Elsevier

Experimental Neurology

Volume 183, Issue 1, September 2003, Pages 220-231
Experimental Neurology

Regular article
FK506 enhances reinnervation by regeneration and by collateral sprouting of peripheral nerve fibers

https://doi.org/10.1016/S0014-4886(03)00173-0Get rights and content

Abstract

We examined the effects of FK506 administration on the degree of target reinnervation by regenerating axons (following sciatic nerve crush) and by collateral sprouts of the intact saphenous nerve (after sciatic nerve resection) in the mouse. FK506-treated animals received either 0.2 or 5 mg/kg/day, dosages previously found to maximally increase the rate of axonal regeneration in the mouse. Functional reinnervation of motor, sensory, and sweating activities was assessed by noninvasive methods in the hind paw over a 1-month period following lesion. Morphometric analysis of the regenerated nerves and immunohistochemical labeling of the paw pads were performed at the end of follow-up. In the sciatic nerve crush model, FK506 administration shortened the time until target reinnervation and increased the degree of functional and morphological reinnervation achieved. The recovery achieved by regeneration was greater overall with the 5 mg/kg dose than with the dose of 0.2 mg/kg of FK506. In the collateral sprouting model, reinnervation by nociceptive and sudomotor axons was enhanced by FK506. Here, the field expansion followed a faster course between 4 and 14 days in FK506-treated animals. In regard to dose, while collateral sprouting of nociceptive axons was similarly increased at both dosages (0.2 and 5 mg/kg), sprouting of sympathetic axons was more extensive at the high dose. This suggests that the efficacy of FK506 varies between subtypes of neurons. Taken together, our findings indicate that, in addition to an effect on rate of axonal elongation, FK506 improves functional recovery of denervated targets by increasing both regenerative and collateral reinnervation.

Introduction

FK506 is an immunosuppressant drug widely used to prevent rejection after solid organ transplantation. FK506 and its nonimmunosuppressant derivatives also exert neuroprotective and neuroregenerative activities (for reviews, see Gold 1999, Gold 2000a, Gold 2000b, Snyder et al 1998. Several experimental studies have shown that administration of FK506 increases the rate of axonal regeneration after axotomy induced by nerve crush Gold et al 1995, Gold et al 1994, Lee et al 2000, Wang et al 1997 or after nerve transection and suture or graft repair Büttemeyer et al 1995, Doolabh and Mackinnon 1999, Fansa et al 1999, Navarro et al 2001. Functional recovery of distal targets, measured by the walking track or by electrophysiological methods, starts earlier in rats and mice treated with FK506, although ultimate levels of recovery are close to those of untreated animals Doolabh and Mackinnon 1999, Fansa et al 1999, Navarro et al 2001. In patients with a hand allograft where FK506 was used to prevent rejection, recovery of sensorimotor functions was faster than expected, this being attributed to an enhancement of axonal regeneration by FK506 Dubernard et al 1999, Jones et al 2000. However, after severe nerve injuries in larger species, including humans, accelerating target reinnervation may be more important for reducing the consequences of denervation (muscle atrophy, loss of sensory receptors, denervation hypersensitivity) (Fu and Gordon, 1995). In this context, the ability of FK506 to increase nerve regeneration following a chronic axotomy (Sulaiman et al., 2002) may be of importance for improving the chances for functional recovery. However, studies on the effects of FK506 on target reinnervation are lacking using any of these models. Furthermore, while FK506 is presently being used in human hand transplantations Dubernard et al 1999, Jones et al 2000 and allograft nerve repairs (Mackinnon et al., 2001), the dose–response needs to be characterized in multiple models to determine its optimal dose for use in clinical practice.

We recently examined the dose dependency for FK506 on the rate of nerve regeneration in the mouse and found a novel bimodal response (Udina et al., 2002). The largest increase in regeneration rate was achieved at a dose of 5 mg/kg, but doses of 0.2 and 2 mg/kg yielded a similar regeneration course through the first week following sciatic nerve crush. In contrast, results with intermediate doses of 0.5 and 1 mg/kg were not different from those of controls. In the rat, the 5 mg/kg dose also maximally accelerates axonal regeneration (Wang et al., 1997), while the 1 mg/kg was less effective; lower dosages were not examined. The efficacy of lower dosages has obvious implications for clinical use for nerve regeneration since low doses (e.g., 0.2 mg/kg) would have less risk of toxicity and would presumably not exhibit immunosuppressant activity Spencer et al 1997, Undre et al 1999.

In the present study, we examined the effects of FK506 on both the speed of axonal elongation and the degree of target reinnervation. Reinnervation of target tissue is achieved by either axonal regeneration from injured nerves or collateral sprouting from intact neurons. In regard to collateral sprouting, one immunophilin ligand (V-10,367) has been shown to increase neurite branching in vitro (Constantini and Isacson, 2000). Therefore, we also investigated the capabilities of FK506 to promote collateral sprouting in vivo. Thus, two models were used to study the effect of FK506 on target reinnervation: nerve regeneration following sciatic nerve crush and collateral sprouting following partial denervation of the mouse hind paw. In both models, we assessed recovery of different targets over a 1-month period in mice receiving FK506 at either 0.2 or 5 mg/kg/day, the two doses previously found as most effective (Udina et al., 2002).

Section snippets

Surgical procedures

Operations were performed under pentobarbital anesthesia (60 mg/kg ip) in 3-month-old female OF1 mice. For nerve regeneration assessment, the sciatic nerve was exposed at the mid thigh and crushed three times in succession with a Dumont No. 5 forceps at a constant point, 45 mm from the tip of the third digit. The saphenous nerve was also cut in the femoral space and a long segment of the distal stump removed to prevent regeneration. The wound was then sutured by layers and disinfected with

Effects of FK506 on nerve regeneration after sciatic nerve crush

Functional tests performed at 7 dpo demonstrated that all nerve-mediated functions were abolished in the denervated hind paw of all animals. The first CMAPs evoked by sciatic nerve stimulation and recorded from plantar muscles reappeared at 15 dpo in most mice and at 18 dpo in two mice in group C. During the following weeks, the latency of the CMAP shortened to close to normal (data not shown) and the amplitude progressively increased to reach mean final values (relative to preoperative values)

Discussion

The present study reveals that FK506 administration enhances the reinnervation of target organs both by regenerating axons following a nerve crush and by collateral sprouts of an intact nerve induced to expand following denervation of its nearby territory. The recovery achieved by regeneration and by collateral sprouting was greater overall with the 5 mg/kg dose than with the dose of 0.2 mg/kg of FK506. Nevertheless, the 0.2 mg/kg dose still produced better results than those found in

Acknowledgements

This work was supported by grants from the Fondo de Investigación Sanitaria (FIS00-0031-02), the Ministerio de Ciencia y Tecnología (SAF2002-04016), and the Acadèmia de Ciències Mèdiques de Catalunya i Balears, Spain. We thank Fujisawa Pharmaceuticals Inc. (Osaka, Japan) for its generous gift of FK506 and the Kerr Company (Romulus, MI) for providing Elasticon. The technical help of Jessica Jaramillo is greatly acknowledged.

References (61)

  • L. Mohiuddin et al.

    Focally administered nerve growth factor suppresses molecular regenerative responses of axotomized peripheral afferents in rats

    Neuroscience

    (1999)
  • X. Navarro et al.

    Effect of age on collateral reinnervation of sweat glands in the mouse

    Brain Res.

    (1988)
  • X. Navarro et al.

    Comparison of the regenerating and reinnervating capabilities of different functional types of nerve fibers

    Exp. Neurol.

    (1994)
  • S.H. Snyder et al.

    Immunophilins in the nervous system

    Neuron

    (1998)
  • O.A.R. Sulaiman et al.

    FK506 increases peripheral nerve regeneration after chronic axotomy but not after chronic Schwann cell denervation

    Exp. Neurol.

    (2002)
  • K. Torigoe

    Distribution of motor nerve sproutings in the mouse gastroenemius muscle after partial denervation

    Brain Res.

    (1985)
  • R.J. Thompson et al.

    PGP 9.5—a new marker for vertebrate neurons and neuroendocrine cells

    Brain Res.

    (1983)
  • N.A. Undre et al.

    Pharmacokinetics of Tacrolimusclinically relevant aspects

    Transplant. Proc.

    (1999)
  • E. Verdú et al.

    Comparison of immunohistochemical and functional reinnervation of skin and muscle after peripheral nerve injury

    Exp. Neurol.

    (1997)
  • J.J. Vilches et al.

    Changes in mouse sudomotor function and sweat gland innervation with aging

    Auton. Neurosci.

    (2002)
  • M.A. Bisby et al.

    GAP-43 mRNA in mouse motoneurons undergoing axonal sprouting in response to muscle paralysis or partial denervation

    Eur. J. Neurosci.

    (1996)
  • B. Bjerre et al.

    Axonal regeneration of peripheral adrenergic neuronseffects of anti-serum to nerve growth factor in mouse

    Cell Tissue Res.

    (1974)
  • A. Brenan et al.

    The demonstration of the cutaneous distribution of saphenous C-fibres using a plasma extravasation technique in the normal rat and following nerve injury

    J. Anat.

    (1988)
  • M.C. Brown et al.

    Nodal and terminal sprouting from motor nerves in fast and slow muscles in the mouse

    J. Physiol.

    (1980)
  • R. Büttemeyer et al.

    Peripheral nerve allograft transplantation with FK506functional, histological, and immunological results before and after discontinuation of immunosuppression

    Ann. Plast. Surg.

    (1995)
  • P. Caroni

    Intrinsic neuronal determinants that promote axonal sprouting and elongation

    Bioessays

    (1997)
  • L.C. Constantini et al.

    Immunophilin ligands and GDNF enhance neurite branching or elongation from developing dopamine neurons in culture

    Exp. Neurol.

    (2000)
  • M. Devor et al.

    Two modes of cutaneous reinnervation following periperal nerve injury

    J. Comp. Neurol.

    (1979)
  • J. Diamond et al.

    Sensory nerves in adult rats regenerate and restore function to the skin independently of endogenous NGF

    J. Neurosci.

    (1992)
  • J. Diamond et al.

    Endogenous NGF and nerve impulses regulate the collateral sprouting of sensory axons in the skin of the adult rat

    J. Neurosci.

    (1992)
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