CAN J OPHTHALMOL—VOL. 44, NO. 4, 2009 427

Effect of intrathecal FK506 administration on intraorbital optic nerve crush: an ultrastructural studyLevent Sarikcioglu,* PhD; Necdet Demir�§ PhD; Yusuf Akar‡ MD; Arife Demirtop§ BSc

ABSTRACT • RÉSUMÉ

Objective: Trauma to the optic nerve caused by fractures of the midface and (or) skull base has been simulated by an optic nerve crush injury model. Because the intraorbital segment of the optic nerve is surrounded by subarach-noidal cerebrospinal fluid and dura mater, we aimed to study the influence of intrathecal tacrolimus (FK506) admin-istration after optic nerve crush injury and to determine its role in optic nerve protection or sparing after injury.

Study Design: Experimental study.Participants: All optic nerves of the animals were included in the study.Methods: A total of 48 female Wistar rats were randomly divided into 4 groups (control, sham operated, FK506 treat-

ed, and vehicle treated). In vehicle- and FK506-treated groups, intrathecal catheter implantation and crush injury to the intraorbital part of the optic nerve were performed and then the animals were treated intrathecally. The optic nerve samples were harvested on the 30th postoperative day. Optic nerve appearances were analyzed qualitatively.

Results: Light and electron microscopic evaluations revealed that numerous damaged myelin residues were present in the vehicle-treated group, whereas fibres of the optic nerve showed a well-shaped appearance in the FK506-treated group.

Conclusion: We propose that such an intrathecal administration route and small-dose regimen should be used to obtain lesser immunosuppression and neurotoxicity and higher protection or sparing after injury.

Objet : Simulation d’un traumatisme du nerf optique sous forme d’écrasement, causé par des fractures de la partie moyenne du massif facial et (ou) à la base du crâne. Comme le segment intraorbitaire du nerf optique est entouré de fluide cérébrospinal sous-arachnoïdien et de dure-mère, l’étude devait porter sur l’influence de l’administration de tracolimus intrathécal (FK506) après l’écrasement du nerf optique et en déterminer le rôle dans la protection ou l’épargne du nerf optique après la blessure.

Nature : Étude expérimentale.Participants : Tous les nerfs optiques des animaux compris dans l’étude.Méthodes : En tout, 48 rates Wistar ont été réparties au hasard en 4 groupes (témoin, pseudo-opéré, traité

au FK506 et traité avec un véhicule). Pour les groupes traités au FK506 et avec un véhiucule, on a pratiqué l’implantation d’un cathétère intrathécal. La blessure d’écrasement dans la partie intraorbitaire du nerf optique a ensuite été produite et, par la suite, les animaux ont été soignés intrathécalement. Les échantillons de nerf optique ont été recueillis le 30e jour après l’opération et on a analysé l’apparence des nerfs optiques.

Résultats : Les évaluations par microscope à la lumière ou électronique ont révélé que de nombreux résidus de myéline endommagée se trouvaient dans le groupe traité avec un véhicule, alors que les fibres du nerf optique semblaient bien formées chez le groupe traité au FK506.

Conclusions : Nous proposons qu’on devrait avoir recours à l’administration intrathécale et au régime à petites doses pour obtenir moins d’immunosuppression et de neurotoxicité et plus de protection ou d’épargne après la blessure.

FK506 (tacrolimus) is a hydrophobic macrocyclic lactone isolated from a fermentation broth of Streptomyces tsuku-

baensis from Mount Tsukuba in northern Japan.1,2 FK506 is a potent immunosuppressant and promotes graft survival in organ transplantation. It has a mechanism of action simi-lar to that of cyclosporin A, but is more potent.3 The use of FK506 is of special interest in ophthalmology, because it may be effective in the treatment of immune-mediated dis-eases, such as corneal graft rejection, keratitis, scleritis, ocular pemphigoid, uveitis, and ocular Behçet disease.4 Although an increased rate of axonal regeneration has been noted in the central and peripheral nervous system, FK506 has some adverse effects, such as nephrotoxicity, hypertension,

hyperesthesia, muscular weakness, insomnia, tremor, photo-phobia, gastrointestinal symptoms, and central ner vous system alterations.4–8 The incidence of side effects may be in-creased when FK506 is administered systemically.4 Further-more, systemically administered drugs penetrate poorly into the intraocular tissues because of the blood– ocular barrier. To avoid systemic side effects, topical application of an agent is preferred to the oral and intravenous routes.4

The optic nerve of the rat is a very vulnerable structure. Trauma to the optic nerve caused by fractures of the mid-face and (or) skull base has been simulated by an optic nerve crush injury model.9 The optic nerve, ontogenetically an outgrowth of the brain, is surrounded by subarachnoid-

From *the Department of Anatomy, †the Department of Histology and Embryology, ‡the Department of Ophthalmology, and §the Electron Microscopy Unit (TEMGA), Akdeniz University Faculty of Medicine, Antalya, Turkey

Originally received Aug. 12, 2008. Revised Oct. 16, 2008Accepted for publication Jan. 1, 2009Published online July 13, 2009

Correspondence to Levent Sarikcioglu, PhD, Department of Anatomy, Akdeniz University Faculty of Medicine, 07070 Antalya, Turkey; sarikcioglul@yahoo.com

This article has been peer-reviewed. Cet article a été évalué par les pairs.

Can J Ophthalmol 2009;44:427–30doi:10.3129/i09-071

Intrathecal FK506 administration—Sarikcioglu et al.

428 CAN J OPHTHALMOL—VOL. 44, NO. 4, 2009

al cerebrospinal fluid and dura mater.10 We hypothesized that a small dose of FK506 should be beneficial to the op-tic nerve recovery because of its administration route. For this reason, we aimed to study the influence of intrathecal FK506 administration after optic nerve crush injury and to determine its effects on optic nerve crush from an ultra-structural point of view.

Methods

AnimalsA total of 48 female Wistar rats (200–250 g) were ran-

domly divided into 4 groups (control, sham operated, FK506 treated, and vehicle treated). The animals were housed in Makrolon cages (5 per cage) and maintained on a 12-hour light–dark cycle. Food and water were provided ad libitum. All procedures were reviewed and approved by the Animal Care and Use Committee of Akdeniz University.

Catheter implantationBefore all surgical operations, the animals were anes-

thetized with intramuscular injection of the mixture of xylazine HCl (10 mg/kg) and ketamine (80 mg/kg). The surgical area was shaved, and swabbed with an antiseptic solution. One longitudinal cutaneous incision was made over the dorsal midline. A small part of the muscles located between the spinous and transverse processes was retracted. The L4-5 or L5-6 vertebral level was adjusted according to the level of the tip of the iliac crest. The insertion needle of a 24-gauge catheter (Neoflon) was pushed forward and the spinal canal was entered through the L4-5 or L5-6 inter-laminar space. During that time, the tail flick reflex was observed. The catheter was pushed in approximately 2 ver-tebral levels. The access port was placed subcutaneously. This implantation technique had been improved in a pilot study before the experimental studies. Additionally, the ver-tebral column of the animal was transected to ensure that the catheter was located in the spinal canal.

The following day, an experienced author (Levent Sarik-cioglu) examined the animals anatomically for impaired motor function to ensure that the catheter itself did not damage the spinal cord. Animals showing impaired motor function were excluded from the study.

Optic nerve crushCrush injury to the intraorbital part of the optic nerve

was performed 1 week after catheter implantation. During that time, the animals recovered from implantation surgery. Neurologically normal rats after catheter implantation were divided into 2 groups (FK506 treated and vehicle treated). These rats were anesthetized with a mixture of ketamine-xylazine and the intraorbital part of their optic nerves was ex-posed after a lateral canthotomy. Three millimetres from the globe, on the left side only, the optic nerve was crushed with a Yasargil aneurysm clip as described previously.11 A sham operation was performed in another group by exposing the

optic nerve, but not crushing it. Then, antibiotic ointment was applied and the animals were allowed to recover.

FK506 administrationFK506 (0.05 mg/kg/day dissolved in sterile saline) was

administered intrathecally from the day of the optic nerve crush to the day of animal sacrifice (postoperative day 30). FK506 administration was performed every day (including the weekends) at 10:00 am. The same volume of saline was administered to the vehicle-treated animals. Before FK506 administration, the catheter’s dead space was measured and then filled with saline. FK506 was administrated through the needle at the entry point.

Light and electron microscopic evaluationsThirty days after optic nerve crush, the animals received

an overdose of chloral hydrate intraperitoneally and were transcardially perfused with phosphate buffer solution. Each optic nerve was re-exposed in all groups. The optic nerve specimen was collected by cutting the optic nerve from the posterior of the crush site to the orbital orifice of the optic canal. Specimens were fixed with 4% glutaralde-hyde in 0.1 mol/L Sorensen’s phosphate buffer solution (pH 7.3) and postfixed with 2% osmium tetroxide in the same buffered solution, and, after dehydratation through a graded ethanol series, embedded in epoxy resin (Araldite CY212, Agar Scientific, Stansted, U.K.). Semithin sections obtained from the middle part of the optic nerve specimens were stained with toluidine blue and examined under a light microscope (Zeiss Axioplan, Carl Zeiss, Oberkochen, Germany). Then, ultrathin sections (40–60 nm) were con-trasted with uranyl acetate and lead citrate and examined with a Zeiss LEO 906E transmission electron microscope.

Results

Application of surgeriesCatheter implantation was the most difficult procedure

of the study. We started to perform the catheter implanta-tion on 24 animals, but in 7 animals, the catheter resulted in a neurological deficit. These animals were excluded from the study and new ones were included until the appropriate catheter implantation was achieved.

As we described in a previous study,11 we reached the optic nerve via the lateral approach. By lateral chantot-omy, a small incision was performed and the optic nerve was exposed, then it was crushed with a Yasargil aneurysm clip. Immediately after the acute compression injury, the crushed area of the optic nerve became very thin, but nerve continuity was not interrupted grossly. After both surgeries, all rats survived and no wound infections were detected.

Light and electron microscopic observationsThe optic nerve consists of both unmyelinated and my-

elinated nerve fibres. Neither myelin debris nor damaged fibres were detected in the control and sham-operated

Intrathecal FK506 administration—Sarikcioglu et al.

CAN J OPHTHALMOL—VOL. 44, NO. 4, 2009 429

groups in low magnification of the electron microscope. Sheaths around the optic nerve showed normal structure. We could not observe endoneural edema or damage in these groups. Myelinated and unmyelinated nerve fibres showed a well-shaped appearance and normal axoplasm (Figs. 1 and 2).

Severe damage was observed in the vehicle-treated group on the 30th postoperative day. Electron microscopic evalua-tions showed that numerous damaged myelin residues were present in the vehicle-treated group. Additionally, nerve crush resulted in severe degradation in the myelinated and unmyelinated fibres of the optic nerve. Normal myelinated fibres were destroyed and the fibres’ borders were not clear. There was an enormous amount of myelin debris and my-elin residue. Normal axon structure, axoplasm, neurofila-ments, and neurotubules could not be seen (Fig. 3).

However, light and electron microscopic observations revealed that a well-shaped appearance on the same post-operative day was present in the FK506-treated group (Figs. 1 and 3). Additionally, evaluation of the cross-section of the optic nerve specimens revealed that there were very small degenerative sites in the optic nerve. These sites located dif-fusely in the optic nerve (Fig. 1). Therefore, we observed less myelin debris in the cytoplasm in the FK506-treated group compared with the vehicle-treated group. Axonal structures, neurofilaments, and neurofibrils were clear and could be defined (Fig. 2).

ConClusions

In this study, we showed that intrathecal administra-tion of FK506 affected the optic nerve after injury of its intraorbital part. Different dosages of FK506 for the re-generation of various nerves have been reported in the literature.12–20 Although Gold21 reported that the optimal dose of FK506 was 5 mg/kg, there is no consensus on the optimal dose of FK506 to achieve the best regeneration

rate. The wide range of these dosages may be due to the administration route or to individual variability in absorp-tion, distribution, and elimination of FK506.

Intrathecal drug delivery places medication directly into the cerebrospinal fluid that surrounds the spinal cord. For instance, morphine delivered directly to the intrathecal space is particularly effective because it does not have to circulate systemically to reach the cerebrospinal fluid and the dorsal horn of the spinal cord. As a result, much smaller doses are needed (e.g., approximately 1/300 of an oral mor-phine dose), and the frequency of side effects is reduced.22,23 For this reason, we selected intrathecal administration of FK506 with a small dose. Compared with the dose (5 mg/kg)

Fig. 1—Light micrographs showing optic nerve appearances in con-trol (A), sham-operated (B), FK506-treated (C), and vehicle-treated (D) groups. White arrows indicate small myelinated fibres, black ar-rows indicate large myelinated fibres, and arrowheads indicate my-elin residues and degenerative sites.

Fig. 2—Electron micrographs showing optic nerve appearances in control and sham-operated groups. In both groups, optic nerve shows normal appearance. (A, B) Electron microscopic appearance of the optic nerve of the control group. (C, D) Electron microscopic appearance of the optic nerve of the sham-operated group. (M, mye-linated fibres.)

Fig. 3—Electron microscopic micrographs showing optic nerve ap-pearance in experimental groups. The optic nerve sections were taken 30 days after nerve crush. In the FK506-treated group, the optic nerve showed well-shaped myelinated fibres. However, in the vehicle-treated group, severe myelin debris was observed. (A, B) Electron microscopic appearance of the optic nerve of the FK506-treated group. (C, D) Electron microscopic appearance of the optic nerve of the vehicle-treated group. White arrows indicate myelin residues. (M, myelinated fibres.)

Intrathecal FK506 administration—Sarikcioglu et al.

430 CAN J OPHTHALMOL—VOL. 44, NO. 4, 2009

proposed by Gold,21 we used a 100-times smaller dose for intrathecal administration.

Gillon et al.24 reported that FK506 did not promote more axonal regeneration into the peripheral nerve graft but did reduce inflammation while not reducing the number of re-generated axons of the retinal ganglion cell, in comparison with the findings in sham-injected animals. In the present study, we showed a protective effect of the FK506 by quali-tative electron microscopic evaluations. We think that fur-ther studies should be performed using specific tracers or intrinsic markers specific to regenerated retinal ganglion cell axons to evaluate the effect of FK506 on axonal re-generation in the optic nerve.

Topical,25,26 intravitreal,26,27 intramuscular,26 subcuta-neous,12 and oral18 application of FK506 have been reported in the literature. Intrathecal administration has not, to our knowledge, been investigated in ocular diseases. In our previ-ous report, intrathecal administration of the FK506 showed beneficial effects to the sciatic nerve recovery.28 It might be possible that FK506 treatment avoids the loss of myelin and therefore plays a protective or sparing role after injury. Be-cause the daily dose of FK506 reported in the literature is very high, we used a small dose (0.05 mg/kg) of FK506. We propose that the intrathecal administration route and dose regimen should be used to obtain lesser immunosup-pression and neurotoxicity and higher protection or sparing after injury.

This study was supported by the Akdeniz University Research Fund. The authors have no proprietary or commercial interest in any materials discussed in this article.

RefeRenCes

1. Kino T, Hatanaka H, Miyata S, et al. FK-506, a novel immuno-suppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiot (Tokyo) 1987;40:1256–65.

2. Goto T, Kino T, Hatanaka H, et al. Discovery of FK-506, a novel immunosuppressant isolated from Streptomyces tsuku-baensis. Transplant Proc 1987;19:4–8.

3. Kawashima H, Fujino Y, Mochizuki M. Effects of a new immuno-suppressive agent, FK506, on experimental autoimmune uveo-retinitis in rats. Invest Ophthalmol Vis Sci 1988;29:1265–71.

4. Sakurai E, Nozaki M, Okabe K, Kunou N, Kimura H, Ogura Y. Scleral plug of biodegradable polymers containing tacrolimus (FK506) for experimental uveitis. Invest Ophthalmol Vis Sci 2003;44:4845–52.

5. Brazis PW, Spivey JR, Bolling JP, Steers JL. A case of bilateral optic neuropathy in a patient on tacrolimus (FK506) therapy after liver transplantation. Am J Ophthalmol 2000;129:536–8.

6. Lake DB, Poole TR. Tacrolimus. Br J Ophthalmol 2003;87:121–2.

7. Mochizuki M, Masuda K, Sakane T, et al. A clinical trial of FK506 in refractory uveitis. Am J Ophthalmol 1993;115:763–9.

8. Sloper CM, Powell RJ, Dua HS. Tacrolimus (FK506) in the treatment of posterior uveitis refractory to cyclosporine. Oph-thalmology 1999;106:723–8.

9. Gellrich NC, Schimming R, Zerfowski M, Eysel UT. Quantifi-cation of histological changes after calibrated crush of the intra-orbital optic nerve in rats. Br J Ophthalmol 2002;86:233–7.

10. Berry M, Bannister LH, Starding SM. Nervous system. In: Wil-liams PLT, Bannister LH, Berry M, et al., eds. Gray’s Anatomy. New York, N.Y.: Churchill Livingstone; 1995:1225–6.

11. Sarikcioglu L, Demir N, Demirtop A. A standardized method to create optic nerve crush: Yasargil eneurysm clip. Exp Eye Res 2007;84:373–7.

12. Campbell G, Holt JK, Shotton HR, Anderson PN, Bavetta S, Lieberman AR. Spontaneous axonal regeneration after optic nerve injury in adult rat. Neuroreport 1999;10:3955–60.

13. Bavetta S, Hamlyn PJ, Burnstock G, Lieberman AR, Anderson PN. The effects of FK506 on dorsal column axons following spinal cord injury in adult rats: neuroprotection and local re-generation. Exp Neurol 1999;158:382–93.

14. Fansa H, Keilhoff G, Altmann S, Plogmeier K, Wolf G, Schneider W. The effect of the immunosuppressant FK 506 on peripheral nerve regeneration following nerve grafting. J Hand Surg [Br] 1999;24:38–42.

15. Feng FY, Ogden MA, Myckatyn TM, et al. FK506 rescues peripheral nerve allografts in acute rejection. J Neurotrauma 2001;18:217–29.

16. Freeman EE, Grosskreutz CL. The effects of FK506 on retinal ganglion cells after optic nerve crush. Invest Ophthalmol Vis Sci 2000;41:1111–5.

17. Gold BG, Katoh K, Storm-Dickerson T. The immunosuppres-sant FK506 increases the rate of axonal regeneration in rat sci-atic nerve. J Neurosci 1995;15:7509–16.

18. Grosskreutz CL, Hanninen VA, Pantcheva MB, Huang W, Poulin NR, Dobberfuhl AP. FK506 blocks activation of the intrinsic caspase cascade after optic nerve crush. Exp Eye Res 2005;80:681–6.

19. Jost SC, Doolabh VB, Mackinnon SE, Lee M, Hunter D. Ac-celeration of peripheral nerve regeneration following FK506 administration. Restor Neurol Neurosci 2000;17:39–44.

20. Wang MS, Zeleny-Pooley M, Gold BG. Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve. J Pharmacol Exp Ther 1997;282:1084–93.

21. Gold BG. FK506 and the role of immunophilins in nerve re-generation. Mol Neurobiol 1997;15:285–306.

22. Gustafsson LL, Wiesenfeld-Hallin Z. Spinal opioid analgesia. A critical update. Drugs 1988;35:597–603.

23. Lamer TJ. Treatment of cancer-related pain: when orally admin-istered medications fail. Mayo Clin Proc 1994;69:473–80.

24. Gillon RS, Cui Q, Dunlop SA, Harvey AR. Effects of immuno-suppression on regrowth of adult rat retinal ganglion cell axons into peripheral nerve allografts. J Neurosci Res 2003;74:524–32.

25. Okada K, Sakata H, Minamoto A, Fujihara M. Effect of FK 506 administered topically versus intramuscularly on suppres-sion of the corneal immune reaction in rats. Ophthalmologica 1996;210:175–9.

26. Akar Y, Yucel G, Durukan A, Yucel I, Arici G. Systemic toxicity of tacrolimus given by various routes and the response to dose reduction. Clin Experiment Ophthalmol 2005;33:53–9.

27. Rosenstiel P, Schramm P, Isenmann S, et al. Differential effects of immunophilin-ligands (FK506 and V-10,367) on survival and regeneration of rat retinal ganglion cells in vitro and after optic nerve crush in vivo. J Neurotrauma 2003;20:297–307.

28. Sarikcioglu L, Duygulu E, Aydin H, Gurer EI, Ozkan O, Tuz-uner S. Effect of intrathecal administration of FK506 after sci-atic nerve crush injury. J Reconstr Microsurg 2006;22:649–54.

Keywords: intrathecal administration, FK506, crush injury, optic nerve, protection