic and histomorphometric results demonstrated the preload FK-506 group had superior results compared with the immediate FK-506 group. Both FK-506 groups were superior to the placebo group. The preload FK-506 demonstrated superior regeneration in mean total nerve fiber counts (p < 0.05), greater percentage neural tissue (p < 0.05), greater mean nerve fiber density (p < 0.05), and lower percentage of debris (p > 0.05). Mean nerve fiber widths were similar in the preload and immediate FK-506 groups but superior to the placebo group. CONCLUSION: These data suggest that enhancement of FK-506's neuroregenerative effect is enhanced when administered before nerve injury such as when performing elective surgery.

FK506 and anti-CD40 ligand in peripheral nerve allotransplantation.

PURPOSE: Immunomodulatory agents are often combined in organ transplantation to minimize toxicity and enhance therapeutic effect. We hypothesized that combining low-dose FK506 with anti-CD40 Ligand (anti-CD40L mAb) would enhance regeneration through peripheral nerve allografts while preserving immune unresponsiveness. METHODS: Eighty Balb/cJ mice underwent tibial nerve grafting and were randomized to 10 groups treated with combinations of anti-CD40L mAb therapy, low-dose FK506 (0.5 mg/kg/day), high-dose FK506 (2 mg/kg/day), and high-dose cyclosporine (25 mg/kg/day). At 3 weeks, histomorphometry and cytokine secretion assays were performed. RESULTS: Animals receiving low-dose FK506 with anti-CD40L mAb exhibited robust nerve regeneration comparable to the isograft and high-dose FK506 allograft groups. Nerve density was significantly increased in the low-dose FK506 with anti-CD40L mAb group compared to animals receiving anti-CD40L mAb alone (p < 0.05). Combining anti-CD40L mAb with high dose cyclosporine decreased nerve fiber counts, nerve density, and percent nerve (p < 0.05). Interferon-gamma production was markedly elevated in untreated allografts compared to all other treatment groups (p < 0.05). Cytokine secretion was intermediate in the low-dose FK506 alone group and suppressed in all remaining groups. CONCLUSION: When combined with anti-CD40L mAb, low-dose FK506 enhances nerve regeneration without disrupting immune unresponsiveness.

Choosing the correct functional assay: a comprehensive assessment of functional tests in the rat.

While there are several ways to quantify peripheral nerve regeneration; the true measure of successful outcome is functional recovery. Functional tests are relatively easily conducted in human subjects; however it is more difficult in a laboratory animal. The laboratory rat is an excellent animal model of peripheral nerve injury and has been used extensively in the field of peripheral nerve research. Due to the intense interest in the rat as an experimental model, functional assays have been reported. In an effort to provide a resource to which investigators can refer when considering the most appropriate functional assay for a given experiment, the authors have compiled and tabulated the available functional tests applicable to various models of rat nerve injury.

Prolonged cold-preservation of nerve allografts.

The goal of this study was to determine the effect of varying durations of cold-preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon-gamma-producing cellular response (untreated allografts) to an absence of response (7-week cold-preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7-week cold-preserved groups when compared to the untreated allograft group. Increasing duration of cold-preservation diminished the cellular immune response. This cold-preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold-preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction.

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration.

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.

Effect of tension on nerve regeneration in rat sciatic nerve transection model.

Excessive tension across a nerve repair is known to impair nerve regeneration. However, it is uncertain whether nerve grafting is necessary when end-to-end repair would result in only mild to moderate tension. This study investigated the effect of tension on nerve regeneration. Sciatic nerves of 48 Lewis rats were transected and then repaired primarily after resection of 0-, 3-, 6-, or 9-mm lengths of nerve. Postoperative tension levels were quantified using a tensometer. Robust nerve regeneration was observed at 4 weeks in all except the 9-mm repair group, which showed lower nerve fiber counts, percent neural tissue, and nerve density (P < 0.05) and decreased functional recovery. These data indicate that modest levels of tension are well tolerated, but nerve regeneration drops precipitously once a critical tension threshold is exceeded. This threshold was between 0.39 and 0.56 N in the model studied, corresponding to a nerve defect between 6 mm and 9 mm.