FK506 Increases the Regeneration of Spinal Cord Axons in a Predegenerated Peripheral Nerve Autograft

Abstract

The authors examined the ability of FK506 to accelerate axonal regeneration of rat spinal cord axons in a peripheral nerve (PN) graft. Predegenerated autografts were produced by transecting the left tibial nerve 1 week prior to spinal cord implantation into the lumbar (L-3-L-4) spinal cord. Rats were given daily injections of either FK506 (5 mg/kg, subcutaneous) or vehicle for 21 days. The PN grafts from FK506-treated rats contained larger sized regenerating axons compared with vehicle-treated controls, and mean axonal areas increased by 25% at 7.5 mm along the PN graft. Fluoro-Gold? retrograde labeling confirmed that the regenerating axons originated from the central nervous system. Unexpectedly, the majority (> 50%) of neurons in the red nucleus were retrogradely labeled in the FK506-treated animals only. The results indicate that FK506 not only accelerates the elongation of spinal cord axons but also promotes regeneration of rubrospinal neurons.     

Effects of intrathecal administration of FK506 after sciatic nerve crush injury

Although various administration routes of FK506 have been published, intrathecal administration of FK506 has not previously been reported in the literature. A daily dose of 0.05 mg/kg of FK506 was given (a small dose compared with those reported in the available literature). The authors used this small dose to obtain lower immunosuppression and neurotoxicity, and a higher axonal regeneration rate. A total number of 40 female Wistar rats were used and randomly divided into four groups: control, sham, FK506-treated, and vehicle-treated. Sciatic nerve regeneration was evaluated by walking track analysis, an electrostimulation test, and light microscopic evaluation. There was a statistically significant difference ( P < 0.05) between FK506-treated and vehicle-treated groups at the end of 6 weeks according to both the walking track analysis and the electrostimulation test. Comparing the stimulus thresholds of the sham and FK506-treated group, no significant difference ( P > 0.05) was observed. Evaluation of the data revealed that FK506 had a beneficial effect on sciatic nerve regeneration.     

FK506 increases the regeneration of spinal cord axons in a predegenerated peripheral nerve autograft

The authors examined the ability of FK506 to accelerate axonal regeneration of rat spinal cord axons in a peripheral nerve (PN) graft. Predegenerated autografts were produced by transecting the left tibial nerve 1 week prior to spinal cord implantation into the lumbar (L-3-L-4) spinal cord. Rats were given daily injections of either FK506 (5 mg/kg, subcutaneous) or vehicle for 21 days. The PN grafts from FK506-treated rats contained larger sized regenerating axons compared with vehicle-treated controls, and mean axonal areas increased by 25% at 7.5 mm along the PN graft. Fluoro-Gold retrograde labeling confirmed that the regenerating axons originated from the central nervous system. Unexpectedly, the majority (>50%) of neurons in the red nucleus were retrogradely labeled in the FK506-treated animals only. The results indicate that FK506 not only accelerates the elongation of spinal cord axons but also promotes regeneration of rubrospinal neurons.     

FK506 rescues peripheral nerve allografts in acute rejection

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.     

Effects of FKBP-12 ligands following tibial nerve injury in rats

The neuroregenerative properties of FK506, an FKBP-12 ligand that inhibits calcineurin, and V-10,367, an FKBP-12 ligand that does not inhibit calcineurin, were evaluated in crush and transection models. Rats were randomly assigned to one of seven groups, including untreated controls and FK506- or V-10,367-treated experimental groups. Following crush or transection nerve injury, animals were assessed with walking tracks, and histomorphometry. FK506-treated animals demonstrated significant functional recovery 11 days following crush and 18 days following transection injury. In untreated and V-10,367 treated animals, nerves recovered 13 days following crush injury, but did not improve significantly prior to sacrifice at 28 days in animals sustaining a transection injury. No statistically significant differences in histomorphometric parameters were identified between any of the groups. The study confirms that FK506 accelerates recovery from tibial nerve injury.     

Effect of FK506 on peripheral nerve regeneration through long grafts in inbred swine

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.     

Effect of sialodacryoadenitis virus infection on axonal regeneration

The effect of sialodacryoadenitis virus (SDAV) infection on axonal regeneration and functional recovery was investigated in male Lewis rats. Animals underwent unilateral tibial nerve transection, immediate repair, and treatment with either FK506 (treated) or control vehicle (untreated). Serial walking track analyses were performed to assess functional recovery. Nerves were harvested for morphometric analysis on postoperative day 18 after an SDAV outbreak occurred that affected the 12 experimental animals. Histomorphometry and walking track data were compared against 36 historical controls. Rats infected with SDAV demonstrated severely impaired axonal regeneration and diminished functional recovery. Total fiber counts, nerve density, and percent neural tissue were all significantly reduced in infected animals (P < 0.05). Active SDAV infection severely impaired nerve regeneration and negated the positive effect of FK506 on nerve regeneration in rats. Immunosuppressive risks must be weighed carefully against the potential neuroregenerative benefits in the treatment of peripheral nerve injuries.     

The effects of FK506 on neurologic and histopathologic outcome after transient spinal cord ischemia induced by aortic cross-clamping in rats

Spinal cord injury is a devastating complication of thoracoabdominal aortic surgery. We investigated the effect of the immunosuppressant FK506, a macrolide antibiotic demonstrated to have neuroprotective effects in cerebral ischemia models, in a rat model of transient spinal cord ischemia. Spinal cord ischemia was induced in anesthetized rats by using direct aortic arch plus left subclavian artery cross-clamping through a limited thoracotomy. Experimental groups were as follows: sham-operation; control, receiving only vehicle; FK506 A, receiving FK506 (1 mg/kg IV) before clamping; and FK506 B, receiving FK506 (1 mg/kg IV) at the onset of reperfusion. Neurologic status was assessed at 24 h and then daily up to 96 h with a 0 to 6 scale (0, normal function; 6, severe paraplegia). Rats were randomly killed at 24, 48, or 96 h, and spinal cords were harvested for histopathology. Physiologic variables did not differ significantly among experimental groups. All control rats suffered severe and definitive paraplegia. FK506-treated rats had significantly better neurologic outcome compared with control. Histopathologic analysis disclosed severe injury in the lumbar gray matter of all control rats, whereas most FK506-treated rats had less injury. These data suggest that FK506 can improve neurologic recovery and attenuate spinal cord injury induced by transient thoracic aortic cross-clamping. IMPLICATIONS: A single dose-injection of the immunosuppressant FK506 significantly improved neurologic outcome and attenuated spinal cord injury induced by transient thoracic aortic cross-clamping in the rat.     

他克莫司对犬急性脊髓损伤神经保护作用的实验研究

目的探讨他克莫司（FK506）对犬急性脊髓损伤的神经保护作用。方法采用Allen＇s法制成犬急性脊髓损伤实验模型。动物随机分为：A组（n=8）,经动脉插管注入生理盐水;B组（n=8）,FK5060.18mg／kg;C组（n=8）．FK5060.3mg／kg。B组、C组均在致伤后2h经动脉插管单次给药。致伤后行脊髓MRI影像学检查、脊髓功能评分、脊髓组织病理观察、神经丝蛋白（NF200）及胶质纤维酸性蛋白（GFAP）的免疫组织化学分析。结果脊髓功能评分C组优于A组（P〈0.05）;B组优于A组,但无统计学差异;MRI显示：脊髓损伤后C组病变范围小,恢复快,B组次之．A组最差。C组NF和GFAP表达高于A组（P〈0.05）,B组与A组无统计学差异。结论局部应用FK506（0.3mg／kg）对急性脊髓损伤具有神经保护作用,可减轻继发损伤,加快脊髓功能的恢复;局部应用FK506对急性脊髓损伤治疗有一定的剂量-效应依赖关系。     

转化生长因子-β1在他克莫司肾毒性中的作用

目的 观察转化生长因子(TGF)-β1在他克莫司大鼠肾毒性中的作用.方法 将SD大鼠24只随机分成对照组、CsA组、FK506组和FK506+Dil组,用药4周后建立起各组大鼠模型.观察各组大鼠的肾功能,应用免疫组织化学技术检测各组大鼠TGF-β1的表达.结果 FK506组大鼠的血肌酐值为(34.17±4.54)μmol/L,肌酐清除率为(0.58±0.39)ml·min-1·100g-1,与对照组比较差异有统计学意义(P<0.05).FK506组TGF-β1的阳性表达率均为100%(6/6),对照组TGF-β1的阳性表达率为16%(1/6),两者差异有统计学意义(P<0.05). 结论 TGF-β1可能介导了FK506引起的肾毒性.     

FK506局部缓释膜片促进周围神经再生的临床应用研究

目的临床观察在神经缝合口周围置入FK506高分子缓释膜片后对神经再生的影响。方法?选取同一时间段(4个月内)，在腕横纹至肘上5cm处因切割伤而导致正中神经或尺神经断伤的急诊病例16例。结合病人是否接受FK506治疗的意愿分为实验组及对照组，每组8例。实验组用9-0无创尼龙缝线将神经两断端作端端缝合，并将含有20mg FK506的高分子可生物降解膜片环绕神经缝合口一圈后用筋膜等软组织覆盖修复的神经和膜片。将膜片制成以1mg／d的速度释放，共20d。对照组仅用同法修复神经。结果术后1周起随访至2年。实验组在术后14周肌电图检测即有新生电位出现，较对照组提前4周。术后3～12个月测定，实验组感觉神经再生速度平均为3．1mm／d，明显快于对照组的1．7mm／d。实验组远期功能恢复的优良率也优于对照组。结论从FK506的药理作用和应用结果分析，FK506具有针对性促进周围神经再生的作用。     

Effect of FK506 and basic fibroblast growth factor on nerve regeneration using a polytetrafluoroethylene chamber for nerve repair

Peripheral nerve repair can be accomplished by using a polytetrafluoroethylene tubular chamber to guide nerve healing and regeneration. In this study, we delivered basic fibroblast growth factor (bFGF) into the chamber for sciatic nerve repair in rats. In addition, the animals were given systemically 1 mg/kg/day FK506 (tacrolimus), a potent immunosuppressant with neurotrophic properties. Nerve regeneration was evaluated by means of a nociceptive test and a grasping test starting 2 weeks postoperatively. Animals that received bFGF and FK506 showed a significantly faster recovery from injury than did the control group. Morphometric analysis at 3 months showed no difference between the two groups in total number of axonal fibers, fiber diameter, fiber density, and myelin:axon ratio. We conclude that the combination of bFGF and low dose FK506 enhances nerve healing in this animal model by accelerating early regrowth but has no effect on the final outcome.     

Effect of FK506 on functional recovery after facial nerve injury in the rat

OBJECTIVE: To examine the effect of the immunosuppressive agent FK506 on the rate of functional recovery of the rat facial nerve after crush injury. METHODS: Forty rats underwent facial nerve crush injury and were randomly assigned to 4 experimental groups: isotonic sodium chloride solution control, FK binding protein 52 (FKBP-52) antibody control, FK506, and FK506 and FKBP-52 antibody. Rats underwent daily recovery testing from postoperative day 9 until postoperative day 21 by videotaping 3 validated variables in this model: blink reflex return, vibrissial fibrillation loss, and return of vibrissial sweeping symmetry. RESULTS: FK506-treated animals demonstrated improved recovery in all 3 variables compared with control animals. The FK506 and FKBP-52 antibody group demonstrated improved recovery of only the return of the blink reflex. CONCLUSIONS: FK506 accelerated functional recovery of facial nerve function after crush injury. Neuroregeneration was inhibited by FKBP-52 antibody in the rat midface but not the upper face. FK506 may be a viable adjuvant treatment for facial nerve neurapraxic injury.     

Chlorpromazine protects rat spinal cord against contusion injury

The protective effect of chlorpromazine on rat spinal cord injury was investigated using a dynamic impact model. A 10 g weight was dropped 5 cm on an impounder placed on the exposed spinal cord at the T-11 level. Changes in potassium concentration on the epidural surface of the injured spinal cord were measured using a combined impounder-K+ electrode assembly. Recovery of motor performance was estimated using the modified Tarlov score. In the injury control (no treatment) group, the recovery was slow. Animals were still paralyzed 4 weeks after injury and none of them could walk; the Tarlov score was 1.88 +/- 0.78 (S.D.). In contrast, the chlorpromazine-treated group (20 mg/kg i.p. 30 min prior to injury) recovered significantly in 4 weeks. Animals could either support body weight or walk with some deficit; the Tarlov score was 4.0 +/- 0.35. Chlorpromazine inhibited potassium efflux from the spinal cord after contusion. Possible mechanisms of protection of neural cells by chlorpromazine are discussed.     

The effect of FK1706 on erectile function following bilateral cavernous nerve crush injury in a rat model

PURPOSE: We investigated the neurotrophic effect of FK1706 on erectile recovery following bilateral cavernous nerve crush injury in a rat model. MATERIALS AND METHODS: A total of 28 male Sprague-Dawley rats were randomly divided into 4 equal groups. Seven animals underwent sham operation and subcutaneous vehicle injection, whereas 21 underwent bilateral cavernous nerve crush injury followed by vehicle injection alone, or by low (0.1 mg/kg) or high (1.0 mg/kg) dose FK1706 treatment. Injections were continued 5 days weekly for 8 weeks. Erectile function was then assessed by cavernous nerve electrostimulation and penile tissue was evaluated immunohistochemically. RESULTS: No erectile dysfunction was identified in the sham treated group (mean maximal intracavernous pressure +/- SEM 106.8 +/- 6.4 cm H(2)O), whereas nerve injury significantly decreased ICP to 17.9 +/- 7.0 cm H(2)O. FK1706 facilitated neural and erectile recovery in a concentration dependent manner with a mean ICP in the high dose FK treatment group of 80.1 +/- 7.8 cm H(2)O compared with 44.1 +/- 12.9 cm H(2)O in the low dose group. Similar stepwise findings were observed using mean area under the curve data. Sham treated animals showed regular axon sizes and shapes with homogenous GAP-43 and neurofilament staining, whereas injured axons showed irregular shapes, sizes and staining patterns. FK1706 treatment restored axon shape and staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide phosphate staining and FK1706 treatment showed a nonsignificant trend toward increased staining. CONCLUSIONS: Bilateral cavernous nerve crush causes reproducible erectile dysfunction, consistent with prior experiments. High dose subcutaneous FK1706 therapy promotes significant neuroregeneration and erectile function recovery.     

Axonal regeneration after cold preservation of nerve allografts and immunosuppression with tacrolimus in mice

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.     

Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60-67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 μm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.     

Neuroprotective effects of FK-506, L-carnitine and azathioprine on spinal cord ischemia-reperfusion injury.

OBJECTIVE: In our experimental study, we aimed to test the effect of FK506, azathioprine and L-carnitine on protection of spinal cord injury due to ischemia-reperfusion. METHODS: Twenty-seven Sprague-Dawley male rats were randomly divided into five groups. They were subjected to spinal cord ischemia by clamping the abdominal aorta for 45 min. Thirty minutes before the aortic clamping, group I received 0.5 mg/kg FK506, group II received 100 mg/kg L-carnitine, group III received 4 mg/kg azathioprine, the fourth group was the control group and received only normal saline injection intravenously and the last group was the sham group. Neurological status was scored by using the Tarlov scoring system. Sections of the lumbar cord were harvested for histopathological grades (1-4), having regard to percentage of the apoptotic cells. RESULTS: Hind-limb motor function had recovered normally 48 h after the operation in all rats which received FK506, azathioprine and L-carnitine prophylactically. In contrast, all rats in the control group had deteriorated to paraplegia by 48 h after the operation (P<0.05). Histopathologic sections in the involved spinal cord segment showed that a greater number of motor neuron cells were preserved and there were less apoptotic cells in the rats that received FK506, azathioprine and L-carnitine than those in control group. CONCLUSIONS: These results suggest that prophylactic use of FK506, azathioprine and L-carnitine protects motor neuron cells from ischemic spinal cord injury.     

FK506对GAP-43和Tau蛋白活性影响的实验研究

目的观察FK506缓释膜片对大鼠神经损伤后生长相关蛋白(Growthassociatedprotein43,GAP43)和Tau蛋白活性的影响。方法将FK506缓释膜片(释放速度为1mg·d,共21d)放置于损伤坐骨神经周围为实验组,损伤坐骨神经周围不放置药物者为对照组。于术后3d,术后1、2、3、4、6和8周共7个时间组,分别取神经缝合口近端和远端的神经段、腰段脊神经节及脊髓腰膨大,用SABC法检测GAP43及Tau蛋白的阳性标记量。结果实验组于术后1～4周已出现GAP43及Tau蛋白表达增强,与对照组相比差异有统计学意义(P<0.01)。结论FK506能增加GAP43及Tau蛋白表达,对周围神经损伤后的神经再生有一定的促进作用。     

Immunophilin ligands FK506 and cyclosporine A improve neurologic and histopathologic outcome after transient spinal cord ischemia in rabbits

BACKGROUND: We comparatively evaluated the protective effect of the immunophilin ligands cyclosporine A (INN: ciclosporin), FK506, and rapamycin on the spinal cord in a rabbit model of transient ischemia. Both cyclosporine A and FK506 inhibit calcineurin, whereas rapamycin does not. METHODS: Thirty-six male New Zealand White rabbits were divided into the following 6 groups: group C, 15 minutes of spinal cord ischemia; group FK, FK506 (1 mg/kg) administered 30 minutes before ischemia; group CsA, cyclosporine A (30 mg/kg) administered 30 minutes before ischemia; group CsA-C, chronic administration of cyclosporine A (20 mg/kg) for 9 days before ischemia; group R, rapamycin (1 mg/kg) administered 30 minutes before ischemia; and group R+FK, rapamycin (1 mg/kg) administered 20 minutes before FK506 pretreatment (1 mg/kg). Group CsA-C was added because the drug does not readily cross the blood-brain barrier. Neurologic function was evaluated by Johnson's 5-point scale at 8, 24, and 48 hours after ischemia, and histopathology was assessed 48 hours after ischemia. RESULTS: At 24 and 48 hours after ischemia, the Johnson score was better in groups FK (4.0 +/- 1.1), R+FK (3 +/- 1.1), and CsA-C (2.7 +/- 1.2) than in group C (0.8 +/- 1.2). Numbers of morphologically intact anterior horn cells were higher in groups FK (31.3 +/- 9.9), R+FK (23.2 +/- 4.5), and CsA-C (18.3 +/- 6.8) than in group C (6.3 +/- 4.3). CONCLUSIONS: FK506 and chronic administration of cyclosporine A, but not rapamycin, protect the spinal cord from transient ischemia. Although these results are compatible with inhibition of calcineurin in the mechanism of neuroprotective action of these drugs, other effects through different pathways cannot be excluded before further study.