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Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour
Neuroscience Letters, 111 (1990) 28--33 Elsevier ScientificPublishers Ireland Ltd.
2~
NSL 06740
Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour M. Gautron l, F. Jazat l, H. Ratinahirana 2, J.J. H a u w 2 a n d G. G u i l b a u d 1 ~Unit~ de Reeherches de Physiopharmacologie du systeme nerveux ( I N S E R M U 1610, Paris (France) and :Laboratoire de Neuropathologie R. Escourolle ( FRA Cl. Bernard), CHU Pitie-Salpetriere, Paris (France)
(Received 18 July 1989; Revised version received 10 November 1989;Accepted 10 November 1989) Key word~." Sciaticnerve; Constriction; Myelination; Pain-related behavior
Morphology or peripheral rnyelinated fibres was analyzed in rats exhibiting hyperalgesia and allodynia with mechanical and thermal stimuli, consecutive to a mononeuropathy induced by 4 loose ligatures around a sciatic nerve. This preliminary study was based on weeks 2-3 after surgery, the time of the maximum alterations of the pain-related behaviour. At this time, contrasting with a marked decrease of the large afferent fibres a consistent number of << 5 #m fibres was pointed out. In addition to their extremely short internodal length, the majority of these fibres had an abnormal g-ratio, thus an abnormal myelin sheath. It is suggested that this group of abnormal fibres might be related to the A6 fibres described in neuromas and involved in pain-related behaviours seen in the mononeuropathic rats.
A m o n o n e u r o p a t h y , induced in the rat by 4 loose ligatures a r o u n d the c o m m o n sciatic nerve, has been recently proposed as a model of experimental pain [1]. The initial study was followed by a n extensive investigation, using several quantified b e h a v i o u r a l nociceptive tests [12], which confirmed the occurrence of hyperalgesia (excessive responses to n o x i o u s stimuli) a n d allodynia (nociceptive reactions to i n n o cuous stimuli) [13], with b o t h mechanical a n d thermal stimulation. The time course of the observed modifications has been clearly established. Alterations in the nociceptive tests begin at week 1 ( W l ) after surgery, are m a x i m u m at W2, recovery starting after W3, a n d being usually complete by W 8 - W 1 0 . It appeared of interest to analyze the m o r p h o l o g y of peripheral sciatic nerve fibres in n e u r o p a t h i c rats in parallel with the pain-related behaviour. This has n o t been pre-
Correspondence." G. Guilbaud, Unit6 de Recherches de Physiopharmacologie du syst6me nerveux,
INSERM U. 1610, 2 Rue d'Alesia, F-75014Paris, France. 0304-3940/90/$ 03.50 © 1990 ElsevierScientificPublishers Ireland Ltd.
29 viously considered, although histological aspects of degeneration and regeneration of rat sciatic nerve after compression or constriction of variable intensity and duration have been extensively studied (refs. in refs. 5, 9, 10). This preliminary study was essentially based on W2 and W3 after surgery, the time of the maximum hyperalgesia and allodynia (in 5 and 3 rats respectively). Data were compared to those from normal rats (n = 4) and from rats sacrificed, at W1 when the hyperalgesia and allodynia commence (n= 1), or at W12 when the animals recover to give normal behavioural reactions to nociceptive stimuli (n = 3). Several characteristics of the myelinated fibres in the ligated nerve segment were measured: degree or degeneration and regeneration, spectrum of diameters, sheath thickness (axon diameter/fibre diameter), and internode lengths. As pain-related behaviours were concerned, experiments were performed in agreement with the IASP ethical guide lines [4]. The neuropathic rats used for the present study (n = 12) were taken from a pool of 133 animals used in an extensive behavioural investigation [12], tested with 3 nociceptive tests before surgery, and then regularly afterwards. Briefly, these tests were the following: (a) the vocalization threshold to paw pressure, a decrease indicating an abnormal reactivity to mechanical stimuli; (b) the threshold temperature inducing a struggle reaction using immersion of the affected paw in a hot water bath of 40, 42, 44, or 46°C which allowed aliodynia and hyperalgesia to hot to be gauged; and finally (c) the time in a 10°C water bath necessary to elicit a struggle reaction, with a decrease indicating allodynia to cold. (This temperature was not perceived as noxious by the experimenter, nor induced nociceptive reactions in normal rats with less than 15 s immersion.) In the sample of rats used for the histological analysis the threshold decrease and time course of the 3 tests were comparable to those observed in the whole population [12], and the abnormalities in the pain-related behaviour from the lesioned paw were maximal at W2. At this time the vocalization threshold to paw pressure, decreased by about 70 g, was 211.0_+ 37.82 g; the struggle threshold to heat decreased by about 4°C, was 41.0 + 2.0°C; the immersion duration for struggle in a 10°C water bath decreased by 4 s was 10.6 + 2.19 s (n = 5). In order to excise the nerve, animals (male Sprague-Dawley rats, 290-650 g) were deeply anesthetized with barbiturate. Sciatic nerve and its terminals were exposed and dissected as far as possible towards the upper thigh and to the ankle. The excised piece or nerve, 3.5~4.5 cm length, was immediately immersed for 24 h in 2.5 % glutaraldehyde in Millonig buffer (pH 7.3, 4°C), then for 1 h in 2 % OsO4. During glutaraldehyde fixation, nerves were divided into several portions for preparation of teased fibres (n = 15/16 nerves) and/ or semi-thin transverse sections (n = 13/16, as described previously [3]: teased samples were selected at random among the nerve fascicles, and examined over at least 4 internodes; 2-/~m-thick transverse sections of epoxy-embedded specimen were stained with Toluidine blue for morphometry of myelinated fibres. Semi-automatic analysis was performed using a Macintosh 512 K microcomputer [15]. The following data were analyzed: density of fibres; distribution of diameters; g-ratio (internal/external diameter) as a function of the external diameter; internodal length as a function of the fibre diameter; according to the simplified classification of Chaunu et al. (ref. 3 adapted from ref. 8), teased fibres were considered as normal, or with axonal degeneration (myelin
ovoids and balls), regeneration (short regular internodes), segmental demyelination (paranodal or internodal), or remyelination (excessive variability of myelin thickness and short irregular internodes) (Fig. 1). Data obtained with nerves of normal rats were roughly comparable to those already reported (refs. in refs. 5, 9, 10). From the various analyses it appeared that the lesion had induced a massive degeneration of the Wallerian type of large myelinated fibres, which was maximum at W2: 1. At this time, 99% of teased fibres (n= 360) showed axonal degeneration almost exclusively with myelin balls (Fig. la). This effect was somewhat less marked at WI or W3 (n = 50 and 273 teased fibres, respectively): at W3 a few signs of regeneration were seen since 2 % of fibres exhibited segmental demyelination and even remyelination (Fig. Ib,c). At W12, 42% of fibres (n=58) could be considered as normal and 50% showed clear signs of axonal regeneration (Fig. l b). 2. Morphometric analysis revealed a dramatic drop in density of large fibres with a diameter above 5/lm, when compared with normal nerves (present study and refs. 9, 10): in marked contrast, there was a consistent group of < < 5/zm fibres with a density around 5000/mm 2. A comparable spectrum was observed at W3. Although the 5 9 / m l fibres began to increase again at W4, the spectrum was not yet totally normal at W 12. 3. Internodal lengths could be measured for only a few teased fibres at W1 and W2, due to the extreme fragility of the nerve. It is however clear that it was greatly shortened for all the remaining fibres whatever their diameter, and that abnormalities were maximum at W2 (mean value lk)r < < 5 / t m fibres was 333.8_+68.4 and 170.3_+ 14.3 t~m at W1 and W2, respectively (P < 0.05 n = 8 in each group, Studenffs t~test).
Fig. 1. Teased fibres preparations. X 660. Bar = 20/~m. a: axonal degeneration with numerous ovoids and balls (example at week (W) 2 after ligature), b: axonal regeneration, with small regenerating fibre indicating axonal degeneration and subsequent regeneration. Note the small regular internodes. Four nodes of Ranvier are indicated by arrows (example at W12 after ligature), c: widening of a node of Ranvier. This condition could be due either to axonal degeneration or to demyelination [13] (example at W3 after ligature).
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Fig. 2. Myelin sheath thickness of sciatic fibres in terms of g-ratio (internal/external diameter) as a function of the external diameter, in normal nerves and at several weeks after the nerve ligatures (fibres of 3 nerves have been pooled in each group). At W2 the g-ratio of the << 5 pm fibres was of 0.7~).8 for 52%, < 0.7 for 40%, and >0.8 for the others: the respective differences between these percentages and those obtained for the few numbers of such fibres in abnormal nerve, are highly significant (e-test P< 1 x 10-9). 4. Scatter diagrams o f the g-ratio (Fig. 2) confirmed the consistent number o f fibres < < 5 p m at W2, and s h o w e d that m o s t o f them had abnormal myelin sheaths (Fig. 3): as c o m p a r e d with normal nerves (see also refs. 9, 10), there was a noticeable dispersion o f g-ratio values (normally concentrated between 0.7 and 0.8). At W3, evolution o f the g-ratio also indicated that regeneration was starting (as in sections 2 and 3). At W12, the g-ratio scattergram was more or less normal. F r o m this preliminary study it seems clear that the 4 loose ligatures placed around the sciatic nerve (still in place at least until W6), induced an acute 'axonopathy' which greatly affected myelinated fibres as seen in the same model [1], and in other lesions based on compression [5, 10]. It seems worth pointing out that pain-related behaviours were m a x i m u m when a consistent number o f < < 5 / ~ m fibres, thus fibres in the A 6 range, could be observed. In addition to their short internodal length, these fibres exhibited a wide range o f g-ratio values, suggesting that m o s t o f them had an abnormal myelin sheath, s o m e being likely atrophic, and others regenerating fibres. The presence o f this group o f abnormal fibres in the A6 range is evocative o f nerve fibres sprouts, observed in n e u r o m a s in various species (refs. in refs. 6, 11), which exhibit intense o n g o i n g electrical activity, abnormal sensitivity to various stimuli and to sympathetic agents, a b n o r m a l contacts or ephapses between axons, (which overall the existence o f potential 'cross talk' between fibres) (refs. in refs. 2, 6, 11). It is difficult to assume that degenerating and regenerating processes are similar in the case o f a partially ligated sciatic nerve, but the present observations suggest this as a possi-
32
Fig. 3. Transverse sections of sciatic nerve at the ligature level: controls in 1 and 2, lesioned nerve at W2 in 3 and 4. Semi-thin sections, To|uidine blue; Nomarski optics. × 120 for 1 and 3, x 300 for 2 and 4 (bars = 100 #m). Note the decrease in myelinated fibre density in 3, and the presence of fibres with thick or very thin (arrows in 4) myelin sheaths in the ligated nerve.
33
bility, and that the abnormal small myelinated fibres could thus be partly responsible for most of the pain-related behaviour observed in these rats. Nevertheless, on the basis of the gate control theory, that nociceptive behaviour could be enhanced by the lack of inhibitory controls on messages driven by the small afferent fibres (refs. in refs. 7, 15) is also possible, due to the marked and prolonged damage of the large nervous fibres. In addition, the involvement of damaged unmyelinated fibres appears to be likely (Basbaum et al., in preparation). The authors wish to thank Y.L. Chen and N. Attal for behavioural tests, F. Roudier for histological preparations, and A.H. Dickenson for English revision. 1 Bennett, G.J. and Xie, Y.K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107. 2 Campbell, J.N., Raja, S.N., Cohen, R.H., Manning, D.C., Khan, A.A. and Meyer, R.A., Peripheral neural mechanisms of nociception. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingston, 1989, pp. 22-45. 3 Chaunu, M.P., Ratinahirana, H., Raphael, M., Henin, D., Leport, C., Brun-Vezinet, F., Leger, J.M., Brunet, P. and Hauw, J.J., The spectrum of changes on 20 nerve biopsies in patients with HIV infection, Muscle Nerve, 12 (1989) 452-459. 4 Committee for research and ethical issues of the IASP. Ethical standards for investigations of experimental pain in animals, Pain, 16 (1983) 109-110. 5 Dahlin, L.B., Nordborg, C. and Lundborg, G., Morphologic changes in nerve cell bodies induced by experimental graded nerve compression, Exp. Neurol., 95 (1987) 611-621. 6 Devor, M., The pathophysiology of damaged nerve. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingston, 1989, pp. 63-81. 7 Dyck, P.J., Lambert, E.H. and O'Brien, P.C., Pain in peripheral neuropathy related to rate and kind of fiber degeneration, Neurology, 26 (1976) 466-471. 8 Dyck, P.J., Karnes, J., Lais, A., Logfren, E.P. and Stevens, J.C., Pathologic alterations of the peripheral nervous system in humans. In P.J. Dyck et al. (Eds.), Peripheral Neuropathy, Saunders, Philadelphia, 1984, Vol. I, pp. 760-870. 9 Friede, R.L. and Beuche, W., A new approach towards analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes, J. Neuropathol. Exp. Neurol., 44 (1985) 60-72. 10 Hildebrand, C., Kocsis, J.D., Berglund, S. and Waxman, S.G., Myelin sheath remodelling in regenerated rat sciatic nerve, Brain Res., 358 (1985) 163-170. 11 J~inig, W., Pathophysiology of nerve following mechanical injury. In R. Dubner et al. (Eds.), Pain Research and Clinical Management, Vol. 3, Proc. Vth World Congress on pain, Elsevier, Amsterdam, 1988, pp. 89-108. 12 Jazat, F., Attal, N., Gautron, M. and Guilbaud, G., Behavioural evidence that a peripheral experimental neuropathy in rat produces abnormal pain sensation, Eur. J. Neurosc., Suppl. (1988) 179. 13 Merskey, H., Lindblom, U., Mumford, J.M., Nathan, P.W., Noordenbos, W. and Sunderland, S., Pain terms. A current list with definitions and notes on usage, Pain, Suppl. 3 (1986) $217-221. 14 Ochoa, J. and Noordenbos, W., Pathology and disorders sensation in local nerve lesions: An attempt at correlation. In J.J. Bonica et al. (Eds.), Advances in Pain Research and Therapy, Vol. 3, Raven, New York, 1979, pp. 67-77. 15 Ratinahirana, H., Duyckaerts, C., Chaunu, M.P., Henin, D. and Hauw, J.J., Un systrme simple d'analyse semi-automatique d'image, Ann. Pathol., 9 (1989) 68-70.
2~
NSL 06740
Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour M. Gautron l, F. Jazat l, H. Ratinahirana 2, J.J. H a u w 2 a n d G. G u i l b a u d 1 ~Unit~ de Reeherches de Physiopharmacologie du systeme nerveux ( I N S E R M U 1610, Paris (France) and :Laboratoire de Neuropathologie R. Escourolle ( FRA Cl. Bernard), CHU Pitie-Salpetriere, Paris (France)
(Received 18 July 1989; Revised version received 10 November 1989;Accepted 10 November 1989) Key word~." Sciaticnerve; Constriction; Myelination; Pain-related behavior
Morphology or peripheral rnyelinated fibres was analyzed in rats exhibiting hyperalgesia and allodynia with mechanical and thermal stimuli, consecutive to a mononeuropathy induced by 4 loose ligatures around a sciatic nerve. This preliminary study was based on weeks 2-3 after surgery, the time of the maximum alterations of the pain-related behaviour. At this time, contrasting with a marked decrease of the large afferent fibres a consistent number of << 5 #m fibres was pointed out. In addition to their extremely short internodal length, the majority of these fibres had an abnormal g-ratio, thus an abnormal myelin sheath. It is suggested that this group of abnormal fibres might be related to the A6 fibres described in neuromas and involved in pain-related behaviours seen in the mononeuropathic rats.
A m o n o n e u r o p a t h y , induced in the rat by 4 loose ligatures a r o u n d the c o m m o n sciatic nerve, has been recently proposed as a model of experimental pain [1]. The initial study was followed by a n extensive investigation, using several quantified b e h a v i o u r a l nociceptive tests [12], which confirmed the occurrence of hyperalgesia (excessive responses to n o x i o u s stimuli) a n d allodynia (nociceptive reactions to i n n o cuous stimuli) [13], with b o t h mechanical a n d thermal stimulation. The time course of the observed modifications has been clearly established. Alterations in the nociceptive tests begin at week 1 ( W l ) after surgery, are m a x i m u m at W2, recovery starting after W3, a n d being usually complete by W 8 - W 1 0 . It appeared of interest to analyze the m o r p h o l o g y of peripheral sciatic nerve fibres in n e u r o p a t h i c rats in parallel with the pain-related behaviour. This has n o t been pre-
Correspondence." G. Guilbaud, Unit6 de Recherches de Physiopharmacologie du syst6me nerveux,
INSERM U. 1610, 2 Rue d'Alesia, F-75014Paris, France. 0304-3940/90/$ 03.50 © 1990 ElsevierScientificPublishers Ireland Ltd.
29 viously considered, although histological aspects of degeneration and regeneration of rat sciatic nerve after compression or constriction of variable intensity and duration have been extensively studied (refs. in refs. 5, 9, 10). This preliminary study was essentially based on W2 and W3 after surgery, the time of the maximum hyperalgesia and allodynia (in 5 and 3 rats respectively). Data were compared to those from normal rats (n = 4) and from rats sacrificed, at W1 when the hyperalgesia and allodynia commence (n= 1), or at W12 when the animals recover to give normal behavioural reactions to nociceptive stimuli (n = 3). Several characteristics of the myelinated fibres in the ligated nerve segment were measured: degree or degeneration and regeneration, spectrum of diameters, sheath thickness (axon diameter/fibre diameter), and internode lengths. As pain-related behaviours were concerned, experiments were performed in agreement with the IASP ethical guide lines [4]. The neuropathic rats used for the present study (n = 12) were taken from a pool of 133 animals used in an extensive behavioural investigation [12], tested with 3 nociceptive tests before surgery, and then regularly afterwards. Briefly, these tests were the following: (a) the vocalization threshold to paw pressure, a decrease indicating an abnormal reactivity to mechanical stimuli; (b) the threshold temperature inducing a struggle reaction using immersion of the affected paw in a hot water bath of 40, 42, 44, or 46°C which allowed aliodynia and hyperalgesia to hot to be gauged; and finally (c) the time in a 10°C water bath necessary to elicit a struggle reaction, with a decrease indicating allodynia to cold. (This temperature was not perceived as noxious by the experimenter, nor induced nociceptive reactions in normal rats with less than 15 s immersion.) In the sample of rats used for the histological analysis the threshold decrease and time course of the 3 tests were comparable to those observed in the whole population [12], and the abnormalities in the pain-related behaviour from the lesioned paw were maximal at W2. At this time the vocalization threshold to paw pressure, decreased by about 70 g, was 211.0_+ 37.82 g; the struggle threshold to heat decreased by about 4°C, was 41.0 + 2.0°C; the immersion duration for struggle in a 10°C water bath decreased by 4 s was 10.6 + 2.19 s (n = 5). In order to excise the nerve, animals (male Sprague-Dawley rats, 290-650 g) were deeply anesthetized with barbiturate. Sciatic nerve and its terminals were exposed and dissected as far as possible towards the upper thigh and to the ankle. The excised piece or nerve, 3.5~4.5 cm length, was immediately immersed for 24 h in 2.5 % glutaraldehyde in Millonig buffer (pH 7.3, 4°C), then for 1 h in 2 % OsO4. During glutaraldehyde fixation, nerves were divided into several portions for preparation of teased fibres (n = 15/16 nerves) and/ or semi-thin transverse sections (n = 13/16, as described previously [3]: teased samples were selected at random among the nerve fascicles, and examined over at least 4 internodes; 2-/~m-thick transverse sections of epoxy-embedded specimen were stained with Toluidine blue for morphometry of myelinated fibres. Semi-automatic analysis was performed using a Macintosh 512 K microcomputer [15]. The following data were analyzed: density of fibres; distribution of diameters; g-ratio (internal/external diameter) as a function of the external diameter; internodal length as a function of the fibre diameter; according to the simplified classification of Chaunu et al. (ref. 3 adapted from ref. 8), teased fibres were considered as normal, or with axonal degeneration (myelin
ovoids and balls), regeneration (short regular internodes), segmental demyelination (paranodal or internodal), or remyelination (excessive variability of myelin thickness and short irregular internodes) (Fig. 1). Data obtained with nerves of normal rats were roughly comparable to those already reported (refs. in refs. 5, 9, 10). From the various analyses it appeared that the lesion had induced a massive degeneration of the Wallerian type of large myelinated fibres, which was maximum at W2: 1. At this time, 99% of teased fibres (n= 360) showed axonal degeneration almost exclusively with myelin balls (Fig. la). This effect was somewhat less marked at WI or W3 (n = 50 and 273 teased fibres, respectively): at W3 a few signs of regeneration were seen since 2 % of fibres exhibited segmental demyelination and even remyelination (Fig. Ib,c). At W12, 42% of fibres (n=58) could be considered as normal and 50% showed clear signs of axonal regeneration (Fig. l b). 2. Morphometric analysis revealed a dramatic drop in density of large fibres with a diameter above 5/lm, when compared with normal nerves (present study and refs. 9, 10): in marked contrast, there was a consistent group of < < 5/zm fibres with a density around 5000/mm 2. A comparable spectrum was observed at W3. Although the 5 9 / m l fibres began to increase again at W4, the spectrum was not yet totally normal at W 12. 3. Internodal lengths could be measured for only a few teased fibres at W1 and W2, due to the extreme fragility of the nerve. It is however clear that it was greatly shortened for all the remaining fibres whatever their diameter, and that abnormalities were maximum at W2 (mean value lk)r < < 5 / t m fibres was 333.8_+68.4 and 170.3_+ 14.3 t~m at W1 and W2, respectively (P < 0.05 n = 8 in each group, Studenffs t~test).
Fig. 1. Teased fibres preparations. X 660. Bar = 20/~m. a: axonal degeneration with numerous ovoids and balls (example at week (W) 2 after ligature), b: axonal regeneration, with small regenerating fibre indicating axonal degeneration and subsequent regeneration. Note the small regular internodes. Four nodes of Ranvier are indicated by arrows (example at W12 after ligature), c: widening of a node of Ranvier. This condition could be due either to axonal degeneration or to demyelination [13] (example at W3 after ligature).
31 1 •
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Fig. 2. Myelin sheath thickness of sciatic fibres in terms of g-ratio (internal/external diameter) as a function of the external diameter, in normal nerves and at several weeks after the nerve ligatures (fibres of 3 nerves have been pooled in each group). At W2 the g-ratio of the << 5 pm fibres was of 0.7~).8 for 52%, < 0.7 for 40%, and >0.8 for the others: the respective differences between these percentages and those obtained for the few numbers of such fibres in abnormal nerve, are highly significant (e-test P< 1 x 10-9). 4. Scatter diagrams o f the g-ratio (Fig. 2) confirmed the consistent number o f fibres < < 5 p m at W2, and s h o w e d that m o s t o f them had abnormal myelin sheaths (Fig. 3): as c o m p a r e d with normal nerves (see also refs. 9, 10), there was a noticeable dispersion o f g-ratio values (normally concentrated between 0.7 and 0.8). At W3, evolution o f the g-ratio also indicated that regeneration was starting (as in sections 2 and 3). At W12, the g-ratio scattergram was more or less normal. F r o m this preliminary study it seems clear that the 4 loose ligatures placed around the sciatic nerve (still in place at least until W6), induced an acute 'axonopathy' which greatly affected myelinated fibres as seen in the same model [1], and in other lesions based on compression [5, 10]. It seems worth pointing out that pain-related behaviours were m a x i m u m when a consistent number o f < < 5 / ~ m fibres, thus fibres in the A 6 range, could be observed. In addition to their short internodal length, these fibres exhibited a wide range o f g-ratio values, suggesting that m o s t o f them had an abnormal myelin sheath, s o m e being likely atrophic, and others regenerating fibres. The presence o f this group o f abnormal fibres in the A6 range is evocative o f nerve fibres sprouts, observed in n e u r o m a s in various species (refs. in refs. 6, 11), which exhibit intense o n g o i n g electrical activity, abnormal sensitivity to various stimuli and to sympathetic agents, a b n o r m a l contacts or ephapses between axons, (which overall the existence o f potential 'cross talk' between fibres) (refs. in refs. 2, 6, 11). It is difficult to assume that degenerating and regenerating processes are similar in the case o f a partially ligated sciatic nerve, but the present observations suggest this as a possi-
32
Fig. 3. Transverse sections of sciatic nerve at the ligature level: controls in 1 and 2, lesioned nerve at W2 in 3 and 4. Semi-thin sections, To|uidine blue; Nomarski optics. × 120 for 1 and 3, x 300 for 2 and 4 (bars = 100 #m). Note the decrease in myelinated fibre density in 3, and the presence of fibres with thick or very thin (arrows in 4) myelin sheaths in the ligated nerve.
33
bility, and that the abnormal small myelinated fibres could thus be partly responsible for most of the pain-related behaviour observed in these rats. Nevertheless, on the basis of the gate control theory, that nociceptive behaviour could be enhanced by the lack of inhibitory controls on messages driven by the small afferent fibres (refs. in refs. 7, 15) is also possible, due to the marked and prolonged damage of the large nervous fibres. In addition, the involvement of damaged unmyelinated fibres appears to be likely (Basbaum et al., in preparation). The authors wish to thank Y.L. Chen and N. Attal for behavioural tests, F. Roudier for histological preparations, and A.H. Dickenson for English revision. 1 Bennett, G.J. and Xie, Y.K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107. 2 Campbell, J.N., Raja, S.N., Cohen, R.H., Manning, D.C., Khan, A.A. and Meyer, R.A., Peripheral neural mechanisms of nociception. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingston, 1989, pp. 22-45. 3 Chaunu, M.P., Ratinahirana, H., Raphael, M., Henin, D., Leport, C., Brun-Vezinet, F., Leger, J.M., Brunet, P. and Hauw, J.J., The spectrum of changes on 20 nerve biopsies in patients with HIV infection, Muscle Nerve, 12 (1989) 452-459. 4 Committee for research and ethical issues of the IASP. Ethical standards for investigations of experimental pain in animals, Pain, 16 (1983) 109-110. 5 Dahlin, L.B., Nordborg, C. and Lundborg, G., Morphologic changes in nerve cell bodies induced by experimental graded nerve compression, Exp. Neurol., 95 (1987) 611-621. 6 Devor, M., The pathophysiology of damaged nerve. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingston, 1989, pp. 63-81. 7 Dyck, P.J., Lambert, E.H. and O'Brien, P.C., Pain in peripheral neuropathy related to rate and kind of fiber degeneration, Neurology, 26 (1976) 466-471. 8 Dyck, P.J., Karnes, J., Lais, A., Logfren, E.P. and Stevens, J.C., Pathologic alterations of the peripheral nervous system in humans. In P.J. Dyck et al. (Eds.), Peripheral Neuropathy, Saunders, Philadelphia, 1984, Vol. I, pp. 760-870. 9 Friede, R.L. and Beuche, W., A new approach towards analyzing peripheral nerve fiber populations. I. Variance in sheath thickness corresponds to different geometric proportions of the internodes, J. Neuropathol. Exp. Neurol., 44 (1985) 60-72. 10 Hildebrand, C., Kocsis, J.D., Berglund, S. and Waxman, S.G., Myelin sheath remodelling in regenerated rat sciatic nerve, Brain Res., 358 (1985) 163-170. 11 J~inig, W., Pathophysiology of nerve following mechanical injury. In R. Dubner et al. (Eds.), Pain Research and Clinical Management, Vol. 3, Proc. Vth World Congress on pain, Elsevier, Amsterdam, 1988, pp. 89-108. 12 Jazat, F., Attal, N., Gautron, M. and Guilbaud, G., Behavioural evidence that a peripheral experimental neuropathy in rat produces abnormal pain sensation, Eur. J. Neurosc., Suppl. (1988) 179. 13 Merskey, H., Lindblom, U., Mumford, J.M., Nathan, P.W., Noordenbos, W. and Sunderland, S., Pain terms. A current list with definitions and notes on usage, Pain, Suppl. 3 (1986) $217-221. 14 Ochoa, J. and Noordenbos, W., Pathology and disorders sensation in local nerve lesions: An attempt at correlation. In J.J. Bonica et al. (Eds.), Advances in Pain Research and Therapy, Vol. 3, Raven, New York, 1979, pp. 67-77. 15 Ratinahirana, H., Duyckaerts, C., Chaunu, M.P., Henin, D. and Hauw, J.J., Un systrme simple d'analyse semi-automatique d'image, Ann. Pathol., 9 (1989) 68-70.