SHORT REPORT Key words: neuropathy Guillain-Barre syndrome experimental autoim- mune neuritis ciliary neurotrophic factor

MUSCLE & NERVE 19:1177-1180 1996

TREATMENT WITH CILIARY NEUROTROPHIC FACTOR DOES NOT IMPROVE REGENERATION IN EXPERIMENTAL AUTOIMMUNE NEURITIS OF THE LEWIS RAT RALF GOLD, MD, JURGEN ZIELASEK, MD, J. MICHAEL SCHRODER, MD, BERND SELLHAUS, MD, JESSE CEDARBAUM, MD,

KLAUS V. TOYKA, MD HANS-PETER HARTUNG, MD, MICHAEL SENDTNER, MD, and

Patients with Guillain-BarrC syndrome (GBS) may run a severe clinical course with profound axonal damage and permanent disabling ~ e a k n e s s . ~ Hence there is a need for therapies aiming at improved regeneration in the peripheral nervous system (PNS) . Pharmacological application of ciliary neuro- trophic factor (CNTF) has been shown to be highly effective in preventing the degeneration of newborn rat facial motoneurons after axotomy18 and to prevent congenital motoneuron degeneration in the pmn, wobbler, and mnd mouse ~ t r a ins .~~ '~~ ' ' Since CNTF is produced in myelinating Schwann cells3 we reasoned that pharmacological application of CNTF might be of benefit to maintain axon caliber and improve re- generation in AT-EAN (adoptive transfer experimen- tal autoimmune neuritis). This animal model resem- bles GBS in many aspect^.^ With a high T cell number transferred a severe and predominantly axonal dis- ease occurs.

From the Department of Neurology and Clinical Research Unit for Multiple Sclerosis (Drs. Gold, Zielasek, Hartung, and Toyka) Clinical Research Unit for Neuroregeneration (Dr Sendtner), Julius-Maximilians-Univer- sitat, Wurzburg. Germany: Department of Neuropathology, Rheinisch- Westfalisch Technische Hochschule, Aachen, Germany (Drs Schroder and Sellhaus); and Regeneron Pharmaceuticals Inc , Tarrytown, New York (Dr. Cedarbaum).

Dr. Cedarbaum is a full-time employee and shareholder in Regeneron Pharmaceuticals Inc.

Acknowledgments: We thank Dr. Wankmuller. Wurzburg. for measuring rat fibrinogen, and Dr. Dittrich, Wurzburg, for testing the biological activity of human recombinant CNTF. This work was supported in part by a grant from Regeneron Inc. and by the Deutsche Forschungsgemeinschaft (TO

Address reprint requests to Klaus V. Toyka, MD, Neurologische Universi- tatsklinik. Josef-Schneider-Strasse 11, 1397080 Wurzburg, Germany.

Accepted for publication March 31, 1996.

0 1996 John Wiley & Sons, Inc

6 1 /8-1).

CCC 0148-639X/96/091177-04

MATERIAL AND METHODS

Human recombinant CNTF12 and vehicle serving as placebo were provided by Regeneron Pharmaceuti- cals (Tarrytown, NY) and tested for activity before experimentation.

Female Lewis rats from the Zentralinstitut fur Ver- suchstierzucht (Hannover, Germany) were 8-10 weeks old and had a body weight of 180-200 g. All experiments were conducted according to Ba- varian state regulations for animal experimenta- tion.

Severe AT-EAN was induced by tail vein injection of 15 X l o6 P2-specific CD4positive activated T cells. These neuritogenic T cells were generated and main- tained as previously described in detail.'3 Animals were weighted and inspected daily for clinical signs. Disease severity was scored as described.6

In a first study, two groups of rats with 6 animals each were injected SC in the neck with 500 pg/kg CNTF or placebo every other day starting from day 4 after cell transfer. Detailed electrophysiological studies were performed as described.'

After 42 days 3 animals per group were perfused transcardially with 2.5% glutaraldehyde following standard protocols.23 For morphometric evaluation of nerve fibers, segments with optimal preservation and cross-sectional orientation were selected to mea- sure the diameters of the 10 largest regenerated nerve fibers. These sectors were photographed with the 40X objective lense (planapochromate) using an Olympus BH2 at a final magnification of 835X.

In a second experiment, three groups of 6 rats each received 300 pg/kg, 1 mg/kg, or 3 mg/kg CNTF SC starting on day 7. They were followed clinically until day 46 when 3 animals per group were sacrificed for morphological studies. Furthermore, erythrocyte

MUSCLE & NERVE September 1996 1177

sedimentation rate and fibrinogen as indicators of inflammatory side effects were measured. In each experiment, another 3 rats per group were followed cljnically up to 100 days into the disease.

RESULTS

In the first set of experiments we compared the effect of CNTF at a dose of 500 pg/kg with placebo. There was no difference in neurological dificit between

C

n v) +

10 N = 6 per group

S.C. injection CNTF every other day iY - 0 10 20 30 40 50 60

n v) +

D T T

N = 6 per group 200 I

180

160

140

4 S.C. injection CNTF every other d L

0 10 20 30 40 50 60

days

* death of emaciated rat in 1 mg group

dars -r- 300 pglkg - 1 mglkg

+ 3 mglkg

FIGURE 1. (A, B) Representative cross-sectional areas of distal sciatic nerves. Regenerated newe fibers in between endoneurial vessels (v), macrophages (m), and some large, preexisting nerve fibers (arrowhead) in the control (A) or the CNTF-treated rat (B). Paraphenylendiamine staining/original magnification, x 1000. (C, D) Disease course and mean body weight of AT-EAN in the different therapy groups. Period of CNTF treatment is indicated at the bottom. Values are given as mean i SD (standard deviation) in groups of 6 animals each.

1178 MUSCLE & NERVE September 1996

treatment and control groups, nor did electrophysio- logical examination of motor nerve conduction ve- locities, sensory nerve conduction velocities, proxi- mal and distal compound motor action potential, and distal F-wave frequency reveal any differences between treatment groups. Blinded morphological evaluation at five different segments from the radicu- lar area to the distal portion showed a gradient from proximal to distal with roughly 70% of fibers being degenerated." In some areas up to 10% of the fibers with intact axons showed different degrees of re- myelination in the treatment and control groups. There were no significant differences of the nerve fiber diameters between the controls and the CNTF- treatment animals (illustrated in Fig. 1A and B). Quantitative analysis of macrophage infiltration re- vealed higher cell numbers at the nerve root level than at the sciatic notch. Again there was no differ- ence between CNTF- and placebo-treated animals.

We reasoned that because of the short half life of CNTF in the circ~lation',~ too little CNTF may reach its presumed target and repeated the experi- ments with slight modifications. Three groups of rats were treated with 300 pg/kg, 1 mg/kg, or 3 mg/kg CNTF. Since the low-dose regimen had proven to be ineffective in the first trial, this dosage was now cho- sen instead of a placebo. Treatment was started on day 7, corresponding to the maximum of disease before regeneration starts. As shown in Figure lC, rats were treated until day 46, when 3 animals per group were sacrificed for morphological analysis. There was no difference between groups in the mean clinical score. Rats receiving 1 mg or 3 mg/kg body weight of CNTF looked generally sick. The surviving rats regained weight much more slowly than the low- dose group, leading to a 10% reduction in body weight at the end of the treatment period (Fig. 1D) (p < 0.01). Morphological analysis showed that all groups had a similar degree of nerve pathology. No difference could be observed between treatment groups with regard to sedimentation rate and fibrin- ogen levels. Clinical recovery of the remaining rats was not different between the groups up to 72 days after cell transfer.

DISCUSSION

Subcutaneous treatment with CNTF did not benefi- cially affect recovery in severe AT-EAN of the Lewis rat at any of the doses tested. Nerve pathology showed profound axonal damage with macrophage infiltra- tion and mild d-myelination of fibers with intact ax- ons as described previously." Recently up-regulation of myelin-related proteins has been described after treatment of experimental autoimmune encephalo-

myelitis with insulin-like growth factor I.zz In our study CNTF did not promote regeneration as shown by morphometric analyses. Although the compli- cated mixture of degenerated, regenerated, and pre- existing myelinated nerve fibers renders a standard- ized morphometric evaluation at the electron microscopic level more difficult than following trans- section or crush lesions of peripheral nerves,I6 no difference could be observed between treatment groups in distal segments of the sciatic nerve. Side effects were remarkable in the higher-dose treatment group as described in recent reports.2~'0~21

The failure of subcutaneous CNTF in this model may be interpreted in various ways: (1) CNTF does not act on inflammation-associated nerve fiber de- generation in contrast to intrinsic motor neuron de- generati~n. '~ (2) Peripheral nerve inflammation leads to release of CNTF from myelinating Schwann cellsz0 sufficient for maximal neuronal responses so that exogenous CNTF cannot improve nerve regener- ation any more. (3) CNTF does not reach its receptor sites in the PNS in sufficient quantity. This latter possibility may be due to proteolytic degradation by inflammatory cells within the peripheral nerves or to peripheral absorption of CNTF as described in detail by Dittrich et a1.2 However, the terminal-phase half- life of CNTF, which ranged from 44 to 71 min in Sprague-Dawley rats after SC administration of doses up to 30 pg/kg (Lakings, Zhan, Lagunowich, et al.; submitted for publication), may be pharmacologi- cally more relevant than the early phase investigated by Dittrich et al.' Unfortunately such data are not yet available, but direct evidence for this is still lacking.

Future studies with CNTF-deficient animals" and specific systems such as intrathecal drug pumpsI5 and implanted slow-release systems' should provide more details on a physiological function of CNTF in periph- eral nerve inflammatory disorders.

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