Neurotoxic damage evokes regenerative responses from adult rat sensory neurones

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Neuroscience Letters, 146 (1992) 48-52 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 09035

Neurotoxic damage evokes regenerative responses from adult rat sensory neurones Janet Winter a, Caroline J. Evison a, Claire O ' B r i e n b'l, L a r r y Benowitz c, R o n a l d M. L i n d s a y a, Peter M u l d e r r y e a n d Clifford W o o l f b "Sandoz Institute for Medical Research, London (UK), ~Department of Anatomy, University College London, London (UK), 'Mailman Research Center, McLean Hospital, Belmont, MA 02178 (USA), aRegeneron, Tarry Town, N Y 10951 (USA) and eMRC Brain Metabolism Unit, Royal Edinburgh Hospital, Edinburgh (UK) (Received 25 March 1992; Revised version received 24 July 1992; Accepted 24 July 1992)

Key words." Capsaicin; Axotomy; Dorsal root ganglion; Regeneration; Culture When adult rats were pre-treated systemically with the excitotoxin, capsaicin, a subset of their dorsal root ganglion (DRG) sensory neurones responded as if they had been axotomised. Firstly, an increased proportion of capsaicin-sensitive D R G neurones (identified by using a cobalt stain) vigorously extended processes in vitro. Additionally, a subset of small dorsal root ganglion neurones expressed elevated levels of the growthassociated protein-43 (GAP-43) in situ. Both vigorous neuritogenesis and elevated GAP-43 levels are characteristics of neurones that are actively regenerating following mechanical damage. Although capsaicin-treated adult DRG neurones show these regenerative responses, their C-fibers fail to regenerate in vivo.

In adult rats, systemic treatment with capsaicin, the pungent ingredient in hot chilli peppers, can cause degeneration of peripheral C-fibre terminals without apparent loss of either the dorsal root ganglion (DRG) neurone cell bodies, the central axon branches in the dorsal roots or the axons in peripheral nerves [6]. Capsaicin-sensitive C-fibres in the skin [5] and the ureter [6] for example, degenerate rapidly after systemic capsaicin treatment [50 mg/kg). Although many sensory neurones regenerate and reinnervate their peripheral targets following a surgical lesion [7, 13], capsaicin-damaged C-fibres seem never to regenerate [14, 15]. The question arises - - does capsaicin-induced damage to sensory fibres fail to provoke a regenerative response from the sensory neurone cell bodies? One characteristic feature of regenerating neurones is their increased propensity for neurite outgrowth in culture. The speed of neurite induction in vitro tends to reflect the state of neuronal growth in vivo, so that developing or mature regenerating neurones grow processes

Correspondence." J. Winter, Sandoz Institute for Medical Research, 5, Gower Place, London WC1E 6BN, UK. Fax: (44) 71-387-4116. ~Present address: Elsevier Trends Journals, 68 Hills Road, Cambridge CB2 I LA, UK.

rapidly in vitro. In contrast mature unlesioned neurones that are capable of regeneration take days rather than hours to elaborate processes in vitro [9]. Thus, surgically axotomised D R G neurones from adult rats also put out neurites in culture more rapidly than control, unlesioned D R G neurones (unpublished observations). The delay in the formation and extension of neurites from mature neurones is probably due in part to the need for re-expression of proteins involved in neurite extension. The best known example is growth-associated protein-43 (GAP-43) (also known as B-50, F1, pp46 and p57, reviewed in refs. 2, 10, 17, 18). Thus, axotomised dorsal root ganglion sensory neurones also show a sustained increase in their GAP-43 synthesis during regeneration [3, 11, 19, 20, 24]. We looked for these changes in animals that had been treated systemically with capsaicin. Sprague Dawley rats (~200 g) were anaesthetized with halothane and injected subcutaneously with a single dose of 50 mg/kg of capsaicin (Sigma) dissolved in a vehicle of 10% ethanol, 10% Tween-80 and 80% saline, or with vehicle alone. Adult rat D R G neurones were dissociated and cultured as described in detail elsewhere [12]. Cell suspensions were plated onto polyornithine and laminincoated tissue Petri dishes in F14 medium with 4% US-G (Ultraser-G, Gibco, a serum substitute). After 24 h in culture, cells were fixed in 4% paraformaldehyde and the

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Fig. 1. Effectof systemiccapsaicin on DRG neurone regeneration in vitro. One day after systemicinjection of adult rats with capsaicin or with vehicle alone, lumbar DRG were dissociated and plated. Capsaicin-sensitiveneurones were identified by the cobalt staining method after 24 hours in culture. A: phase contrast micrograph of 5 neurones from a control animal, 4 without neurites, and (B) 3 of these are capsaicin sensitive (i.e. they contain cobalt sulphide which is dark under bright-field illumination), and are marked by large arrowheads. C: phase contrast, 6 neurones from a capsaicin treated animal, 4 have long neurites (marked by small arrowheads) and of these (D) all are capsaicin sensitive (dark, and marked by large arrowheads). Asterisks in A and B mark groups of non-neuronal cells. Bar = 50/.tm.

number o f process-bearing neurones were counted using phase contrast microscopy. Neurones with neurites greater than 2 cell body diameters in length were scored as positive. Prior to fixation, in some o f the cultures capsaicin-sensitive neurones were identified using a cobalt uptake method, as previously described [21, 22]. Ten p m cryostat sections of 4% paraformaldehyde-fixed ganglia were stained overnight with sheep anti GAP-43 [3] at a final concentration o f 1-1000 diluted in 10% horse serum, 0.1% Triton X-100 in PBS, followed by biotinylated anti-sheep antibodies and streptavidin fluorescein (Amersham). An increased proportion of neurite-forming D R G neurones was seen when cells were taken from rats that had been pretreated with 50 mg/kg of capsaicin 24 h prior to sacrifice. In the capsaicin-pretreated group a

mean of 74% (n=5, over 400 neurones counted/point) of the dissociated lumbar D R G neurones responded with a rapid and extensive neurite outgrowth during the first 24 h in culture, compared to only 31.8% D R G neurones obtained from control, vehicle injected, animals (Table I). These 2 groups were compared using a M a n n - W h i t ney test and were shown to be significantly different at the level of 0.05. We also found similarly enhanced neurite outgrowth in vitro when animals were treated with capsaicin (50 mg/kg) 2 weeks prior to sacrifice, with 67.5% (n=5, at least 200 neurones counted/point) of the neurones from the capsaicin treated group and only 26.3% of the neurones from the control group forming processes over 2 cell body diameters in length after 24 h. Again this represents a significant increase in neurones with neurites in the capsaicin-pretreated animals com-

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Fig. 2. The effect of surgical axotomy or systemic capsaicin treatment on expression of GAP-43 immunoreactivity in DRG neurones in situ. A: cryostat section of a control L4 ganglion from a vehicle-treated animal (14 days) stained with anti-GAP-43 antibody. Very little staining can be seen in neurons. One weakly stained neurone is indicated with an arrow. B: 14 days after sciatic nerve transection, GAP-43 staining can be seen in many neurones, both small (small arrows) and large (large arrow) in an L4 ganglion. C: 14 days after capsaicin treatment strong GAP-43 staining can be seen in many small DRG neurones (three are indicated by small arrows) in an L4 ganglion. Bar = 50 gm.

pared to control animals. There was no significam difference, however, between the 1 day and the t 4 day capsaicin-treated groups; so the regenerative response was still maximal at 2 weeks post capsaicin treatment. The increased neurite o u t g r o w t h response induced by capsaicin was specific. Following systemic capsaicin treatment the day before dissociation and culture, the n u m b e r o f capsaicin-sensitive D R G neurones with TABLE I PERCENTAGE OF NEURITE-BEARING DRG NEURONES FROM CAPSAICIN-TREATEDANIMALS Neurones were taken from animals that had been treated with capsaicin I or 14 days previously. After 24 h in culture, they were scored for absence or presence of neurites.

1 2 3 4 5 Mean

Day 1

Day 14

control

capsaicin control

capsaicin

36.5 32.2 31.8 25.5 23.8 31.8%*

63.3 74.0 78.2 75.3 72.6 74%*

67.5 65.2 69.4 84.5 58.7 67.5%**

23.4 26.3 36.8 19.2 31.6 26.3%**

*Significantly different at the 0.05 level (Mann-Whitney test). **Significantly different at the 0.05 level (Mann-Whitney test).

neurites increased approximately 6 fold (to 62% f r o m 13% in controls, Table II, Fig. 1) whereas the p r o p o r t i o n o f capsaicin-insensitive neurones with neurites remained constant. The percentage o f capsaicin-sensitive neurones identified by cobalt uptake was similar in D R G cultures from animals that had been treated with either vehicle (mean -- 63.3, n--3) or capsaicin (mean = 65, n=3) 24 h prior to sacrifice, suggesting that there is no rapid cell death or loss o f capsaicin sensitivity. G A P - 4 3 in D R G cryostat sections was visualised by immunofluorescence. Very little G A P - 4 3 staining was seen in D R G f r o m control animals (Fig. 2A). F o r comparison, G A P - 4 3 labelling o f D R G neurones is shown 2 weeks after a x o t o m y by sciatic nerve transection (Fig. 2B), when D R G neurones o f all sizes are k n o w n to express GAP-43. Two weeks after systemic capsaicin treatment, intense G A P - 4 3 staining was still present (Fig. 2C), mainly in small D R G neuronal cell bodies. In light o f the observation reported here, that systemic treatment o f adult rats with 50 mg/kg o f capsaicin evokes a sustained regenerative response (for at least 2 weeks after treatment) f r o m D R G neurones, it is perhaps surprising that target reinnervation by capsaicin-damaged C-fibres does not occur. A l t h o u g h there are some signs o f abortive sprouting within the nerve after local application [16], both local and systemic capsaicin-induced lesion, unlike mechanical nerve lesion, seem to cause a per-

51 TABLE II PERCENTAGE OF CAPSAICIN-SENSITIVE OR -INSENSITIVE NEURONES WITH NEURITES Neurones were taken from animals that had been treated with capsaicin 1 day previously. After 24 h in culture, they were scored for capsaicin sensitivity by cobalt uptake and for absence or presence of neurites.

1 2 3 4 5 6 7 Mean

Cobalt positive

Cobalt negative

control

capsaicin

control

capsaicin

18.0 27.2 5.4 12.9 6.3 8.0 14.0 12.9%*

66.7 64.5 62.1 60.2 48.5 76.0 57.0 62.1%*

40.5 40.4 34.5 31.9 33.0 42.0 33.0 34.5%**

43.8 33.3 40.4 41.0 29.4 30.0 39.0 39%**

*Significantly different at the 0.05 level (Mann-Whitney test). **Not significantly different at the 0.05 level (Mann-Whitney test).

axotomy. One clue may lie in data obtained from the mouse strain C57BL/Ola, where DRG sensory neurones regenerate poorly following peripheral nerve lesion [4]. The distal portions of the cut peripheral nerves in the Ola animals show little sign of 'Wallerian degeneration' for several weeks, i.e. there is poor macrophage infiltration, no glial proliferation, and myelin and axons remain intact. These changes, when they occur in a normal nerve, may support sensory axon regeneration (reviewed in ref. 8). A recent in vitro study demonstrating that dissociated, cultured adult sensory neurones will grow on cryostat sections of predegenerated but not unlesioned peripheral nerve supports this view [1]. If damage to myelinated axons is necessary to evoke these changes [8], degeneration of capsaicin-sensitive fibres, the vast majority of which are unmyelinated C-fibres, may not be sufficient. We therefore suggest that although capsaicin-damaged sensory neurones have a regenerative potential, the environment of the peripheral nerve is not permissive for regrowth. Part of this work has previously been published in abstract form [23]. manent

We would like to thank Martin Raft and Stuart Bevan for helpful discussion, and Jackie Donoghue for technical assistance. 1 Bedi, K.S., Winter, J., Berry, M. and Cohen, J. Adult dorsal root ganglion neurons extend neurites on predegenerated but not on normal peripheral nerves in vitro, European J. Neurosci., 4 (1992) 193200.

2 Benowitz, L.I. and Routenberg, A., A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity, Trends Neurosci., 10 (1987) 527-531. 3 Bisby, M.A., Dependence of GAP-43 (B50, F1) transport on axonal regeneration in rat dorsal root ganglion cells, Brain Res., 458 (1988) 157-161. 4 Brown, M.C., Perry, V.H., Lunn, E.R., Gordon, S. and Heumann, R., Macrophage dependence of peripheral sensory nerve regeneration: possible involvement of nerve growth factor, Neuron, 6 ( 1991) 359-370. 5 Chung, K., Klein, C.M. and Coggeshall, R.E., The receptive part of the primary afferent axon is most vulnerable to systemic capsaicin in adult rats, Brain Res., 511 (1990) 222-226. 6 Chung, K., Schwen, R.J. and Coggeshall, R.E., Ureteral axon damage following subcutanous administration ofcapsaicin in adult rats, Neurosci. Lett., 53 (1985) 221-226. 7 Diamond, J., Foerster, A., Holmes, M. and Coughlin, M., Sensory nerves in adult rats regenerate and restore sensory function to the skin independently of endogenous NGF, J. Neurosci., 12 (1992) 1467-1476. 8 Fawcett, J.W. and Keynes, R.J., Peripheral nerve regeneration, Annu. Rev. Neurosci., 13 (1990) 43-60. 9 Fawcett, J.W., Intrinsic neuronal determinants of regeneration, Trends Neurosci., 15 (1992) 5-8. 10 Gordon-Weeks, ER., GAP-43 - What does it do in the growth cone?, Trends Neurosci., 12 (1989) 363-365. 11 Hoffman, E, Expression of GAP-43, a rapidly transported growthassociated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons, J. Neurosci., 9 (1989) 893-897. 12 Lindsay, R.M., Nerve growth factors (NGE BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurones, J. Neurosci., 8 (1988)2394-2405. 13 Lisney, S.J.W., Regeneration of unmyelinated axons after injury of mammalian peripheral nerve, Quart. J. Exp. Physiol., 74 (1989) 757- 784. 14 Lynn, B., Pini, A. and Baranowski, R., Injury of somatosensory afferents by capsaicin: selectivity and failure to regenerate. In Effects of Injury on Trigeminal and Spinal Somatosensory Systems, L.M. Pubols and B.J. Sessle (Eds.), Alan R. Liss, New York, 1987, pp. 115-124. 15 Pini, A., Baranowsky, R. and Lynn, B., Long-term reduction in the number of C-fibre nociceptors following capsaicin treatment of a cutaneous nerve in adult rats, Eur. J. Neurosci., 2 (1990) 8%97. 16 Pini, A. and Lynn, B., C-fibre function during the 6 weeks following brief application of capsaicin to a cutaneous nerve in the rat, Eur. J. Neurosci., 3 (1991) 274-284. 17 Skene, J.H.E, Axonal growth-associated protein, Annu. Rev. Neurosci., 12 (1989) 127-156. 18 Skene, JH.P. and Willard, M., Axonally transported proteins associated with growth in rabbit central and peripheral nervous system, J. Cell Biol., 89 (1981) 96-103. 19 Tetzlaff, W., Zwiers, H., Lederis, K., Cassar, L. and Bisby, M.A., Axonal transport and localization of B-50/GAP-43-1ike immunoreactivity in regenerating sciatic and facial nerves of the rat, J. Neurosci., 9 (1989) 1303-1313. 20 Van der Zee, C.E.E.M., Nielander, H.B., Vors, J.P., Lopes da Silva, S., Verhaagen, J., Oestreicher, A.B., Schrama, L.H., Schotman, P. and Gispen, W.H., Expression of growth-associated protein B-50 (GAP-43) in dorsal root ganglia and sciatic nerve during regenerative sprouting, J. Neurosci., 9 (1989) 3505-3512.

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43 production and neurite outgrowth from rat dorsal root ganglion neurones, Neurosci. Lett., Suppl. 38 (1990) $34. 24 Woolf, C.J., Reynolds, M.L., Molander, C., O'Brien, C., Lindsay, R.M. and Benowitz, L.I., GAP-43, a growth-associated protein, appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury, Neuroscience 34 (1990) 465478.