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Old September 24th, 2001, 21:58
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1999.11.15 Neurotrophic effect of FK 506 on peripheral nerve regeneration

by Pramod K. Nelluri, MD*, Robert Lyons*, and Uli K. Chettipally, MD, MPH, FACP+.

* From the Division of Plastic and Reconstructive Surgery, University of Texas, Health Science Center at San Antonio, Texas, and the + Department of Emergency Medicine, Kaiser Permanente Medical Center, San Francisco, California.


[abstract] [introduction] [methods] [results] [discussion] [conclusion] [references]

ABSTRACT

Traumatic nerve injuries of the upper extremity seldom regain full pre - injury level of function. This poor functional recovery can be attributed to irreversible distal muscle atrophy due to prolonged denervation. The new immunosuppressive agent FK 506 has been experimentally shown to enhance axonal regeneration and shorten the time to functional recovery. We conducted a systematic review of randomized controlled trials of FK 506 experimental studies in rat nerve injury models to evaluate and determine the significance of the results. Methods: A search of MEDLINE databases, PUBMED and OVID of English-language articles published from 1990 to 1999 was conducted using the terms ‘FK 506," "Tacrolimus," FKBP-12 ligands," and "Peripheral nerve regeneration." A manual search was also conducted. Six randomized controlled trials, four using the rat sciatic nerve crush model and two using a nerve graft model, were identified. Two reviewers assessed the trials independently. Results: Morphometric measurements such as mean axonal area, axonal density and number of nerve fibers noted more than 50% increase in all experimental animals when compared to controls. In one study using a minimum dose of 1 mg/kg body weight, the density of nerve fibers increased by 175% in experimental animals compared to controls. Functional recovery, which was analysed by walking track methods, showed a two day earlier recovery in experimental animals. All the studies showed statistically significant results, except for one study. Conclusions: FK 506 has been experimentally proven to be a potent nerve growth stimulator. However, additional well designed studies with blinded outcome assessment in larger animal models where axonal growth has to travel a greater distance from its proximal injury level would give further credibility to our conclusion. Given the multiple side effects and known complications of FK 506, seen in both humans and rats, how much these statistically significant results can be clinically beneficial might be answered by further studies focusing on risk/benefit ratios. Nelluri PK, Lyons R, Chettipally UK. Neurotrophic effect of FK 506 on peripheral nerve regeneration – a systematic review of controlled trials and analysis. Internet Medical Journal 15-November-1999;3(20). http://www.medjournal.com

INTRODUCTION

Peripheral nerve damage, such as that caused by trauma, cannot always be repaired primarily and often needs nerve grafting. Patients suffering from these injuries are often devastated and rarely return to pre-injury employment or avocations. Treatments which enhance the rate and/or degree of axonal recovery across a peripheral nerve injury and/or gap and yield superior clinical outcomes, will result in less patient suffering, disability, fewer lost days of work. The past decade has seen numerous experiments to identify neutrotrophic factors such as nerve growth factor (NGF), alpha melanocyte –stimulating hormone (a-MSH) and corticotropin (ACTH), among others that influence the direction and maturation of the regenerating nerve (1-7). FK 506 (tacrolimus), isolated from streptomyces tsukubaensis (8,9), and widely used as an immunosuppressant, is an agent recently demonstrated to enhance axonal regeneration in rats (10). In addition, in vitro studies have shown this agent to induce neurite outgrowth in spinal sensory neurons (11). This study utilizes a systematic review of all randomized controlled trials using FK 506 on rat sciatic nerve models to assess the results of these experiments and summarize the available evidence.

METHODS

[Literature Search] [Study Selection] [Analysis of Studies] Literature Search A computer search of MEDLINE databases PUBMED and OVID using the following MeSH headings was done: "FK 506," "nerve regeneration," "tacrolimus," "FKBP-12," "peripheral nerve regeneration" and "nerve growth factors (NGF)." The search was limited to English-language publications on experimental rat models. A manual search was then conducted of relevant text books, abstracts and appropriate citations. Unpublished trials were not reviewed.Study Selection (Table 1)Inclusion criteria were: 1) prospective randomized trials on experimental rat sciatic nerve models evaluating the effect of FK 506 and 2) outcome measures: a) walking track analysis and b) morphometric analysis. Studies done with allografts and xenografts were excluded due to their potential variability as confounders. Six studies were identified, (Table 1). Four utilized rat sciatic nerve crush models and two used nerve graft models. All the trials were evaluated by two reviewers, and any differences were resolved by consensus. An attempt was made to conduct the evaluation in the manner proposed by Detsky et al (12) even though this study was purely based on trials conducted on rats.Analysis of Studies Our question was, does FK 506 significantly enhance axonal regeneration in experimental animals. The research hypothesis is that regardless of the mechanism, FK 506 enhances morphologic and functional recovery in rats with damaged sciatic nerves. Studies 1,2,and 3, which are from a single center, used a sciatic nerve crush model and evaluated the morphometric measures at 18 days (10,13,14). In addition, in study 3, different doses of the drug administered subcutaneously (s.c.) were compared to evaluate their effect. In all these studies, the animals were sacrificed on day 18 and the histological evaluation was performed in the soleus nerve distal to the area of nerve crush. Study 4 (15), which utilized the same model with the same duration of observation, however, harvested the crush site for their morphometric measurements. Moreover, the route of the drug administration in this study was local application rather than systemic in contrast to the other trials reviewed. The remaining two studies (16,17), created a nerve gap model and while study 5 inserted a posterior tibial nerve isograft, study 6 used the same nerve segment orthotopically. The route of drug administration in study 5 was intraperitoneal. An average of 3 - 5 animals per group were studied in the first 4 studies as compared to the larger sample sizes of 8 –15 in the latter two studies. Again, there was some overlapping of animals in different groups in the first three studies.

RESULTS (Table 2)

Though the outcome measures examined by the above studies were the same, namely, walking track and morphometric analytic methods, the units of measurement differed in all the studies. In view of the data in studies 5 and 6 summarized as histograms, we calculated a rough estimate in these studies. Study 1 which examined the density of nerve fibers/5000m m2 showed a substantial 175% increase in the FK 506 group when compared to the control group. Similar measures shown in study 5 as fibers/mm2 were about 290% more nerve fibers in the experimental group than controls. Axonal diameter and the axonal cross sectional area increased more than 50% in all of the studies.(Table 2). Study 1 demonstrated a two day earlier functional recovery in the drug treated group when compared to the controls, whereas study 5 examining the full functional recovery observed a difference of one week in favor of the FK 506 group. The histologic and walking track parameters observed in all the above studies were statistically significant except in study 5.

DISCUSSION

Complete restoration of function following peripheral nerve injury is not yet a reality. The reason for such permanent deficits, is irreversible muscle atrophy due to prolonged denervation. Current research in neurobiology focuses on the identification and manipulation of the biochemical and cellular microenvironment that influences nerve regeneration. FK 506 is a potent immunosuppressant drug. Its new therapeutic value in regeneration of axonal growth was first demonstrated in a rat model by Gold et al in 1994 (10). Since then there have been several studies using FK 506 in evaluating the functional recovery and enhanced histologic regeneration in the experimental rat model. In our systematic review of these studies, all demonstrated a uniform enhancement of nerve regeneration in drug induced animals when compared to the controls. Study 3, (Table 1) which compared three different doses evaluating nerve regeneration, observed a maximum beneficial effect in the 5 mg group when compared to either 2mg or 10mg groups. Similar enhanced morphometric effects and functional results were shown in study 5 with 1mg/kg given intraperitoneally. A comparative dose dependence study in rats with 1mg/kg and 10mg/kg doses, for 14 days, by Sakai et al, observed high mortality in all the animals in the higher dose group (18). In another study, evaluating the nephrotoxicity of FK 506 in rats, doses as low as 0.5 to 0.8 mg/kg/day showed significant changes in renal function at 4 weeks (19). However, some encouraging results were shown by Study 6, using as little as 0.3mg - 0.6mg s.c. This study showed an increase of about 76% in axon counts measured per 10,000 m m2 at 2 weeks. An interesting but intriguing observation noted in both studies 5 and 6 was that the beneficial effects demonstrated early on at 7 and 2 weeks  were absent at 10.5 and 6 weeks respectively. Whether this phenomenon is related to the neurotoxicity or peripheral neuropathy of FK 506 observed in some clinical studies remains to be seen(20,21). Similarly, study 5 which used a larger sample size of 15 and 10 animals in the respective groups observed enhancement of axonal growth at 7 weeks but did not show statistically significant results in terms of total number of fibers and mean axonal areas. We feel, the tibial nerve graft used in this case could have been a possible confounder for the difference in the results. Studies 1,2 and 3 which used onset of movement and actual walking to measure the functional recovery in the lesioned limb, showed earlier walking by two days in the experimental group when compared to the controls. Additional studies are needed to improve upon this modest difference which may not be clinically significant as noted by the authors themselves in study 1. Similar early functional recovery was shown in study 5 as well, but how these statistically significant results can be clinically beneficial is a question to be answered. While all of this evidence offers considerable encouragement in the evolution of an ideal neurotrophic agent, multiple side effects and complications of immunosuppressive agents noted in rats and humans (22,23) require additional studies showing meaningful and beneficial results in favour of nerve regeneration. Lastly, study 4, which used the local route for drug application is another useful technique to avoid the systemic effects of the drug.

CONCLUSION

Experimental evidence in the rat sciatic nerve model shows a beneficial effect of FK 506 in terms of earlier functional recovery and superior histological parameters of fiber maturation and axonal regeneration. However, the question that arises in the case of peripheral nerve regeneration is whether the risks of toxic effects of FK 506 are acceptable. Although it may be the case that the inference resulting from the above experiments--- that there is a real effect of the agent--- is statistically significant, it is our opinion that a truly convincing case for or against the use of FK 506 in nerve repair cannot be made unless additional studies show risk/ benefit ratios. Studies showing equivalent effects with FK 506 analogues, which lack immunosuppressive effect, are an attractive proposition, as are studies using the local route for drug application. It is our suggestion that additional, well-designed, randomized -controlled trials in larger animals where the distance a regenerating axon has to travel is greater from the proximal nerve injury would provide beneficial information. Lastly, trials of longer duration focussing on risk/ benefit ratio and blinded outcome assessment would lend further credibility to the conclusion that FK 506 and its analogues enhance nerve regeneration.  

REFERENCES

  1. Varon S, Manthorpe M, Williams LR. Neuronotrophic and neurite-promoting factors and their clinical potentials. Dev Neurosci 1983;6:73-100
  2. Landor AD. Molecules that make axons grow. Mol Neurobiol 1987;1:213-45.
  3. Bijlsma, WA, Jennekens FG, Schotman P, Gispen WH. Effects of corticotropin(ACTH) on recovery of sensorimotor function in the rat: structure activity study. Euro J Pharmacol 1981;76:73-9.
  4. Bijlsma WA, Jennekens FG, Schotman P,Gispen WH, Neurotrophic factors and regeneration in the peripheral nervous system. Psychoneuroendocrinolgy 1984;9:199-215.
  5. De Koning P, Gispen WH. Rationale for the use of Melanocortins in Neural Damage. In DG Stein and B Sabel (Ed) Pharmacological approaches to the treatment of Brain and Spinal Cord Injury. Plenum Press, New York, 1987;233-58.
  6. Strand FL and Kung TT. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1980:1;135-8.
  7. Strand FL, Rose KJ, King JA, Segarra AC and Zuccarelli LA, ACTH modulation of nerve development and regeneration. Prog Neurobiol 1989;33:45-85.
  8. Kino T, Hatanaka H, Hashimoto M, et al. FK 506 , a novel immunosuppressant isolated from streptomyces I, Fermentation, isolation and physiochemical and biological characteristics. J Antibiotics 1987;40:1249-55.
  9. Kino T, Hatanaka H, Miyata S, et al. FK 506 , a novel immunosuppressant isolated from streptomyces II. Immunosuppressive effect of FK 506 in vitro. J Antibiotics 1987;40:1256-65.
  10. Gold BG, Dickerson TS, Austin DR. The immunosuppressant FK 506 increases functional recovery and nerve regeneration following peripheral nerve injury. Restor Neurol Neurosci 1994;6:287-96.
  11. Lyons WE, George EB, Dawson TM , Steiner JP, Snyder SH. Immunosuppressant FK 506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia. Proc Natl Acad Sci USA 1994;91:3191-5.
  12. Detsky AS, Naylor CD, O’Rourke K, McGeer A, L’Abbe KA. Incorporating variations in the quality of individual randomized trials into meta-analysis. J Clin Epidemiol 1992;45:255-65.
  13. Gold BG, Katoh K, Dickerson TS. The immunosuppressant FK 506 Increases the rate of axonal regeneration in rat sciatic nerve. The J of Neurosci 1995;15(11):7509-16.
  14. Wang MS, Pooley MZ, Gold BG. Comapritive dose dependence study of FK 506 and Cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve. J Pharm Exper Therap 1997;282(2):1084-97.
  15. Steiner JP, Connolly MA, Valentine HL, et al. Neurotropic actions of nonimmunosuppressive analogues dgrugs FK 506, rapamycin,and cyclosporin. A Nature Medicine. 1997;3(4):421-8.
  16. Doolabh VB, Mackinon SE. FK 506 accelerates functional recovery following nerve grafting in a rat model. Plast Reconstr Surg 1999;103(7):1928-36.
  17. Fansa H, Keilhoff S, Plogmeier K, Wolf G, Schneider W. The effect of the immunosuppressant FK 506 on peripheral nerve regeneration following nerve grafting. J Hand Surg (British & European) 1999;24B:1:38-42.
  18. Sakai K, Date I, Yoshimoto Y, et al. The effect of a new immunosuppressant agent , FK 506 on xenogeneic neural transplantation in rodents. Brain Res 1991;565:167-70.
  19. Nielson FT, Leyssac PP, Kemp E, Starklint H, Dieperink H. Nephrotoxicity of FK 506 in the rat. Studies on glomerular and tubular function and on the relationship between efficacy and toxicity. Nephrol Dial Transplant. 1995;10:334-40.
  20. Mizisin AP, Powell HC. Toxic neuropathies.Current opinion in Neurology 1995;8:367-71.
  21. Ayers RCS, Dousset B, Wixon S, et al. Peripheral neurotoxicity with tacrolimus. Lancet 1994;343:862-3.
  22. Shapiro R, Fung JJ, Jain AB, Parks P, Todo S, Strazl TE, The side effects of FK 506 in humans. Transplant Proc 1990;22:35-6.
  23. Green MD, Michaels MG. Tacrolimus: effects and side effects. Pediatr Infect Dis J 1999;18(4):372-3.

Table 1 SUMMARY OF TRIALS
STUDIES MODEL DOSE ROUTE n= *
1. Gold et al (10) Sciatic Nerve Crush 1.0 mg/kg s.c. 5
2. Gold et al (13) Sciatic Nerve Crush 1.0 mg/kg s.c. v**
3. Wang et al (14) Sciatic Nerve Crush 2, 5, 10 mg/kg s.c. 3/5 +
4. Steiner et al (15) Sciatic Nerve Crush 1.0 mg/kg local 5
5. Doolabh et al (16) Tibial Nerve Isocrush 1.0 mg/kg i.p. 15/10
6. Fansa et al (17) Autograft 0.3, 0.6 mg/kg s.c. 8

*number of rats in each group, **variable number of rats in each group, + control/experimental, s.c. = subcutaneous, i.p. = intraperitoneal

Table 2 SUMMARY OF THE RESULTS

STUDY YEAR MA * E/C ** % INCREASE
1 1994 DNF 22/8 fibres/5000 µm2 175
2 1995 MAA (T) 1.0/0.6 µm2
(L) 2.7/1.4 µm2
66.66
92
3 1997  MAA (2 mg) 1.1/0.6 µm2
(5 mg) 1.3/0.6 µm2
(10 mg) 1.2/0.6 µm2
88.33
116.66
100
4 1995 AD
MAA
1.99/1.56 µm2
4.16/2.74 µm2
21.78
51.82
5 f 1999 % FIBERS n/a 56.25
6 f 1999 AXC
M/A
n/a 76.92
2.5
*MA-morphometric measurements, **E/C- experimental /controls, (mg)-different doses, (T) total axonal area, (L)- larger sized axons, MAA-mean axonal area, DNF-density of nerve fibers, AD – axonal density, AXC- axonal count/10,000m m2, M/A – myelin/axon ratio. f - data summarized as histograms. 
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