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Neuropeptide changes persist in spinal cord despite resolving hyperalgesia in a rat model of mononeuropathy
Brain Research 743 Ž1996. 102–108
Research report
Neuropeptide changes persist in spinal cord despite resolving hyperalgesia in a rat model of mononeuropathy R. Munglani a
a, )
, S.M. Harrison b, G.D. Smith b, C. Bountra b, P.J. Birch b, P.J. Elliot b, S.P. Hunt
c
UniÕersity Department of Anaesthesia, UniÕersity of Cambridge Clinical School, Box 93, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK b Glaxo-Wellcome, Gunnelswood Road, SteÕenage, Herts SG1 2NY, UK c DiÕision of Neurobiology, Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK Accepted 13 August 1996
Abstract We have previously described the changes in spinal cord neuropeptides in the unilateral sciatic chronic constriction injury ŽCCI. model of Bennett and Xie w Pain, 33 Ž1988. 87–108x at 28 days, a time of maximum mechanical hyperalgesia. In this study we examine the same model 100–120 days post injury by which time resolution of the hyperalgesia and peripheral nerve injury has occurred according to previous studies. Rats underwent either CCI of the sciatic nerve Ž n s 12. or else sham operation Ž n s 8. which involved exposure but no ligation of the nerve. Mechanical hyperalgesia was assessed with a Ugo-Basile analgesymeter and immunohistochemistry performed on the spinal cord sections of the animals and quantified using a confocal microscope. At this late time point CCI rats were no longer significantly mechanically hyperalgesic compared to the sham animals Ž P G 0.09.. However, examination of the lumbar spinal cord revealed the following changes. Ži. The neuropeptides substance P ŽSP. Ž P - 0.0001. and galanin Ž P - 0.003. both showed decreases of about 30% ipsilaterally in immunoreactivity in laminae 1 and 2 of the dorsal horn compared to the sham operated animals. Žii. Calcitonin gene-related peptide ŽCGRP. and neuropeptide Y ŽNPY. in laminae 1 and 2 showed no significant changes compared to sham animals. Žiii. NPY levels in laminae 3 and 4 of the spinal cord showed a 15% increase in immunoreactivity compared to sham animals Ž P s 0.008..These results indicate that changes in neuronal markers in the spinal cord can persist after apparent resolution of a peripheral nerve injury. We suggest that these changes may form a substrate for subsequent development of abnormal pain states. Keywords: Neuropeptide; Hyperalgesia; Spinal cord; Pain; Neuropathic; Model; Rat; Nerve injury; Chronic constriction injury
1. Introduction Bennett and Xie described in 1988 a peripheral nerve ligation model in the rat which has many of the features of a neuropathic pain state in humans w1,4,20x. In this chronic constriction injury ŽCCI. model, the sciatic nerve on one side is loosely ligated with chromic cat gut. Following this, oedema of the nerve causes restriction of the perineural blood supply and damage to the peripheral axons. There is damage to both myelinated and unmyelinated peripheral axons, but the damage is greater to the myelinated fibres w7,28x. This damage is accompanied by hyperalgesia and allodynia which develop over the following 7–10 days. The unilateral mechanical and thermal hyperalgesia in this model peaks at 14–28 days and persists for up to 28–80 days w1,4,15,24x. The appearance of the peripheral nerves )
Corresponding author. Fax: q44 Ž1223. 21-7223; E-mail: [email protected]
returns to normal after the resolution of the hyperalgesia and usually by 80 days w7,11x. As well as the peripheral nerve changes, the CCI model is accompanied by neurochemical changes in the spinal cord and dorsal root ganglia. These neurochemical changes have been well described at 14–28 days, a time when the model displays marked hyperalgesia w3,6,10,15,24,27,29x. Here we report that the resolution of the hyperalgesia is not accompanied by complete restitution of neuropeptide levels in the spinal cord. 2. Materials and methods 2.1. Treatment of animals Strict attention was paid to the IASP guidelines on the use of animals in these studies w40x. In all the studies Glaxo-bred, random hooded rats Žmale, 100–200 g. were used. The animals were housed in solid bottom cages with
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 2 6 - 8
R. Munglani et al.r Brain Research 743 (1996) 102–108
sawdust litter, four to a cage, and allowed food and water ad libitum. Illumination was automatically controlled giving 14 h light from 05.00–19.00 h. Mononeuropathy was induced according to the method of Bennett and Xie w4x. Animals were anaesthetised with isoflurane Ž2% in 1 lrmin O 2 , 2 lrmin N2 O., the sciatic nerve was exposed in mid-thigh, freed of connective tissue and four ligatures of 4.0 chromic gut were tied loosely around the nerve followed by clips to skin. Survival times were 100 or 120 days. For sham-operation, the sciatic nerve was exposed, but not manipulated. In both groups the wound was closed in layers and secured with suture clips. Animals received loose ligation or sham-operation on one limb only. In both groups, the contralateral limb remained unoperated and served as a further control. Thus both ligated and sham operated animals have an operated and an unoperated side. 2.2. Assessment of behaÕiour To assess the mechanical hyperalgesia, paw withdrawal thresholds measured in grams Žg. of ipsilateral and contralateral limbs were determined using an Analgesymeter ŽUgo-Basile, Italy.. Increasing pressure Ž20 grs up to 500 g max.. was applied to each paw in turn, chosen at random, until a withdrawal flexion was elicited. The test requires that the rat is completely free to remove its paw from the increasing pressure at any time. Applying the device to a human finger only caused a sensation of firm pressure. Hyperalgesia, was defined as by Bennett and Xie w4x, i.e. D PWL g s ipsilateral paw withdrawal threshold Žg. y contralateral paw withdrawal threshold Žg.. Animals were checked regularly. The ligated group displayed characteristic behaviour w1,4x and testing at 28 days confirmed hyperalgesia was established. Final testing at 100–120 days was followed by terminal anaesthesia within 5 min. 2.3. Histological analysis Animals were terminally anaesthetised with saturated aqueous chloral hydrate Ž2 ml i.p.. and perfused intracardially with 200 ml of normal saline followed by 400 ml of freshly depolymerized paraformaldehyde, 4% in 0.1 M sodium phosphate buffer ŽPB., pH 7.4. Spinal cords and the lumbar 4r5 DRG were removed and post-fixed in the latter solution for 2 h and washed overnight in 0.1 M PB containing 30% sucrose and 0.01% sodium azide. The 4th and 5th lumbar segments were identified and cut on a freezing microtome to produce 40-m m-thick sections and saved serially in batches of ten. Sections of spinal cord were incubated free-floating overnight in rabbit anti serum to either SP Žgift of P. Emson., CGRP Žgift of K. Stemini., NPY Žgift of P. Emson., NK1 Žgift of S.R. Vigna. or galanin Žgift of J. Polak.. These sections were processed then washed in PB for 30 min and incubated with anti rabbit IgG ŽVector-BA1000; Vector Laboratories, Peter-
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borough, UK. and subsequently either with an avidin-biotin-horseradish peroxidase kit ŽABC-HRP; Vector-SK6100. if examining for NK1, or directly for immunofluorescence using avidin-FITC ŽVector-A2001. if examining for SP, CGRP, galanin or NPY. Sections were rinsed after both incubations in 0.1 M PB for 30 min and if processed with ABC-HRP, finally reacted with diaminobenzidine-peroxidase kit ŽDAB; Vector-SK4100. to reveal a brown reaction product. Omission of first antibody abolished the immunostaining. Lectin histochemistry followed the protocol of Williams et al. w34x. Processing was done in large batches with tissue from all groups to minimise errors caused by variations in staining methods. Quantification of changes in SP, CGRP, galanin and NPY staining in the superficial dorsal horn were performed using a MRC 600 Bio-Rad confocal laser microscope and FITC immunohistochemistry as previously described w24x. Measurements were made in laminae 1–2 and also laminae 3–4 of the dorsal horn if appropriate using a measurement area of 2738 m m2 . Initial adjustment of the imaging system software was done to keep the fluorescent measurement signals in the linear part of the range Žpixel luminosity range 50–200., but once adjusted, the same settings were used for all measurements with a particular neuropeptide. Five spinal cord sections per neuropeptide measurement per animal were examined. The unoperated sides of both ligation and sham animals were examined qualitatively and quantitatively to look for contralateral effects, and if appropriate the results were then expressed as a percentage change in peptide immunoreactivity in operated side compared to the unoperated side for each animal. All measurements were performed blind to the behavioural measurements and drug treatment of the animal. 2.4. Statistical analysis The data were analysed with Statview 4.1 ŽAbacus concepts, Berkeley, CA. on a Macintosh Quadra 610 computer. A Mann-Whitney test was applied to the data with a Bonferroni correction as appropriate and statistical significance was considered at the 5% level. Numerical data are displayed as means " S.E.M.
3. Results A total of 20 animals were studied behaviourally and histologically, six CCI and four sham animals at 100 days and the same number again at 120 days. 3.1. BehaÕioural results We found that the control Žunoperated. paw withdrawal latencies ŽPWLs. of all animals were also statistically similar Ž P s 0.7, Mann-Whitney. and the control PWL was also indistinguishable to naive animals’ PWLs Ždata
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not shown.. Hyperalgesia scores are therefore presented as difference in paw withdrawal threshold between the operated-unoperated paws in grams Ž D PWL g .. We also found that the behaviour of animals did not change between the time points of 100 and 120 days and so the data from these two time points were pooled before further analysis. At 28 days CCI animals displayed typical behaviour as previously described including guarding, elevation and spontaneous withdrawal of the affected limb, and furthermore the affected paw showed trophic changes w1,4x. None of these changes were seen in the sham group. As in our previous study, testing the animals at 28 days showed significant hyperalgesia compared to sham animals Žligated animals D PWL g s y80 " 20, sham animal D PWL g s 0 " 4. Ž P s 0.002, Mann-Whitney.. However, by 100–120 days there was no significant difference in mechanical hyperalgesia scores between the sham Ž D PWL g s y15 " 10. and ligated group Ž D PWL g s y46 " 19. Ž P ) 0.09, MannWhitney.. 3.2. Changes in neuronal markers For SP, CGRP, galanin and NPY, changes were statistically indistinguishable for the two time points and so the
results have been pooled before further analysis. The results of a quantitative analysis of changes in SP, CGRP, galanin and NPY in both CCI and sham animals at 100–120 days using immunofluorescence and confocal microscopy are shown in Fig. 1. The immunostaining on the unoperated side of both ligated and sham animals appeared indistinguishable from each other and revealed no statistical difference in intensity, and we therefore chose to present the changes in neuropeptide staining as a percentage of the contralateral staining intensity w24x. We found there were decreases in SP Ž P - 0.0001, Mann-Whitney. and galanin immunostaining Ž P ) 0.003, Mann-Whitney. in laminae 1–2 of the dorsal horn compared to the sham group ŽFig. 1.. Laminae 1–2 CGRP Ž P ) 0.3, Mann-Whitney. and NPY Ž P ) 0.8, Mann-Whitney. revealed no significant change compared to the sham group. In contrast NPY in laminae 3–4 showed a significant increase in immunoreactivity compared to that seen in the sham animals Ž P - 0.008, Mann-Whitney.. Photomicrographs of the CCI animals only are presented in Fig. 2, as the sham groups revealed no significant changes. At 100–120 days the dorsal horn of the spinal cord showed little or no qualitative decrease in lectin IB4 binding in the area corresponding to the innervation of the sciatic nerve w8,30x ŽFig. 2.. Sham animals showed no change in lectin binding Ždata not shown.. A qualitative analysis of the NK1 staining showed a typical pattern of fine immunoreactive dendrites and cell bodies mainly in laminae 1 and 3. In the ligated group, there was a moderate increase in the density of this staining ipsilateral to the injury compared to the contralateral side in the region corresponding to the decrease in SP staining and also the innervation of the sciatic nerve ŽFig. 2.. NK1 receptor staining showed no discernible change in the sham animals Ždata not shown.. 4. Discussion 4.1. Summary of findings The CCI animals showed typical behavioural changes at 28 days and no significant remaining hyperalgesia at 100– 120 days post injury. The resolution of the hyperalgesia is comparable to that seen in previous studies w1,4,7,11x. Despite the lack of hyperalgesia, significant decreases in SP and galanin staining were seen in laminae 1–2, and an increase in laminae 3–4 of NPY, all compared to sham animals.
Fig. 1. Results of a quantitative analysis of the peptide changes at 100–120 days in the CCI model. Results are expressed as percentage change in immunoreactive staining in the dorsal horn corresponding to the injured or sham-operated sciatic nerve against the unoperated contralateral side. Only laminae 1–2 SP, galanin and laminae 3–4 NPY show significant changes compared to sham operated animals at the 100–120 day time point Ž ) .. See text for exact P-values.
4.2. Do changes in spinal cord markers eÕer recoÕer after peripheral nerÕe injury? Peptide changes in the spinal cord have previously been examined at long time points after sciatic nerve injury. One study showed that if the nerve is crushed rather than transected Ži.e. the myelin sheaths are still intact., there
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was a drop in SP staining in the spinal cord which was maximal at 14–15 days and recovered by about 35 days Žas assessed by visual inspection. w2x. In contrast to the
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effects of crush injury, complete peripheral nerve transection produced a profound loss of staining in laminae 1–2 of the dorsal horn, which still persisted at 35 days, the last
Fig. 2. Changes in dorsal horn immunoreactivity at 100–120 days post-chronic constriction injury ŽCCI.. The right hand panels show the control side of the dorsal horn of the spinal cord whilst those on the left show the lesioned side. Lectin and NK1 were not amenable to quantitative analysis and are therefore shown with DAB staining and bright field microscopy. The rest were quantified after fluorescent immunostaining. In the top left-hand panel the laminae of the spinal cord are indicated. At 100–120 days injury there was recovery of Lectin IB4 Ža marker of non-peptide containing primary afferent C-fibres. in laminae 1–2 to near normal levels. Substance P still showed some depletion in the medial part of the dorsal horn where the sciatic nerve enters Žarrows. w30x. The decrease in SP staining was mirrored by a small increase in the NK1 receptor expression shown by a coarsening of the immunostaining Žarrows.. There was a mismatch between the expression of SP which occurs in laminae 1–2 and of the receptor NK1 which is present in laminae 1 and 3 with very little in lamina 2 w5x. Calcitonin gene-related peptide ŽCGRP. showed only little change compared to the control side. Galanin, which is present in both primary afferent neurones and in neurones intrinsic to the spinal cord, showed decreases in intensity of staining Žarrows.. In contrast, NPY showed clear evidence of an increase on the lesioned side. Scale bar s 100 m m.
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time point examined w2x. Other authors have examined the dorsal horn at later time points after transection and shown that there seems to be some recovery of SP staining in the spinal cord by 6 months but this remained incomplete even at 15 months w12,31x. In man, a decrease in SP staining was found in the lumbar spinal cord in a patient who had had a leg amputation many years previously w16x. These studies indicate that decreases in spinal cord neuropeptides are still found long after peripheral nerve transection, although an degree of recovery may take place. In the CCI model, we previously reported a 60% decrease in substance SP staining at 28 days, and in the present study the decrease was only 30% in substance P, suggesting some degree of recovery w24x; the source of this recovery is likely to be the remaining primary afferents which survived the initial injury w12x. CGRP, which is present almost exclusively in primary afferent fibres in naive animals, did not show a significant decrease compared to sham animals. That SP and CGRP show differential effects in response to nerve injury has been previously described and may be explained perhaps in terms of varying rates of protein synthesis, turnover and release and the possibly the presence of intra-spinal sprouting of CGRP containing primary afferents w10,22,24x. The average increase in laminae 3–4 NPY staining in this study was about 15%, which is much less than 120% increase seen at 28 days w24x. The upregulation in NPY is mainly seen in medium to large size neuronal cell bodies in the DRG, which give rise to the myelinated primary afferents which also terminate in laminae 3–4 of the spinal cord w32,33,39x. The observation that some of the myelinated fibres in the peripheral nerve still appeared abnormal would be a possible explanation for the small persistent increase in NPY seen in our study w11x. Against this, however, is the finding there were only occasional c-Junpositive cells in the dorsal root ganglia of these long-term animals, indicating that regeneration was complete Žunpublished findings.. In this study there was a small decrease in galanin staining at 100 days, though at 28 days we saw no significant changes. Previous studies have indicated variable changes in galanin after nerve injury Ža peptide with both excitatory and inhibitory effects w13x.. Qualitative examination of the spinal cord revealed that there was little or no decrease in lectin IB4 staining at 100 days ŽFig. 2.. In contrast, this same marker at 28 days post nerve ligation did show significant decreases in staining, implying that recovery of staining had taken place in the interval confirming previous findings after axotomy w9,24x. Qualitatively the antibody against NK1 protein Žwhich binds to part of the receptor for Substance P w5x., seemed to show an increased staining in the superficial laminae of the dorsal horn. This may represent compensatory upregulation of the NK1 receptor secondary to the decrease in SP levels and is confirmed by electrophysiological studies which show increased sensitivity to SP after nerve injury w36x.
As discussed in the Section 1, many of the signs of hyperalgesia disappear well before the histology of the peripheral nerve has returned to normal, and in this study we found that there are changes in the spinal cord that persist well beyond the duration of hyperalgesia in the CCI model. 4.3. What is the relationship between the hyperalgesia and the spinal cord changes? These findings raise a number of questions. Firstly, what is the origin of the hyperalgesia in this model, and secondly what are the implications of the spinal cord changes? In the CCI model, there is damage to a large proportion of myelinated and some unmyelinated peripheral axons in this model which is reflected in changes of peptide levels within the spinal cord. Our previous study examining neuropeptide changes in the spinal cord at 28 days post-injury as well as studies of the peripheral nerve pathology in CCI suggest that the hyperalgesia seemed to be related to the degree of myelinated large fibre loss ŽA b and A d fibres. as suggested by the increase in spinal cord NPY w7,11,24,28x. However a background of unmyelinated small fibre loss ŽC-fibre. also seems to be necessary as all hyperalgesic animals at 28 days showed some degree of SP loss in the spinal cord w24x. The imbalance of the large versus small fibres in relation to pain control is reminiscent of the ‘gate theory’ of pain which stressed large fibre modification of small fibre induced activity within the dorsal horn of the spinal cord w23x. These findings of peripheral fibre imbalance in CCI suggest similar mechanisms may operate in chronic pain states. Our previous findings also suggest that the initiation of the hyperalgesia also seems to be related to the electrical discharge produced at the time of CCI induction Žthe ‘injury discharge’., since the subsequent hyperalgesia is markedly delayed if the effect of the injury discharge is blocked by topical pre-emptive local anaesthetic on the sciatic nerve, or systemic MK-801 or clonidine w24,37x. In addition, a factor produced at the site of peripheral nerve injury and transported in to the spinal cord may also contribute to the hyperalgesic state since blockade of peripheral fast axonal transport by topical colchicine attenuates the hyperalgesia w38x. Ectopic discharges also occur within 3 days at the site of nerve injury and in the neuronal cell bodies in the dorsal root ganglia of the injured animal, and are also suggested to maintain the chronic pain state w18,19x. As well as the peripheral contributions, central mechanisms may also contribute to the hyperalgesic state including ectopic nerve discharges in the spinal cord w21,35x. The most parsimonious explanation of our findings in this paper is that the peptide changes are not related to the pain behaviour. However, in view of the known function of neuropeptides in the modulation of sensory transmission
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w13,26x, we suggest that the central changes in neuropeptide expression detailed in this paper could be viewed as either maladaptive, or as an adaptive response to the to the peripheral insult which tend to oppose the developing hyperalgesia. We have shown that an antibody against NK1 receptor protein, the receptor for the excitatory neuromodulator substance P, shows persistent increase in staining in the superficial laminae after resolution of the hyperalgesia. This may represent residual sensitivity to SP in the long term w36x. However, we also saw a decrease in SP levels in the spinal cord Žan excitatory neurotransmitter. and an increase in NPY – a neuropeptide that has been shown to have analgesic activity. The function of NPY as a centrally acting analgesic may be increased after peripheral nerve injury Žfor further discussion see references in w25x.. Both the changes in SP and NPY in changes might serve to reduce the magnitude of the hyperalgesia seen w13,14x. In conclusion, we report that the resolution of the hyperalgesia occurs before the central nerve changes have fully resolved. We cautiously suggest that long-term peptidergic changes seen within the central axons of injured peripheral nerves, particularly NPY, may serve to mask a residual hyperalgesia after a peripheral nerve injury. In addition the injury itself, despite apparent peripheral resolution, may lead to increased vulnerability of the nervous system to subsequent injury and the development of chronic pain states, for which there is some clinical evidence w17x.
Acknowledgements Work presented previously in abstract form: wMunglani, R., Harrison, S., Smith, G., Bountra, C., Elliot, P.J., Birch, P.J., Hunt, S.P., Persistent changes in neuronal markers in a mononeuropathic rat model despite resolution mechanical hyperalgesia, Brain Research Association Abstracts Õol. 12, Oxford, 1995, p. 76x. Financial support from Glaxo-Wellcome Research and Development is gratefully acknowledged. We thank all the staff of the animal house at Glaxo-Wellcome Research and Development, Ware, Herts., for care and behavioural testing of the animals.
References w1x Attal, N., Jazat, F., Kayser, V. and Gilbaud, G., Further evidence for pain related behaviours in a model of unilateral peripheral neuropathy, Pain, 41 Ž1990. 235–251. w2x Barbut, D., Polak, J.M. and Wall, P.D., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Res., 205 Ž1981. 289–298. w3x Bennett, G.J., Kajander, K.C., Sahara, Y., Iadarola, M.J. and Sugimoto, T., Neurochemical and anatomical changes in the dorsal horn of rats with an experimental painful peripheral neuropathy. In F. Cervero, G.J. Bennett and S.M. Headley ŽEds.., Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 463–472.
107
w4x Bennett, G.J. and Xie, Y.-K., A peripheral neuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 Ž1988. 87–108. w5x Brown, J.L., Liu, H., Maggio, J.E., Vigna, S.R., Mantyh, P.W. and Basbaum, A.I., Morphological characterisation of a substance P receptor-immunoreactive neurones in the rat spinal cord and trigeminal nucleus caudalis, J. Comp. Neurol., 356 Ž1995. 327–344. w6x Cameron, A.A., Cliffer, K.D., Dougherty, P.M., Willis, W.D. and Carlton, S.M., Changes in lectin, GAP-43 and neuropeptide staining in the rat superficial dorsal horn following experimental peripheral neuropathy, Neurosci. Lett., 131 Ž1991. 249–252. w7x Coggeshall, R.E., Dougherty, P.M., Pover, C.M. and Carlton, S.M., Is large myelinated fibre loss associated with hyperalgesia in a model of experimental peripheral neuropathy in the rat? Pain, 52 Ž1993. 233–242. w8x Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn, Brain Res., 190 Ž1980. 17–28. w9x Fitzgerald, M. and Vrbova, G., Plasticity of acid phosphatase ŽFRAP. afferent terminal fields and of dorsal horn cell growth in the neonatal rat, J. Comp. Neurol., 240 Ž1985. 414–422. w10x Garrison, C.J., Dougherty, P.M. and Carlton, S.M., Quantitative analysis of Substance P and calcitonin gene-related peptide immunohistochemical staining in the dorsal horn of neuropathic MK-801 treated rats, Brain Res., 607 Ž1993. 205–214. w11x Guilbaud, G., Gautron, M., Jazat, F., Ratinahirana, H., Hassig, R. and Hauw, J.J., Time course of degeneration and regeneration of myelinated nerve fibres following chronic loose ligatures of the rat sciatic nerve: Can nerve lesions be linked to the abnormal pain-related behaviours? Pain, 53 Ž1993. 147–158. w12x Himes, B.T. and Tessler, A., Death of some dorsal root ganglion neurones and plasticity of others following sciatic nerve section in adult and neonatal rats, J. Comp. Neurol., 284 Ž1989. 215–230. w13x Hokfelt, T., Zhang, X. and WiesenfeldHallin, Z., Messenger plasticity in primary sensory neurones following axotomy and its functional implications, Trends Neurosci., 17 Ž1994. 22–30. w14x Hua, X.Y., Boublik, J.H., Spicer, M.A., Rivier, J.E. and Yaksh, T.L., The antinociceptive effect of spinally administered neuropeptide Y: systemic studies on structure–activity relationships, J. Pharmacol. Exp. Ther., 258 Ž1991. 243–248. w15x Hunt, S., Smith, G., Bond, A., Munglani, R., Thomas, T. and Elliot, P., Changes in Immediate Early Genes, Neuropeptides Immunoreactivity and other neuronal markers in the spinal cord and dorsal root ganglion in a rat model of Neuropathic pain. In R. Schmidt and H.G. Schaible ŽEds.., Neuropeptides, Nociception and Pain, Chapman Hall, 1994, pp. 329–349. w16x Hunt, S.P., Rossor, M.N., Emson, P.C. and Clement-Jones, V., Substance P and enkephalins in spinal cord after limb amputation, Lancet, i Ž1982. 1023. w17x Jensen, T.S., Borge, K. and Nielesen, J., Immediate and longterm phantom limb pain in amputees: incidence, clinical characteristics and relationship to pre-amputation limb pain, Pain, 21 Ž1985. 267–278. w18x Kajander, K.C. and Bennett, G.J., Onset of a painful peripheral neuropathy in rat: A partial and differential deafferentation and spontaneous discharge in Ab and Ad primary afferent neurones, J. Neurophysiol., 68 Ž1992. 734–744. w19x Kajander, K.C., Wakisaka, S. and Bennett, G.J., Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat, Neurosci. Lett., 138 Ž1992. 225– 228. w20x Kupers, R.C., Nuytten, D., De, C.M. and Gybels, J.M., A time course analysis of the changes in spontaneous and evoked behaviour in a rat model of neuropathic pain, Pain, 50 Ž1992. 101–111. w21x Laird, J. and Bennett, G.J., An electrophysiological study of dorsal horn neurones in the spinal cord of rats with an experimental peripheral neuropathy, J. Neurophysiol., 69 Ž1993. 2072–2085.
108
R. Munglani et al.r Brain Research 743 (1996) 102–108
w22x McNeill, D.L., Carlton, S.M. and Hulsebosch, C., Intra-spinal sprouting of calcitonin gene related peptide containing primary afferents after deafferentation in the rat, Exp. Neurol., 114 Ž1991. 321–329. w23x Melzack, R. and Wall, P.D., Pain mechanisms: a new theory, Science, 150 Ž1965. 971–979. w24x Munglani, R., Bond, A., Smith, G., Harrison, S., Elliot, P.J., Birch, P.J. and Hunt, S.P., Changes in neuronal markers in a mononeuropathic rat model: relationship between Neuropeptide Y, pre-emptive drug treatment and long term mechanical hyperalgesia, Pain, 63 Ž1995. 21–31. w25x Munglani, R., Hudspith, M. and Hunt, S.P., Therapeutic potential of NPY, Drugs, 52 Ž1996. 371–389. w26x Munglani, R., Hunt, S. and Jones, J.G., Spinal cord and chronic pain. In L. Kaufmann ŽEd.., Anaesthesiology ReÕiews, Vol. 12, Chruchill Livingstone, 1996, pp. 53–76. w27x Nahin, R.L., Ren, K., De Leon, M. and Ruda, M., Primary sensory neurones exhibit altered gene expression in a rat model of neuropathic pain, Pain, 58 Ž1994. 95–108. w28x Nuytten, D., Kupers, R., Lammens, M., Dom, R., Van, H.J. and Gybels, J., Further evidence for myelinated as well as unmyelinated fibre damage in a rat model of neuropathic pain, Exp. Brain Res., 91 Ž1992. 73–78. w29x Sugimoto, T., Bennett, G.J. and Kajander, K.C., Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: Effects of a chronic constriction injury, transection, and strychnine, Pain, 42 Ž1990. 205–213. w30x Swett, J.E. and Woolf, C.J., The somatotopic organisation of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 Ž1985. 66–77. w31x Tessler, A., Himes, B.T., Krieger, N.R., Murray, M. and Gold-
w32x
w33x
w34x
w35x w36x w37x
w38x
w39x
w40x
berger, M.E., Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord, Brain Res., 332 Ž1985. 209–218. Wakisaka, S., Kajander, K.C. and Bennett, C.J., Effects of peripheral nerve injuries and tissue inflammation on the levels of neurpoptide Y-like immunoreactivity in rat primary afferent neurones, Brain Res., 598 Ž1992. 349–352. Wakisaka, S., Kajander, K.C. and Bennett, G.J., Increased neuropeptide Y ŽNPY.-like immunoreactivity in rat sensory neurones following peripheral axotomy, Neurosci. Lett., 124 Ž1991. 200–203. Williams, S., Evan, G. and Hunt, S.P., C-fos induction in the spinal cord after peripheral nerve lesion, Eur. J. Neurosci., 3 Ž1991. 887–894. Woolf, C., Evidence for a central component of post injury pain, Nature, 306 Ž1983. 686–688. Wright, D.M. and Roberts, M.H.T., Supersensitivity to a substance P analogue following dorsal root section, Life Sci., 22 Ž1978. 19–24. Yamamoto, T., Shimoyama, N. and Mizuguchi, T., Role of the injury discharge in the development of thermal hyperaesthesia after sciatic nerve constriction in the rat, Anaesthesiology, 79 Ž1993. 993–1002. Yamamoto, T. and Yaksh, T.L., Effects of colchicine applied to the peripheral nerve on thermal hyperalgesia evoked with chronic nerve constriction, Pain, 55 Ž1993. 227–233. Zhang, X., Bean, A.J., Wiesenfeld-Hallin, Z. and Hokfelt, T., Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord. IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptiderpeptide histidine isoleucine, Neuroscience, 64 Ž1995. 917–941. Zimmerman, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 Ž1983. 109–110.
Research report
Neuropeptide changes persist in spinal cord despite resolving hyperalgesia in a rat model of mononeuropathy R. Munglani a
a, )
, S.M. Harrison b, G.D. Smith b, C. Bountra b, P.J. Birch b, P.J. Elliot b, S.P. Hunt
c
UniÕersity Department of Anaesthesia, UniÕersity of Cambridge Clinical School, Box 93, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK b Glaxo-Wellcome, Gunnelswood Road, SteÕenage, Herts SG1 2NY, UK c DiÕision of Neurobiology, Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK Accepted 13 August 1996
Abstract We have previously described the changes in spinal cord neuropeptides in the unilateral sciatic chronic constriction injury ŽCCI. model of Bennett and Xie w Pain, 33 Ž1988. 87–108x at 28 days, a time of maximum mechanical hyperalgesia. In this study we examine the same model 100–120 days post injury by which time resolution of the hyperalgesia and peripheral nerve injury has occurred according to previous studies. Rats underwent either CCI of the sciatic nerve Ž n s 12. or else sham operation Ž n s 8. which involved exposure but no ligation of the nerve. Mechanical hyperalgesia was assessed with a Ugo-Basile analgesymeter and immunohistochemistry performed on the spinal cord sections of the animals and quantified using a confocal microscope. At this late time point CCI rats were no longer significantly mechanically hyperalgesic compared to the sham animals Ž P G 0.09.. However, examination of the lumbar spinal cord revealed the following changes. Ži. The neuropeptides substance P ŽSP. Ž P - 0.0001. and galanin Ž P - 0.003. both showed decreases of about 30% ipsilaterally in immunoreactivity in laminae 1 and 2 of the dorsal horn compared to the sham operated animals. Žii. Calcitonin gene-related peptide ŽCGRP. and neuropeptide Y ŽNPY. in laminae 1 and 2 showed no significant changes compared to sham animals. Žiii. NPY levels in laminae 3 and 4 of the spinal cord showed a 15% increase in immunoreactivity compared to sham animals Ž P s 0.008..These results indicate that changes in neuronal markers in the spinal cord can persist after apparent resolution of a peripheral nerve injury. We suggest that these changes may form a substrate for subsequent development of abnormal pain states. Keywords: Neuropeptide; Hyperalgesia; Spinal cord; Pain; Neuropathic; Model; Rat; Nerve injury; Chronic constriction injury
1. Introduction Bennett and Xie described in 1988 a peripheral nerve ligation model in the rat which has many of the features of a neuropathic pain state in humans w1,4,20x. In this chronic constriction injury ŽCCI. model, the sciatic nerve on one side is loosely ligated with chromic cat gut. Following this, oedema of the nerve causes restriction of the perineural blood supply and damage to the peripheral axons. There is damage to both myelinated and unmyelinated peripheral axons, but the damage is greater to the myelinated fibres w7,28x. This damage is accompanied by hyperalgesia and allodynia which develop over the following 7–10 days. The unilateral mechanical and thermal hyperalgesia in this model peaks at 14–28 days and persists for up to 28–80 days w1,4,15,24x. The appearance of the peripheral nerves )
Corresponding author. Fax: q44 Ž1223. 21-7223; E-mail: [email protected]
returns to normal after the resolution of the hyperalgesia and usually by 80 days w7,11x. As well as the peripheral nerve changes, the CCI model is accompanied by neurochemical changes in the spinal cord and dorsal root ganglia. These neurochemical changes have been well described at 14–28 days, a time when the model displays marked hyperalgesia w3,6,10,15,24,27,29x. Here we report that the resolution of the hyperalgesia is not accompanied by complete restitution of neuropeptide levels in the spinal cord. 2. Materials and methods 2.1. Treatment of animals Strict attention was paid to the IASP guidelines on the use of animals in these studies w40x. In all the studies Glaxo-bred, random hooded rats Žmale, 100–200 g. were used. The animals were housed in solid bottom cages with
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 2 6 - 8
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sawdust litter, four to a cage, and allowed food and water ad libitum. Illumination was automatically controlled giving 14 h light from 05.00–19.00 h. Mononeuropathy was induced according to the method of Bennett and Xie w4x. Animals were anaesthetised with isoflurane Ž2% in 1 lrmin O 2 , 2 lrmin N2 O., the sciatic nerve was exposed in mid-thigh, freed of connective tissue and four ligatures of 4.0 chromic gut were tied loosely around the nerve followed by clips to skin. Survival times were 100 or 120 days. For sham-operation, the sciatic nerve was exposed, but not manipulated. In both groups the wound was closed in layers and secured with suture clips. Animals received loose ligation or sham-operation on one limb only. In both groups, the contralateral limb remained unoperated and served as a further control. Thus both ligated and sham operated animals have an operated and an unoperated side. 2.2. Assessment of behaÕiour To assess the mechanical hyperalgesia, paw withdrawal thresholds measured in grams Žg. of ipsilateral and contralateral limbs were determined using an Analgesymeter ŽUgo-Basile, Italy.. Increasing pressure Ž20 grs up to 500 g max.. was applied to each paw in turn, chosen at random, until a withdrawal flexion was elicited. The test requires that the rat is completely free to remove its paw from the increasing pressure at any time. Applying the device to a human finger only caused a sensation of firm pressure. Hyperalgesia, was defined as by Bennett and Xie w4x, i.e. D PWL g s ipsilateral paw withdrawal threshold Žg. y contralateral paw withdrawal threshold Žg.. Animals were checked regularly. The ligated group displayed characteristic behaviour w1,4x and testing at 28 days confirmed hyperalgesia was established. Final testing at 100–120 days was followed by terminal anaesthesia within 5 min. 2.3. Histological analysis Animals were terminally anaesthetised with saturated aqueous chloral hydrate Ž2 ml i.p.. and perfused intracardially with 200 ml of normal saline followed by 400 ml of freshly depolymerized paraformaldehyde, 4% in 0.1 M sodium phosphate buffer ŽPB., pH 7.4. Spinal cords and the lumbar 4r5 DRG were removed and post-fixed in the latter solution for 2 h and washed overnight in 0.1 M PB containing 30% sucrose and 0.01% sodium azide. The 4th and 5th lumbar segments were identified and cut on a freezing microtome to produce 40-m m-thick sections and saved serially in batches of ten. Sections of spinal cord were incubated free-floating overnight in rabbit anti serum to either SP Žgift of P. Emson., CGRP Žgift of K. Stemini., NPY Žgift of P. Emson., NK1 Žgift of S.R. Vigna. or galanin Žgift of J. Polak.. These sections were processed then washed in PB for 30 min and incubated with anti rabbit IgG ŽVector-BA1000; Vector Laboratories, Peter-
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borough, UK. and subsequently either with an avidin-biotin-horseradish peroxidase kit ŽABC-HRP; Vector-SK6100. if examining for NK1, or directly for immunofluorescence using avidin-FITC ŽVector-A2001. if examining for SP, CGRP, galanin or NPY. Sections were rinsed after both incubations in 0.1 M PB for 30 min and if processed with ABC-HRP, finally reacted with diaminobenzidine-peroxidase kit ŽDAB; Vector-SK4100. to reveal a brown reaction product. Omission of first antibody abolished the immunostaining. Lectin histochemistry followed the protocol of Williams et al. w34x. Processing was done in large batches with tissue from all groups to minimise errors caused by variations in staining methods. Quantification of changes in SP, CGRP, galanin and NPY staining in the superficial dorsal horn were performed using a MRC 600 Bio-Rad confocal laser microscope and FITC immunohistochemistry as previously described w24x. Measurements were made in laminae 1–2 and also laminae 3–4 of the dorsal horn if appropriate using a measurement area of 2738 m m2 . Initial adjustment of the imaging system software was done to keep the fluorescent measurement signals in the linear part of the range Žpixel luminosity range 50–200., but once adjusted, the same settings were used for all measurements with a particular neuropeptide. Five spinal cord sections per neuropeptide measurement per animal were examined. The unoperated sides of both ligation and sham animals were examined qualitatively and quantitatively to look for contralateral effects, and if appropriate the results were then expressed as a percentage change in peptide immunoreactivity in operated side compared to the unoperated side for each animal. All measurements were performed blind to the behavioural measurements and drug treatment of the animal. 2.4. Statistical analysis The data were analysed with Statview 4.1 ŽAbacus concepts, Berkeley, CA. on a Macintosh Quadra 610 computer. A Mann-Whitney test was applied to the data with a Bonferroni correction as appropriate and statistical significance was considered at the 5% level. Numerical data are displayed as means " S.E.M.
3. Results A total of 20 animals were studied behaviourally and histologically, six CCI and four sham animals at 100 days and the same number again at 120 days. 3.1. BehaÕioural results We found that the control Žunoperated. paw withdrawal latencies ŽPWLs. of all animals were also statistically similar Ž P s 0.7, Mann-Whitney. and the control PWL was also indistinguishable to naive animals’ PWLs Ždata
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not shown.. Hyperalgesia scores are therefore presented as difference in paw withdrawal threshold between the operated-unoperated paws in grams Ž D PWL g .. We also found that the behaviour of animals did not change between the time points of 100 and 120 days and so the data from these two time points were pooled before further analysis. At 28 days CCI animals displayed typical behaviour as previously described including guarding, elevation and spontaneous withdrawal of the affected limb, and furthermore the affected paw showed trophic changes w1,4x. None of these changes were seen in the sham group. As in our previous study, testing the animals at 28 days showed significant hyperalgesia compared to sham animals Žligated animals D PWL g s y80 " 20, sham animal D PWL g s 0 " 4. Ž P s 0.002, Mann-Whitney.. However, by 100–120 days there was no significant difference in mechanical hyperalgesia scores between the sham Ž D PWL g s y15 " 10. and ligated group Ž D PWL g s y46 " 19. Ž P ) 0.09, MannWhitney.. 3.2. Changes in neuronal markers For SP, CGRP, galanin and NPY, changes were statistically indistinguishable for the two time points and so the
results have been pooled before further analysis. The results of a quantitative analysis of changes in SP, CGRP, galanin and NPY in both CCI and sham animals at 100–120 days using immunofluorescence and confocal microscopy are shown in Fig. 1. The immunostaining on the unoperated side of both ligated and sham animals appeared indistinguishable from each other and revealed no statistical difference in intensity, and we therefore chose to present the changes in neuropeptide staining as a percentage of the contralateral staining intensity w24x. We found there were decreases in SP Ž P - 0.0001, Mann-Whitney. and galanin immunostaining Ž P ) 0.003, Mann-Whitney. in laminae 1–2 of the dorsal horn compared to the sham group ŽFig. 1.. Laminae 1–2 CGRP Ž P ) 0.3, Mann-Whitney. and NPY Ž P ) 0.8, Mann-Whitney. revealed no significant change compared to the sham group. In contrast NPY in laminae 3–4 showed a significant increase in immunoreactivity compared to that seen in the sham animals Ž P - 0.008, Mann-Whitney.. Photomicrographs of the CCI animals only are presented in Fig. 2, as the sham groups revealed no significant changes. At 100–120 days the dorsal horn of the spinal cord showed little or no qualitative decrease in lectin IB4 binding in the area corresponding to the innervation of the sciatic nerve w8,30x ŽFig. 2.. Sham animals showed no change in lectin binding Ždata not shown.. A qualitative analysis of the NK1 staining showed a typical pattern of fine immunoreactive dendrites and cell bodies mainly in laminae 1 and 3. In the ligated group, there was a moderate increase in the density of this staining ipsilateral to the injury compared to the contralateral side in the region corresponding to the decrease in SP staining and also the innervation of the sciatic nerve ŽFig. 2.. NK1 receptor staining showed no discernible change in the sham animals Ždata not shown.. 4. Discussion 4.1. Summary of findings The CCI animals showed typical behavioural changes at 28 days and no significant remaining hyperalgesia at 100– 120 days post injury. The resolution of the hyperalgesia is comparable to that seen in previous studies w1,4,7,11x. Despite the lack of hyperalgesia, significant decreases in SP and galanin staining were seen in laminae 1–2, and an increase in laminae 3–4 of NPY, all compared to sham animals.
Fig. 1. Results of a quantitative analysis of the peptide changes at 100–120 days in the CCI model. Results are expressed as percentage change in immunoreactive staining in the dorsal horn corresponding to the injured or sham-operated sciatic nerve against the unoperated contralateral side. Only laminae 1–2 SP, galanin and laminae 3–4 NPY show significant changes compared to sham operated animals at the 100–120 day time point Ž ) .. See text for exact P-values.
4.2. Do changes in spinal cord markers eÕer recoÕer after peripheral nerÕe injury? Peptide changes in the spinal cord have previously been examined at long time points after sciatic nerve injury. One study showed that if the nerve is crushed rather than transected Ži.e. the myelin sheaths are still intact., there
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was a drop in SP staining in the spinal cord which was maximal at 14–15 days and recovered by about 35 days Žas assessed by visual inspection. w2x. In contrast to the
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effects of crush injury, complete peripheral nerve transection produced a profound loss of staining in laminae 1–2 of the dorsal horn, which still persisted at 35 days, the last
Fig. 2. Changes in dorsal horn immunoreactivity at 100–120 days post-chronic constriction injury ŽCCI.. The right hand panels show the control side of the dorsal horn of the spinal cord whilst those on the left show the lesioned side. Lectin and NK1 were not amenable to quantitative analysis and are therefore shown with DAB staining and bright field microscopy. The rest were quantified after fluorescent immunostaining. In the top left-hand panel the laminae of the spinal cord are indicated. At 100–120 days injury there was recovery of Lectin IB4 Ža marker of non-peptide containing primary afferent C-fibres. in laminae 1–2 to near normal levels. Substance P still showed some depletion in the medial part of the dorsal horn where the sciatic nerve enters Žarrows. w30x. The decrease in SP staining was mirrored by a small increase in the NK1 receptor expression shown by a coarsening of the immunostaining Žarrows.. There was a mismatch between the expression of SP which occurs in laminae 1–2 and of the receptor NK1 which is present in laminae 1 and 3 with very little in lamina 2 w5x. Calcitonin gene-related peptide ŽCGRP. showed only little change compared to the control side. Galanin, which is present in both primary afferent neurones and in neurones intrinsic to the spinal cord, showed decreases in intensity of staining Žarrows.. In contrast, NPY showed clear evidence of an increase on the lesioned side. Scale bar s 100 m m.
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time point examined w2x. Other authors have examined the dorsal horn at later time points after transection and shown that there seems to be some recovery of SP staining in the spinal cord by 6 months but this remained incomplete even at 15 months w12,31x. In man, a decrease in SP staining was found in the lumbar spinal cord in a patient who had had a leg amputation many years previously w16x. These studies indicate that decreases in spinal cord neuropeptides are still found long after peripheral nerve transection, although an degree of recovery may take place. In the CCI model, we previously reported a 60% decrease in substance SP staining at 28 days, and in the present study the decrease was only 30% in substance P, suggesting some degree of recovery w24x; the source of this recovery is likely to be the remaining primary afferents which survived the initial injury w12x. CGRP, which is present almost exclusively in primary afferent fibres in naive animals, did not show a significant decrease compared to sham animals. That SP and CGRP show differential effects in response to nerve injury has been previously described and may be explained perhaps in terms of varying rates of protein synthesis, turnover and release and the possibly the presence of intra-spinal sprouting of CGRP containing primary afferents w10,22,24x. The average increase in laminae 3–4 NPY staining in this study was about 15%, which is much less than 120% increase seen at 28 days w24x. The upregulation in NPY is mainly seen in medium to large size neuronal cell bodies in the DRG, which give rise to the myelinated primary afferents which also terminate in laminae 3–4 of the spinal cord w32,33,39x. The observation that some of the myelinated fibres in the peripheral nerve still appeared abnormal would be a possible explanation for the small persistent increase in NPY seen in our study w11x. Against this, however, is the finding there were only occasional c-Junpositive cells in the dorsal root ganglia of these long-term animals, indicating that regeneration was complete Žunpublished findings.. In this study there was a small decrease in galanin staining at 100 days, though at 28 days we saw no significant changes. Previous studies have indicated variable changes in galanin after nerve injury Ža peptide with both excitatory and inhibitory effects w13x.. Qualitative examination of the spinal cord revealed that there was little or no decrease in lectin IB4 staining at 100 days ŽFig. 2.. In contrast, this same marker at 28 days post nerve ligation did show significant decreases in staining, implying that recovery of staining had taken place in the interval confirming previous findings after axotomy w9,24x. Qualitatively the antibody against NK1 protein Žwhich binds to part of the receptor for Substance P w5x., seemed to show an increased staining in the superficial laminae of the dorsal horn. This may represent compensatory upregulation of the NK1 receptor secondary to the decrease in SP levels and is confirmed by electrophysiological studies which show increased sensitivity to SP after nerve injury w36x.
As discussed in the Section 1, many of the signs of hyperalgesia disappear well before the histology of the peripheral nerve has returned to normal, and in this study we found that there are changes in the spinal cord that persist well beyond the duration of hyperalgesia in the CCI model. 4.3. What is the relationship between the hyperalgesia and the spinal cord changes? These findings raise a number of questions. Firstly, what is the origin of the hyperalgesia in this model, and secondly what are the implications of the spinal cord changes? In the CCI model, there is damage to a large proportion of myelinated and some unmyelinated peripheral axons in this model which is reflected in changes of peptide levels within the spinal cord. Our previous study examining neuropeptide changes in the spinal cord at 28 days post-injury as well as studies of the peripheral nerve pathology in CCI suggest that the hyperalgesia seemed to be related to the degree of myelinated large fibre loss ŽA b and A d fibres. as suggested by the increase in spinal cord NPY w7,11,24,28x. However a background of unmyelinated small fibre loss ŽC-fibre. also seems to be necessary as all hyperalgesic animals at 28 days showed some degree of SP loss in the spinal cord w24x. The imbalance of the large versus small fibres in relation to pain control is reminiscent of the ‘gate theory’ of pain which stressed large fibre modification of small fibre induced activity within the dorsal horn of the spinal cord w23x. These findings of peripheral fibre imbalance in CCI suggest similar mechanisms may operate in chronic pain states. Our previous findings also suggest that the initiation of the hyperalgesia also seems to be related to the electrical discharge produced at the time of CCI induction Žthe ‘injury discharge’., since the subsequent hyperalgesia is markedly delayed if the effect of the injury discharge is blocked by topical pre-emptive local anaesthetic on the sciatic nerve, or systemic MK-801 or clonidine w24,37x. In addition, a factor produced at the site of peripheral nerve injury and transported in to the spinal cord may also contribute to the hyperalgesic state since blockade of peripheral fast axonal transport by topical colchicine attenuates the hyperalgesia w38x. Ectopic discharges also occur within 3 days at the site of nerve injury and in the neuronal cell bodies in the dorsal root ganglia of the injured animal, and are also suggested to maintain the chronic pain state w18,19x. As well as the peripheral contributions, central mechanisms may also contribute to the hyperalgesic state including ectopic nerve discharges in the spinal cord w21,35x. The most parsimonious explanation of our findings in this paper is that the peptide changes are not related to the pain behaviour. However, in view of the known function of neuropeptides in the modulation of sensory transmission
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w13,26x, we suggest that the central changes in neuropeptide expression detailed in this paper could be viewed as either maladaptive, or as an adaptive response to the to the peripheral insult which tend to oppose the developing hyperalgesia. We have shown that an antibody against NK1 receptor protein, the receptor for the excitatory neuromodulator substance P, shows persistent increase in staining in the superficial laminae after resolution of the hyperalgesia. This may represent residual sensitivity to SP in the long term w36x. However, we also saw a decrease in SP levels in the spinal cord Žan excitatory neurotransmitter. and an increase in NPY – a neuropeptide that has been shown to have analgesic activity. The function of NPY as a centrally acting analgesic may be increased after peripheral nerve injury Žfor further discussion see references in w25x.. Both the changes in SP and NPY in changes might serve to reduce the magnitude of the hyperalgesia seen w13,14x. In conclusion, we report that the resolution of the hyperalgesia occurs before the central nerve changes have fully resolved. We cautiously suggest that long-term peptidergic changes seen within the central axons of injured peripheral nerves, particularly NPY, may serve to mask a residual hyperalgesia after a peripheral nerve injury. In addition the injury itself, despite apparent peripheral resolution, may lead to increased vulnerability of the nervous system to subsequent injury and the development of chronic pain states, for which there is some clinical evidence w17x.
Acknowledgements Work presented previously in abstract form: wMunglani, R., Harrison, S., Smith, G., Bountra, C., Elliot, P.J., Birch, P.J., Hunt, S.P., Persistent changes in neuronal markers in a mononeuropathic rat model despite resolution mechanical hyperalgesia, Brain Research Association Abstracts Õol. 12, Oxford, 1995, p. 76x. Financial support from Glaxo-Wellcome Research and Development is gratefully acknowledged. We thank all the staff of the animal house at Glaxo-Wellcome Research and Development, Ware, Herts., for care and behavioural testing of the animals.
References w1x Attal, N., Jazat, F., Kayser, V. and Gilbaud, G., Further evidence for pain related behaviours in a model of unilateral peripheral neuropathy, Pain, 41 Ž1990. 235–251. w2x Barbut, D., Polak, J.M. and Wall, P.D., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Res., 205 Ž1981. 289–298. w3x Bennett, G.J., Kajander, K.C., Sahara, Y., Iadarola, M.J. and Sugimoto, T., Neurochemical and anatomical changes in the dorsal horn of rats with an experimental painful peripheral neuropathy. In F. Cervero, G.J. Bennett and S.M. Headley ŽEds.., Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum, New York, 1989, pp. 463–472.
107
w4x Bennett, G.J. and Xie, Y.-K., A peripheral neuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 Ž1988. 87–108. w5x Brown, J.L., Liu, H., Maggio, J.E., Vigna, S.R., Mantyh, P.W. and Basbaum, A.I., Morphological characterisation of a substance P receptor-immunoreactive neurones in the rat spinal cord and trigeminal nucleus caudalis, J. Comp. Neurol., 356 Ž1995. 327–344. w6x Cameron, A.A., Cliffer, K.D., Dougherty, P.M., Willis, W.D. and Carlton, S.M., Changes in lectin, GAP-43 and neuropeptide staining in the rat superficial dorsal horn following experimental peripheral neuropathy, Neurosci. Lett., 131 Ž1991. 249–252. w7x Coggeshall, R.E., Dougherty, P.M., Pover, C.M. and Carlton, S.M., Is large myelinated fibre loss associated with hyperalgesia in a model of experimental peripheral neuropathy in the rat? Pain, 52 Ž1993. 233–242. w8x Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn, Brain Res., 190 Ž1980. 17–28. w9x Fitzgerald, M. and Vrbova, G., Plasticity of acid phosphatase ŽFRAP. afferent terminal fields and of dorsal horn cell growth in the neonatal rat, J. Comp. Neurol., 240 Ž1985. 414–422. w10x Garrison, C.J., Dougherty, P.M. and Carlton, S.M., Quantitative analysis of Substance P and calcitonin gene-related peptide immunohistochemical staining in the dorsal horn of neuropathic MK-801 treated rats, Brain Res., 607 Ž1993. 205–214. w11x Guilbaud, G., Gautron, M., Jazat, F., Ratinahirana, H., Hassig, R. and Hauw, J.J., Time course of degeneration and regeneration of myelinated nerve fibres following chronic loose ligatures of the rat sciatic nerve: Can nerve lesions be linked to the abnormal pain-related behaviours? Pain, 53 Ž1993. 147–158. w12x Himes, B.T. and Tessler, A., Death of some dorsal root ganglion neurones and plasticity of others following sciatic nerve section in adult and neonatal rats, J. Comp. Neurol., 284 Ž1989. 215–230. w13x Hokfelt, T., Zhang, X. and WiesenfeldHallin, Z., Messenger plasticity in primary sensory neurones following axotomy and its functional implications, Trends Neurosci., 17 Ž1994. 22–30. w14x Hua, X.Y., Boublik, J.H., Spicer, M.A., Rivier, J.E. and Yaksh, T.L., The antinociceptive effect of spinally administered neuropeptide Y: systemic studies on structure–activity relationships, J. Pharmacol. Exp. Ther., 258 Ž1991. 243–248. w15x Hunt, S., Smith, G., Bond, A., Munglani, R., Thomas, T. and Elliot, P., Changes in Immediate Early Genes, Neuropeptides Immunoreactivity and other neuronal markers in the spinal cord and dorsal root ganglion in a rat model of Neuropathic pain. In R. Schmidt and H.G. Schaible ŽEds.., Neuropeptides, Nociception and Pain, Chapman Hall, 1994, pp. 329–349. w16x Hunt, S.P., Rossor, M.N., Emson, P.C. and Clement-Jones, V., Substance P and enkephalins in spinal cord after limb amputation, Lancet, i Ž1982. 1023. w17x Jensen, T.S., Borge, K. and Nielesen, J., Immediate and longterm phantom limb pain in amputees: incidence, clinical characteristics and relationship to pre-amputation limb pain, Pain, 21 Ž1985. 267–278. w18x Kajander, K.C. and Bennett, G.J., Onset of a painful peripheral neuropathy in rat: A partial and differential deafferentation and spontaneous discharge in Ab and Ad primary afferent neurones, J. Neurophysiol., 68 Ž1992. 734–744. w19x Kajander, K.C., Wakisaka, S. and Bennett, G.J., Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat, Neurosci. Lett., 138 Ž1992. 225– 228. w20x Kupers, R.C., Nuytten, D., De, C.M. and Gybels, J.M., A time course analysis of the changes in spontaneous and evoked behaviour in a rat model of neuropathic pain, Pain, 50 Ž1992. 101–111. w21x Laird, J. and Bennett, G.J., An electrophysiological study of dorsal horn neurones in the spinal cord of rats with an experimental peripheral neuropathy, J. Neurophysiol., 69 Ž1993. 2072–2085.
108
R. Munglani et al.r Brain Research 743 (1996) 102–108
w22x McNeill, D.L., Carlton, S.M. and Hulsebosch, C., Intra-spinal sprouting of calcitonin gene related peptide containing primary afferents after deafferentation in the rat, Exp. Neurol., 114 Ž1991. 321–329. w23x Melzack, R. and Wall, P.D., Pain mechanisms: a new theory, Science, 150 Ž1965. 971–979. w24x Munglani, R., Bond, A., Smith, G., Harrison, S., Elliot, P.J., Birch, P.J. and Hunt, S.P., Changes in neuronal markers in a mononeuropathic rat model: relationship between Neuropeptide Y, pre-emptive drug treatment and long term mechanical hyperalgesia, Pain, 63 Ž1995. 21–31. w25x Munglani, R., Hudspith, M. and Hunt, S.P., Therapeutic potential of NPY, Drugs, 52 Ž1996. 371–389. w26x Munglani, R., Hunt, S. and Jones, J.G., Spinal cord and chronic pain. In L. Kaufmann ŽEd.., Anaesthesiology ReÕiews, Vol. 12, Chruchill Livingstone, 1996, pp. 53–76. w27x Nahin, R.L., Ren, K., De Leon, M. and Ruda, M., Primary sensory neurones exhibit altered gene expression in a rat model of neuropathic pain, Pain, 58 Ž1994. 95–108. w28x Nuytten, D., Kupers, R., Lammens, M., Dom, R., Van, H.J. and Gybels, J., Further evidence for myelinated as well as unmyelinated fibre damage in a rat model of neuropathic pain, Exp. Brain Res., 91 Ž1992. 73–78. w29x Sugimoto, T., Bennett, G.J. and Kajander, K.C., Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: Effects of a chronic constriction injury, transection, and strychnine, Pain, 42 Ž1990. 205–213. w30x Swett, J.E. and Woolf, C.J., The somatotopic organisation of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 Ž1985. 66–77. w31x Tessler, A., Himes, B.T., Krieger, N.R., Murray, M. and Gold-
w32x
w33x
w34x
w35x w36x w37x
w38x
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berger, M.E., Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord, Brain Res., 332 Ž1985. 209–218. Wakisaka, S., Kajander, K.C. and Bennett, C.J., Effects of peripheral nerve injuries and tissue inflammation on the levels of neurpoptide Y-like immunoreactivity in rat primary afferent neurones, Brain Res., 598 Ž1992. 349–352. Wakisaka, S., Kajander, K.C. and Bennett, G.J., Increased neuropeptide Y ŽNPY.-like immunoreactivity in rat sensory neurones following peripheral axotomy, Neurosci. Lett., 124 Ž1991. 200–203. Williams, S., Evan, G. and Hunt, S.P., C-fos induction in the spinal cord after peripheral nerve lesion, Eur. J. Neurosci., 3 Ž1991. 887–894. Woolf, C., Evidence for a central component of post injury pain, Nature, 306 Ž1983. 686–688. Wright, D.M. and Roberts, M.H.T., Supersensitivity to a substance P analogue following dorsal root section, Life Sci., 22 Ž1978. 19–24. Yamamoto, T., Shimoyama, N. and Mizuguchi, T., Role of the injury discharge in the development of thermal hyperaesthesia after sciatic nerve constriction in the rat, Anaesthesiology, 79 Ž1993. 993–1002. Yamamoto, T. and Yaksh, T.L., Effects of colchicine applied to the peripheral nerve on thermal hyperalgesia evoked with chronic nerve constriction, Pain, 55 Ž1993. 227–233. Zhang, X., Bean, A.J., Wiesenfeld-Hallin, Z. and Hokfelt, T., Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord. IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptiderpeptide histidine isoleucine, Neuroscience, 64 Ž1995. 917–941. Zimmerman, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 Ž1983. 109–110.