Effects of FK506 on regeneration and macrophages in injured rat sciatic nerve

Effects of FK506 [5.0 mg/kg body weight (BW), subcutaneous, daily] on nerve regeneration and presence of macrophages in lesioned rat sciatic nerves were studied. Models of autologous nerve graft or a nerve crush lesion were used and regeneration was evaluated by immunocytochemistry (also used to detect ED1/ED2 macrophages) and sensory pinch reflex test, respectively. Treatment with FK506 did not increase regeneration distance or regeneration rate in the autologous nerve grafts. However, regeneration distances after nerve crush were significantly longer following treatment with FK506. The number of macrophages (ED1/ED2) in nerve grafts increased over time, but treatment with FK506 had limited effects only in the presence of ED2 macrophages. Present and previously published studies may imply that there is a time-related and type-of-injury-related profile of FK506's pro-regenerative effect.     

Effects of tacrolimus (FK506) on sciatic nerve regeneration in rats

BACKGROUND: Tacrolimus (FK506) protects peripheral nerves located in damaged regions by inhibiting T lymphocyte proliferation and activation.OBJECTIVE: To evaluate the effect of FK506 on promoting regeneration of rat sciatic nerve. DESIGN, TIME AND SETTING: A randomized, controlled, animal study was performed at the Laboratory of the Department of Orthopedic Surgery, Dalian Medical University, China, from September 2007 to September 2008.MATERIALS: A total of 60 adult, male, Sprague-Dawley rats were equally and randomly divided into model, local administration and systemic administration groups. All rats received a neurotomy of bilateral sciatic nerves to establish models of nerve regeneration chambers. The powder and injection of FK506 were supplied by Fujisawa Pharmaceutical, Japan.METHODS: The regeneration chambers of the model group were infused with 0.2 mL saline. The systemic group were injected with 0.2 mL saline, followed by daily subcutaneous injections of FK506 (1 mg/kg), for 14 days. The local administration group was infused with 0.2 mL FK506 (1 μg/mL).MAIN OUTCOME MEASURES: Local immune response was observed using hematoxylin-eosin staining. Myelinated nerve fiber number, myelin sheath and nerve fiber thickness were observed using toluidine blue staining. Wet weight of gastrocnemius was evaluated. Compound muscle action potential amplitude, latency, and conduction time were recorded, and motor nerve conduction velocity was calculated using electrophysiology.RESULTS: The total number of myeiinated nerve fibers in the local and systemic administration groups was significantly higher than in the model group. The density of myelinated nerve fibers, myelin sheath thickness and mean axon diameter were significantly increased in the systemic administration group compared with the model group (P < 0.05). Lymphocyte infiltration was decreased in the local and systemic administration groups compared with the model group. The wet weight of rat gastrocnemius in the local and systemic administration groups were significantly greater compared with the model group (P<0.05). Motor nerve conduction velocity was the fastest in the systemic administration group, and the slowest in the model group. Compound muscle action potential amplitude was larger in the systemic administration group compared with the local administration and model groups (P<0.05).CONCLUSION: Systemic administration of FK506 can promote regeneration of rat sciatic nerve and recovery of neural function. Systemic administration produced better regeneration and recovery of function than local administration of FK506.     

FK506在神经损伤修复中的应用研究进展

FK506作为强效免疫抑制剂在临床上已得到广泛应用.而随后的研究发现FK506还具有神经损伤修复的促进作用,如今对神经损伤修复的促进作用是FK506研究的热点之一.本文从FK506对于神经损伤修复相关细胞的不同作用、对于不问类型神经损伤的作用、剂量效应、FK506应用中存在的缺陷等方面对其研究现状进行简要综述.FK506,又称他克莫司,1984年被发现于链霉菌属,是一种大环内酯类抗生素.作为一种FDA批准的强效免疫抑制剂,其效力是环孢霉素A(Ciclosporin A,CsA)的10～100倍,广泛应用于预防同种异体肾、肝,心、骨髓等器官或组织移植所产生的排斥反应.     

Comparative dose-dependence study of FK506 on transected mouse sciatic nerve repaired by allograft or xenograft

We evaluated the effects of FK506, at doses of 0.2, 2, and 5 mg/kg/day, on the response to nerve grafts implanted in outbred mice. A 6 mm long segment of the sciatic nerve was transected and repaired by autograft (the same segment resected), allograft (from another mouse), or xenograft (from a rat nerve). The regenerating nerves were harvested after 3 weeks and studied under light and electron microscope. Allografts of animals treated with the 5 mg/kg/day dose of FK506 appeared similar to those from autografts, demonstrating an equivalent number of myelinated fibers. In mice treated with the 2 mg/kg/day dose, regeneration was slightly hindered, as indicated by the reduced number of myelinated fibers. In contrast, in mice given a 0.2 mg/kg/day dose of FK506, allografts were not different from untreated allografts; both groups showed a marked rejection response with only few unmyelinated axons and no myelinated fibers. Xenografts showed a more severe rejection than allografts, with a marked inflammatory cell reaction throughout the graft. In contrast, in mice treated with the 5 mg/kg/day dose, xenografts exhibited a mild cell reaction and a greater number of regenerated myelinated fibers. In conclusion, effective axonal regeneration is achieved with FK506 administration at doses of 5 mg/kg/day through allografts and, partially, through xenografts.     

Comparison of continuous and discontinuous FK506 administration on autograft or allograft repair of sciatic nerve resection

An immunosuppressant drug that also possesses neuroregenerative properties, FK506 enhances the rate of axonal regeneration and improves recovery after nerve lesions. Nevertheless, prolonged immunosuppression may not be justified to assure the success of nerve regeneration. In this study, we compare the effects of continuous and discontinuous FK506 treatment on regeneration and reinnervation after sciatic nerve resection repaired with autologous or allogenic grafts in the mouse. For each type of repair, one group received FK506 (5 mg/kg) for 4 months, whereas a second group was treated with FK506 at 5 mg/kg for 5 weeks followed by 3 mg/kg for 4 weeks; a control group received saline only. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hind paw over 4 months after operation. Morphological analysis of the regenerated nerves was performed at the termination of the study. Autografts and allografts treated with sustained FK506 (5 mg/kg) reached high levels of reinnervation and followed a course of recovery faster than controls. The numbers of myelinated fibers also were similar. Allografts without immunosuppression demonstrated a slower rate of regeneration, exhibiting lower final levels of recovery compared with other groups and containing fewer numbers of regenerating myelinated fibers. Withdrawal of immunosuppressant therapy resulted in a decline in the degree of reinnervation in all functions tested during the third month, with stabilization between the third and fourth months. The number of regenerated myelinated fibers in the group was significantly lower than in autografts. Thus, continuous or discontinuous FK506 administration slightly accelerated the rate of reinnervation in autografts. In allograft repair, FK506 significantly enhanced both the rate and degree of regeneration and recovery, but its withdrawal resulted in graft rejection, a marked deterioration in function, and loss of regenerating fibers.     

Effects of FK506 on nerve regeneration and reinnervation after graft or tube repair of long nerve gaps.

We compared the effects of FK506 administration on regeneration and reinnervation after sciatic nerve resection and repair with an autologous graft or with a silicone tube leaving a 6-mm gap in the mouse. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and sweating functions in the hindpaw over 4 months after operation. Morphometric analysis of the regenerated nerves was performed at the end of follow-up. The nerve graft allowed for faster and higher levels of reinnervation in the four functions tested than silicone tube repair. Treatment with FK506 (for the first 9 weeks only) resulted in a slight, although not significant, improvement of the onset of reinnervation and of the maximal degree of recovery achieved after autografting. The recovery of pain sensibility and of the compound nerve action potentials in the digital nerves, which directly depend on axonal regeneration, showed better progression with FK506 than reinnervation of muscles and sweat glands, which require reestablishment of synaptic contacts with target cells. The myelinated fibers in the regenerated nerve showed a more mature appearance in the FK506-treated rats. However, FK506 showed a marginal effect in situations in which regeneration was limited, as in a silicone tube bridging a 6-mm gap in the mouse sciatic nerve. In conclusion, treatment with FK506 improved the rate of functional recovery after nerve resection and autograft repair.     

FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat

We examined the efficacy of FK 506 in reducing tissue damage after spinal cord injury in comparison to methylprednisolone (MP) treatment. Rats were subjected to a photochemical injury (T8) and were given a bolus of MP (30 mg/kg), FK 506 (2 mg/kg), or saline. An additional group received an initial bolus of FK 506 (2 mg/kg) followed by daily injections (0.2 mg/kg intraperitoneally). Functional recovery was evaluated using open-field walking, inclined plane tests, motor evoked potentials (MEPs), and the H-reflex response during 14 days postoperation (dpo). Tissue sparing and glial fibrillary acidic protein (GFAP), biotinylated tomato lectin LEC, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 1 beta (IL-1 beta) immunoreactivity were quantified in the injured spinal cord. FK 506-treated animals demonstrated significantly better neurologic outcome, higher MEP amplitudes, and lower H-wave amplitude compared to that of saline-treated rats. In contrast, administration of MP did not result in significant differences with respect to the saline-treated group. Histologic examination revealed that tissue sparing was largest in FK 506-treated compared to saline and MP-treated animals. GFAP and COX-2 reactivity was decreased in animals treated with FK 506 compared to that in animals given MP or saline, whereas IL-1 beta expression was similarly reduced in both FK 506- and MP-treated groups. Microglia/macrophage response was reduced in FK 506 and MP-injected animals at 3 dpo, but only in MP-treated animals at 7 dpo with respect to saline-injected rats. Repeated administrations of FK 506 improved functional and histologic results to a greater degree than did a single bolus of FK 506. The results indicate that FK 506 administration protects the damaged spinal cord and should be considered as potential therapy for treating spinal cord injuries.     

Bimodal dose-dependence of FK506 on the rate of axonal regeneration in mouse peripheral nerve

FK506 has been shown to enhance the rate of axonal regeneration after peripheral nerve lesions. However, quite variable doses of FK506 have been used in different animal studies. We examined the dose-dependence of FK506 on the rate of axonal regeneration after crush lesion of the mouse sciatic nerve. Mice received daily subcutaneous injections of FK506 at 0.2, 0.5, 1, 2, 5, or 10 mg/kg for 7 days after lesioning. A control group was injected with saline. The distance that regenerative axons advanced from the crush site was measured by the pinch test at 2, 4, and 7 days. Regenerating axons reached greater mean distances in all FK506-treated groups compared to the control group. The fastest regeneration rate was found at 5 mg/kg (12% increase over controls), although the 0.2 and 2 mg/kg doses achieved similar regeneration rates. In contrast, intermediate doses (0.5 and 1 mg/kg) and a higher dose (10 mg/kg) were not different from controls. Calcitonin gene-related peptide immunohistochemical labeling of regenerating axons yielded similar results to those found with the pinch test. Based on our finding of a double peak in the dose-response for FK506, it is hypothesized that at least two mechanisms of action (perhaps corresponding to distinct functional binding sites) are evoked at different concentrations of the drug to accelerate nerve regeneration. These results have clinical implications for the pharmacological treatment of nerve injuries while avoiding immunosuppressive effects and for the design of related drugs with more specific activities.