Search

Introduction/aims: Repaired nerve injuries can fail to achieve functional recovery. Therapeutic options beyond surgery, such as systemic tacrolimus (FK506) and electrical stimulation (E-stim), can improve recovery. We tested whether dual administration of FK506 and E-stim enhances regeneration and recovery more than either therapeutic alone., Methods: Rats were randomized to four groups: E-stim, FK506, FK506 + E-stim, and repair alone. All groups underwent tibial nerve transection and repair. Two sets of animals were created to measure outcomes of early nerve regeneration using nerve histology (n = 36) and functional recovery (n = 42) (21- and 42-day endpoints, respectively). Functional recovery was measured by behavioral analyses (walking track and grid walk) and, at the endpoint, muscle mass and force., Results: Dual E-stim and FK506 administration produced histomorphometric measurements of nerve regeneration no different than either therapeutic alone. All treatments were superior to repair alone (FK506, P < .0001; E-stim, P < .05; FK506 + E-stim, P < .05). The E-stim and FK506 + E-stim groups had improved behavioral recovery compared with repair alone (at 6 weeks: E-stim, P < .05; FK506 + E-stim, P < .01). The FK506 group had improved recovery based on walking-track analysis (at 6 weeks: P < .001) and muscle force and mass (P < .05). The concurrent use of both therapies ensured earlier functional recovery and decreased variability in functional outcomes compared with either therapy alone, suggesting a moderate benefit., Discussion: Dual administration of FK506 and E-stim showed minimal additive effects to further improve regeneration or recovery compared with either therapy alone. The data suggest the combination of FK506 and E-stim appears to combine the relative strengths of each therapeutic., (© 2022 Wiley Periodicals LLC.)

FK-506 accelerates nerve regeneration and improves functional recovery in vivo; its immunosuppressive properties, however, limit its clinical utility. Geldanamycin (GA), a non-immunosuppressive agent, shares a common binding target (heat shock protein 90) with FK-506 and may accelerate nerve regeneration through a similar mechanism. GA has been shown to augment neurite outgrowth in vitro but has not been tested in vivo. The current study investigated the effect of GA on the rate of axonal regeneration and functional recovery following peripheral nerve injury. In the first experiment, Thy1-GFP transgenic rats underwent serial transmuscular imaging to quantify the rate of axonal regeneration following saphenous nerve crush injury. In subsequent experiments, Lewis rats underwent tibial nerve crush or transection-and-repair injuries and were assessed for functional recovery by walking track analysis. All animals were randomized to receive daily administration of FK-506 (2mg/kg), GA (0.2mg/kg), or a control vehicle (dimethyl sulfoxide, 1 mL/kg) starting 3 days prior to injury. Both GA and FK-506 significantly increased the rate of axonal regeneration following crush injury in Thy1-GFP rats. In Lewis rats undergoing tibial nerve crush injury, earlier functional recovery occurred at day 5 and day 6 in animals treated with FK-506 and GA respectively, vs. day 13 for controls. Over a truncated 21-day timeframe, Lewis rats undergoing tibial nerve transection-and-repair injury and treated with FK-506 regained function at day 16, whereas those treated with GA or the control vehicle did not regain normal function. GA-treated animals, however, did exhibit significant functional improvement vs. controls. The current study demonstrated that GA accelerates axonal regeneration and enhances functional recovery in vivo. Its ability to increase the rate at which peripheral axons regenerate is comparable to that of FK-506. GA, however, did not match the performance of FK-506 in injury models where Wallerian degeneration (WD) is ongoing in the distal stump. This provides evidence that FK-506 accelerates axonal regeneration through two parallel mechanisms: the first being its well-established effect on neurons; the second is likely a newly described, as-yet poorly defined mechanism that affects WD. Finally, given the decrease in observed toxicity with GA administration, it might be a suitable non-immunosuppressive alternative to FK-506 for accelerating peripheral nerve regeneration in cases of clinical nerve injury., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: The slow rate of nerve regeneration following injury can cause extended muscle denervation, leading to irreversible muscle atrophy, fibrosis, and destruction of motor endplates. The immunosuppressant FK506 (tacrolimus) has been shown to accelerate the rate of nerve regeneration and functional recovery. However, the toxic and immunosuppressive properties of FK506 make it undesirable for long-term use., Objective: To take advantage of the regeneration-enhancing effects of FK506 but avoid the potential adverse effects of long-term administration, the current study evaluates and quantifies the efficacy of short-term FK506 treatment in rat models., Methods: Clinically relevant transection and graft models were evaluated, and walking track analysis (WTA) was used to evaluate functional recovery. FK506 was administered for 5 and 10 days post transection injury and 10 and 20 days post graft injury. Both groups involving a short course were compared with the continuous administration group., Results: In the transection model, FK506 was administered for 5 and 10 days postoperatively. WTA demonstrated that 10 days of FK506 administration was sufficient to reduce functional recovery time by 29% compared with negative controls. In the graft model, FK506 was administered for 10 and 20 days postoperatively. Short treatment courses of 10 and 20 days reduced recovery time by 15% and 21%, respectively, compared with negative controls. Analysis of blood-nerve barrier (BNB) integrity demonstrated that FK506 facilitated early reconstitution of the BNB., Conclusions: The results of this study indicate that short-term FK506 delivery following nerve injury imparts a significant therapeutic effect.

Numerous small-animal studies have demonstrated that FK506 enhances nerve regeneration and accelerates functional recovery after nerve injury. However, no experimental study has corroborated these neuroregenerative effects in larger animals. This study investigated the effects of FK506 on nerve regeneration in inbred miniature swine. Eight animals received 8-cm ulnar nerve autografts and allografts. Treated animals received 0.1 to 0.4 mg/kg FK506 injections twice weekly to maintain immunosuppressive serum FK506 levels. At 24 weeks posttransplant, nerve grafts were harvested for histomorphometric analysis. Mixed lymphocyte cultures demonstrated alloreactivity in 1 treated animal and all untreated animals. In autografts, mean fiber count, nerve density, and percent neural tissue were doubled with FK506 therapy. In allografts, significant neuroregeneration was observed in animals treated with FK506, whereas untreated animals had no regeneration. Treatment with FK506 resulted in a trend toward enhanced axonal regeneration through nerve autografts and allografts in a large-animal model with defined histocompatibility barriers.

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.

Objectives/hypothesis: The immunosuppressive agent FK506 has been shown in many studies to enhance nerve regeneration and to accelerate functional recovery after immediate nerve repair. However, in clinical practice the diagnosis and treatment of patients with peripheral nerve injuries is often delayed. The study investigated whether FK506 would retain its neuroregenerative properties when nerve repair and initiation of FK506 therapy were delayed for 7 days., Study Design: In vivo laboratory study., Methods: Thirty-two Lewis rats underwent tibial nerve transection and were randomly assigned to four experimental groups: immediate repair with FK506 treatment, immediate repair without FK506 treatment, 7-day delayed repair with FK506 treatment, and 7-day delayed repair without FK506 treatment. Treated animals received daily subcutaneous injections of 2 mg/kg FK506. Serial walking track measurements were performed at 14, 16, and 18 days after nerve repair. On day 18 after repair, peripheral nerves were injected with a fluorescent tracer for retrograde labeling. On day 21, peripheral nerves and spinal cords were harvested for histomorphometric analysis and motor neuron cell body counts, respectively., Results: Animals that underwent immediate repair with FK506 had significantly higher fiber counts and percentages of nerve than the other three groups (P <.05) but did not show statistically significant earlier functional recovery. The remaining three groups had intermediate levels of nerve regeneration that were not significantly different. Retrograde abled motor neurons counts were decreased in animals with delayed nerve repair that received no FK506 (P <.05)., Conclusion: In a rat tibial nerve transection model, the neuroregenerative effects of FK506 diminished markedly when repair and initiation of FK506 therapy were delayed by 7 days.

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.

The immunosuppressive agent FK-506 has a well-described neuroregenerative effect that is mediated by a mechanism independent of calcineurin inhibition. FK-506 levels that fall below the threshold for immunosuppression could therefore potentially enhance nerve regeneration while minimizing toxicity. The purpose of this study was to characterize the dose-dependent effects of FK506 on T-cell proliferation, and establish a subimmunosuppressive dosing regimen for FK-506 in mice. Forty BALB/cJ mice were randomized to four groups corresponding to 0, 0.25, 0.5, or 1.0 mg/kg/day doses of FK-506. Ten days postoperatively, animals were sacrificed, and mixed lymphocyte reaction assays were performed to quantify the immune response to nerve allografts. Mice receiving 0.25 and 0.5 mg/kg/day of FK-506 exhibited a robust T-cell proliferation response, with stimulation indices approaching those of untreated animals. Mice treated with 1.0 mg/kg/day of FK-506 demonstrated significantly decreased T-cell proliferation. These results establish 0.5 mg/kg/day as an upper limit for subimmunosuppressive FK-506 administration., (Copyright 2003 Wiley-Liss, Inc.)

Prevention and control of graft rejection remain essential in the investigation of peripheral nerve allotransplantation. Although use of cyclosporin A (CsA) has been shown to suppress successfully the rejection of nerve allografts, limited information exists concerning use of this drug to arrest rejection in progress, and thereby effect salvage of these grafts. The aim of this study was to determine the efficacy of CsA in the treatment of ongoing acute rejection of peripheral nerve allografts. Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals and were divided into five groups: group 1 received isografts and no CsA treatment (n = 8), group 2 received allografts with continuous CsA therapy (n = 10), group 3 received allografts with no treatment (n = 7), group 4 received allografts with initiation of CsA therapy delayed until 3 weeks after the procedure (n = 11), and group 5 received allografts with an interrupted course of CsA (n = 15). All grafts were harvested at 10 weeks. Histomorphometric analysis demonstrated comparable nerve regeneration in groups 1 and 2 and good regeneration in group 3 animals, despite cellular infiltrate suggestive of rejection. At 3 weeks after surgery, group 4 animals showed early rejection and significantly less neuroregeneration than positive controls at 10 weeks after delayed initiation of CsA therapy. Finally, group 5 animals showed early regeneration at 3 weeks but significantly lesser regeneration by 10 weeks after interruption of therapy. In this experimental protocol, CsA was ineffective in rescuing histologically proven rejection in progress.

The FKBP-12-binding ligand FK506 has been successfully used to stimulate nerve regeneration and prevent the rejection of peripheral nerve allografts. The immunosuppressant rapamycin, another FKBP-12-binding ligand, stimulates axonal regeneration in vitro, but its influence on nerve regeneration in peripheral nerve isografts or allografts has not been studied. Sixty female inbred BALB/cJ mice were randomized into six tibial nerve transplant groups, including three isograft and three allograft (C57BL/6J) groups. Grafts were left untreated (groups I and II), treated with FK506 (groups III and IV), or treated with rapamycin (groups V and VI). Nerve regeneration was quantified in terms of histomorphometry and functional recovery, and immunosuppression was confirmed with mixed lymphocyte reactivity assays. Animals treated with FK506 and rapamycin were immunosuppressed and demonstrated significantly less immune cell proliferation relative to untreated recipient animals. Although every animal demonstrated some functional recovery during the study, animals receiving an untreated peripheral nerve allograft were slowest to recover. Isografts treated with FK506 but not rapamycin demonstrated significantly increased nerve regeneration. Nerve allografts in animals treated with FK506, and to a lesser extent rapamycin, however, both demonstrated significantly more nerve regeneration and increased nerve fiber widths relative to untreated controls. The authors suggest that rapamycin can facilitate regeneration through peripheral nerve allografts, but it is not a neuroregenerative agent in this in vivo model. Nerve regeneration in FK506-treated peripheral nerve isografts and allografts was superior to that found in rapamycin-treated animals. Rapamycin may have a role in the treatment of peripheral nerve allografts when used in combination with other medications, or in the setting of renal failure that often precludes the use of calcineurin inhibitors such as FK506.

Object: The purpose of this study was to combine the immunosuppressive and neuroregenerative effects of tacrolimus (FK506) with cold preservation of peripheral nerve allografts to maximize axonal regeneration across short peripheral nerve gaps., Methods: Ninety-six male C3H mice were randomized to six groups, which were composed of animals with isografts (Group 1, positive control), allografts (Group 2, negative control), allografts treated with subtherapeutic doses of FK506 without and with cold preservation (Groups 3 and 4), and allografts treated with therapeutic doses of FK506 without and with cold preservation (Groups 5 and 6). Results were determined using walking-track data and histomorphometric measurements. Three weeks postoperatively, animals treated with therapeutic doses of FK506 after receiving cold-preserved allografts demonstrated accelerated functional recovery relative to all other groups. In addition, histomorphometric parameters in these animals (1,257 +/- 847 total axons, 6.7 +/- 3.3% nerve tissue, 11.8 +/- 6.5% neural debris, 8,844 +/- 4,325 fibers/mm2 nerve density, and 2.53 +/- 0.25 microm fiber width) were the same as or better than in all other groups. The parameters of percent nerve tissue (p < 0.016), nerve density (p < 0.038), and percent neural debris (p < 0.01) were statistically significantly better than those in all other groups, including Group 1 (isograft, positive control)., Conclusions: The combination of FK506 treatment with cold preservation of nerve allografts resulted in functional and histomorphometric recovery superior to that with either modality alone.

This study investigated the ability of the immunosuppressant FK506 to reverse nerve allograft rejection in progress. Eighty-four Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals. Allografts were left untreated for either 7, 10, or 14 days before receiving daily subcutaneous FK506 injections (2 mg/kg). Time-matched control animals received either an isograft, an allograft with continuous FK506, or an allograft with no postoperative FK506 therapy. All animals underwent weekly evaluation of nerve function by walking track analysis. Experimental group animals were sacrificed either immediately prior to initiation of FK506 therapy (days 7, 10, or 14), after 2 weeks of immunosuppressive treatment, or 8 weeks postsurgery. Histomorphometric analysis, consisting of measurements of total number of nerve fibers, neural density, and percent of neural debris, demonstrated a statistically significant increase in regeneration in the isograft group relative to the untreated allograft group within 28 days of transplantation. Grafts harvested from animals receiving 2 weeks of FK506 after 7 or 10 days of rejection were histomorphometrically similar to time-matched isografts. By contrast, grafts from animals receiving 2 weeks of FK506 following 14 days without therapy resembled untreated allografts and demonstrated significant histomorphometric differences from isografts at the corresponding time point. Analysis of walking track data confirmed that relative to untreated allografts, functional recovery was hastened in animals receiving an isograft, or allograft treated with FK506. This study demonstrated that when started within 10 days of graft placement, FK506 could reverse nerve allograft rejection in rats evaluated following 2 weeks of treatment.

Lymphocyte migration into nerve allografts was measured to estimate the cyclosporine A (CsA) dose required to suppress rejection. Twelve outbred sheep received daily subcutaneous CsA at 0, 5, 10, or 15 mg/kg/day for 2 weeks prior to implantation of multiple heterotopic subcutaneous nerve grafts. Lymphocyte migration was determined after 7 days by an intravenous pulse of autologous 111indium-labeled lymphocytes and subsequent quantitation of gamma radioactivity in nerve tissue (CPM/g, mean +/- SEM). Measurement by radioimmunoassay revealed a dose-dependent increase in blood cyclosporine levels. Lymphocyte migration into autografts (404+/-44) was significantly less than migration into allografts (16,554+/-2,049), in control animals (P < 0.01). A dose-dependent inhibition of lymphocyte migration into nerve allografts was observed with counts of 7,662+/-1,692, 4,083+/-1,112, and 1,561+/-232 in sheep receiving 5, 10, or 15 mg/kg/day of CsA, respectively. Daily CsA administration produced effective blood levels and immunosuppression sufficient to inhibit lymphocyte migration into nerve allografts.

This study evaluated the immunosuppressive effect of monoclonal antibodies against cell surface molecules in a murine peripheral nerve allograft model. After nerve allografting, 18 recipients were treated with both anti-intercellular adhesion molecule-1 (ICAM-1) and anti-lymphocyte function-associated molecule-1 (LFA-1) monoclonal antibodies in low or high dose. Nerve allografts were harvested at 8 weeks for histologic and morphometric evaluation. Recipients were subsequently challenged with skin grafts at 9 weeks and a cytotoxic assay at 12 weeks. The majority of the antibody-treated allografts (13 of 18) showed excellent regeneration comparable to the autografts with preservation of the normal nerve architecture and scant cellular infiltrate. All untreated allografts demonstrated severe structural disorganization with cellular infiltrate consistent with acute rejection. In the high dose group, the mean skin graft survival time from nerve donor mice, but not third-party mice, was significantly prolonged. (17.5 vs. 11.3 days). Similarly, the cytotoxic activity against nerve donor alloantigen was significantly suppressed. These preliminary findings suggest that antibody therapy alone can facilitate nerve regeneration in a murine nerve allograft model.