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Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury
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NeuroscienceLetters186(1995)184-188
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Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury Keith C. Kajander a,b,*, Jiangyan Xu a aDepartment of Oral Science, 17-252 Moos Tower, 515 Delaware Street S.E., Universityof Minnesota, Minneapolis, MN 55455-0329, USA bDepartment of CeU Biology and Neuroanatomy, and Program in Neuroscience, Universityof Minnesota, Minneapolis, MN 55455-0329, USA
Received 14 October 1994; revisedversionreceived4 January 1995; accepted4 January 1995
Abstract
Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury. Keywords: Chronic constriction injury; Hyperalgesia; Nerve compression syndromes; Neuralgia; Neuroma; Neuropeptides; Radio-
immunoassay; Sciatic nerve
In the past few years, several animal models have been introduced for evaluating changes in the nervous system following peripheral nerve injury [6]. Of these models, the chronic constriction injury of the sciatic nerve (CCI) has become the focus of a great deal of recent research [4]. The CCI appears to injure selectively axons in the sciatic nerve; there is massive degeneration of large myelinated axons but only partial degeneration of thinly myelinated and unmyelinated axons [3,6]. As axonal degeneration occurs in the nerve, neurochemical alterations occur in the dorsal horn of the lumbar spinal cord in segments that receive intraspinal termination's of the sciatic nerve. For example~ calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir), as evaluated using immunocytochemical techniques, is decreased in the ipsilateral dorsal horn at 10, 20, and 28 days after the CCI [5,7]. Bennett et al. [5] confirmed these immunocytochemical findings using radioimmunoassay (RIA). However, a recent study reported that immunocytochemical * Corresponding author (to Departmentof Oral Science), Tel.: + 1 612 6260632; Fax: +1 612 6262651; E-mail: [email protected].
labeling for CGRP-ir was not decreased in the ipsilateral spinal cord at either 7 or 14 days after the CCI [8]. These inconsistencies suggest that further evaluation of alterations in CGRP-ir and SP-ir within the spinal cord is needed. In addition, previous studies have focused on short-term (7-28 days) alterations while changes at rimes later than these have not been evaluated. We decided to re-examine this issue using RIA, and we included additional groups not used in the previous studies. The major goal of this study was to determine levels of CGRP-ir and SP-ir in the dorsal spinal tord at a time-point long after induction of the CCI (60 days), but it also was desirable to re-evaluate peptide levels round shortly after the injury (10 days). Peptide levels at these two rimes were compared to each other, to peptide levels from unoperated control animals, and also to peptide levels seen after transection of the sciatic nerve. Thirty, adult, maie Sprague-Dawley rats (275-350 g) were randomly divided into rive groups. Rats in two groups had the left sciatic nerve transected and were sacrificed 10 (n = 6) and 31 (n = 6) days later. Rats in two other groups had the CCI induced on the left sciatic nerve and were sacrificed 10 (n = 5) and 60 (n = 7) days later.
0304-3940/95/$09.50 © 1995 E|sevier Science lreland Ltd. Ail rights reserved SSDI 0304-3940(95)1 1294-4
185
K.C. Kajander, J. Xu I Neuroscience Letters 186 (1995) 184-188
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Fig. 1. Histograms illustrating changes in CGRP-ir foliowing transection or CCI of the left sciatic nerve. Bars represent means + standard error of the means for CGRP-ir in the L4-L5 segments of the spinal dorsal quadrants. Contra, quadrant eontralateral to the injury; Ipsi, quadrant ipsilateral to the injury; C, unoperated eontrol group. Asterisk (*) indicates significant difference on the relevant side from the unoperated eonrol group. Triangle (A) indicates significant difference on relevant side between 31-day and 10-day transection groups or on relevant side between 60-day and 10-day CCI groups. Circle (O) indicates significant difference between relevant side of 10-day transection and 10-day CCI groups or between relevant side of 31-day transection and 60-day CCI groups. Diamond (~) indicates significant difference between sides in the same experimental group. *P < 0.05, **P < 0.01, ZXp< 0.05, zXZXp< 0.01, Op < 0.05, OOp < 0.01, ~ P < 0.05, <>~P < 0.01. Rats in the fifth group served as unoperated controls (n = 6); two of these rats were sacrificed at the same rime as rats in the two 10-day groups, and four were sacrificed at 60 days. Ail rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). For rats in the transection groups, the left sciatic nerve was exposed, two ligatures were tied tightly about it (5 mm apart), and a short length of the nerve ( 3 4 mm) was removed from between the ligatures. The CCI was produced according to the protocol of Bennett and Xie [4]. The left sciatic nerve was slightly constricted by tying four chromic gut ligatures. At the time of sacrifice, rats were deeply anesthetized (80 mg/kg i.p.) and decapitated. As described previously [10], the spinal cord was forcibly ejected, and the approximate lumbar 4-5 (L4-5) spinal segments were isolated and divided into left dorsal, left ventral, right dorsal, and right ventral quadrants by two perpendicular cuts through the central canal. Tissue samples were quickly frozen in multi-well plates and stored at - 8 0 ° C until assayed. The RIA procedures are described elsewhere [20]. Tissue samples from ail groups were blindly assayed simultaneously in duplicate. Total protein levels, as determined using the method of Lowry, were used to normalize pep-
tide levels between samples. Only results from the dorsal quadrants are reported here. After a significant two-way A N O V A (treatment x rime) for each peptide, Duncan's multiple range test was performed to examine for differences in means. We specified Duncan's test to evaluate six comparisons: (1) side-to-side (i.e. ipsilateral versus contralateral) differences within each group; (2) differences between the four treatment groups and the unoperated control group; (3) differences between the 10-day transection group and the 31-day transection group; (4) differences between the 10day CCI group and the 60-day CCI group ((3) and (4) represent a change over time); (5) differences between the 10-day transection group and the 10-day CCI group; and (6) differences between the 31-day transection group and the 60-day CCI group. Ail comparisons, except (1), were made between the same side (e.g. ipsilateral versus ipsilateral). Significance levels were set for at least P < 0.05 for ail tests. No side-to-side differences (comparison (1)) existed in either CGRP-ir or SP-ir in the unoperated control group (Figs. 1 and 2; data from the two rats sacrificed with the 10-day groups and from the four rats sacrificed with the 60-day group are combined). Significant side-to-side differences occurred in the two transection groups. Levels of CGRP-ir and SP-ir decreased ipsilateral to transection 10 days after injury (P < 0.01; Figs. 1 and 2). Ipsilateral CGRP-ir remained significantly lower 31 days after tran-
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Fig. 2. Histogramsillustrating changes in SP-ir following transeefion or CCI of the left seiatic nerve. Bars reprcsent means + standard error of the means for SP-ir in the L4-L5 segments of the spinal dorsal quadrants. Sec Fig. 1 for abbreviations. Asterisk (*) indicatcs significant diffcrencc on relevant side from thc unoperated control group. Triangle (A) indicatcs significant difference on relevant side betwoen 31-day and 10-day transection groups. Diamond (~) indicatcs significant differencc betwoen sides in the saine exp¢rimental group. *P < 0.05, **P < 0.01, AZ~p< 0.01, ~(>P < 0.01.
186
K.C. Kajander, J. Xu / Neuroscience Letters 186 (1995) 184-188
section (P < 0.05; Fig. 1). No side-to-side differences in either peptide occurred 10 or 60 days after the CCI. Peptide levels in the injury groups were compared to levels in the unoperated control group (comparison (2)). Levels of CGRP-ir (P < 0.01; Fig. 1) and SP-ir (P < 0.05; Fig. 2) decreased ipsilaterally 10 days after transection. Levels of CGRP-ir and SP-ir decreased bilaterally 31 days after transection ( P < 0.01; Figs. 1 and 2). Ten days after the CCI, peptide levels were not different from control values. However, 60 days after the CCI, levels of CGRP-ir were decreased contralaterally ( P < 0.05; Fig. 1), and levels of SP-ir were decreased bilaterally (P < 0.05; Fig. 2). Changes in peptide levels also were evaluated over time within the same injury group (comparisons (3) and (4)). Levels of CGRP-ir and SP-ir decreased bilaterally 31 days after nerve transection compared to levels at 10days (P<0.01; Figs. 1 and 2). Thus, both peptides decreased bilaterally over time after transection. At 60 days after the CCI, levels of CGRP-ir were decreased ipsilaterally (P < 0.05; Fig. 1), but levels of SP-ir were unchanged compared to levels at 10 days. Comparisons also were made between transection and CCI groups at 10 days (comparison (5)) and between the transection group at 31 days and the CCI group at 60 days (comparison (6)). After transection, ipsilateral levels of CGRP-ir at 10 and 31days were lower than 10 and 60 days after the CCI (P < 0.01; Fig. 1). However, contralateral levels of CGRP-ir 10 days after transection were higher than 10 days after the CCI (P < 0.05; Fig. 1), but contralateral levels of CGRP-ir at 31 days after transection were lower than 60 days after the CCI (P < 0.05; Fig. 1). No significant differences existed in SP-ir between the transection and CCI groups. The main purpose of this study was to evaluate levels of CGRP-ir and SP-ir in the dorsal spinal cord at a timepoint long after induction of the CCI. Sixty days was chosen for this purpose because in the original CCI report by Bennett and Xie [4] rats were still hyperalgesic at 60 days. In addition, CGRP-ir and SP-ir had not been evaluated this long after induction of the CCI. Two major findings emerged from this study. First, levels of CGRP-ir and SP-ir decreased in the dorsal quadrant of the spinal cord ipsilateral to either a transection injury or the CCI. This decrease was much more pronounced following the transection injury. In addition, levels of CGRP-ir and SPir decreased further with increasing time after the injury. Second, the decrease in peptides was bilateral 31 days after transection, and both peptides were reduced on the contralateral side of the dorsal spinal cord 60 days after the CCI. We included several groups of rats in the experimental design for the following reasons. A 10-day CCI group was included because we wanted to compare our peptide data with other data previously reported for the CCI [5,7,8]. The unoperated control group was included to
serve as a negative control in the assays (i.e. this group represents normal levels of peptides), while two transection groups were included to serve as positive controls (i.e. we expected these groups would represent the greatest change from normal peptide levels). Previous reports indicate that SP-ir is reduced in the spinal cord by 5 7 days after nerve transection, while peak reduction is reached at 15-31 days [1,11,17,18]. We included the 10day transection group to make a direct comparison with the levels seen 10 days after the CCI. The 31-day transection group was included because we believed this nerve injury at this time time-point would provide the maximal decrease in spinal peptide levels [1,17]. This group provided the standard by which we could assess extent of decrease seen 60 days after the CCI. Thus, inclusion of an unoperated control group and two different transection groups provided levels of CGRP-ir and SP-ir to compare with levels seen 10 and 60 days after the CCI. It is well documented that SP-ir decreases in the ipsilateral dorsal spinal cord following peripheral nerve transection, and our findings of SP-ir from ipsilateral cord confirm the results of others [1,11,17,18]. Whether CGRP-ir decreases in the ipsilateral dorsal spinal cord after nerve transection is not as clear [11,18]. Villar et al. [18] reported that CGRP-ir was not decreased at times up to 14 days after transection of the sciatic nerve (cf. their Fig. 7 for what appears to be a decrease, however). On the other hand, Klein et al. [11] reported that CGRP-ir was decreased at 60 days after transection of the sciatic nerve. They used an experimental paradigm that was more complex than a simple transection procedure, but nonetheless their data indicated that CGRP-ir decreased in the ipsilateral dorsal spinal cord following sciatic nerve transection. Our data demonstrating that CGRP-ir decreased following sciatic nerve transection are the first to show these changes occur at times as early as 10 and 31 days after the injury. It also is interesting to note that this decrease in CGRP-ir became bilateral by 31 days. Additional research is needed to substantiate and further evaluate these decreases in CGRP-ir following transection of a peripheral nerve. Changes in CGRP-ir and SP-ir following the CCI are not as well documented as those following transection. Three reports have been published on CGRP-ir and SP-ir after induction of the CCI [5,7,8]. Ail three indicated that SP-ir decreased, but this decrease was not very large, ranging from 15 to 21% [5]. Two of these reports indicated that CGRP-ir also decreased [5,7], but the most recent one, in which quantitative image-analysis techniques were used to evaluate immunocytochemical data, found that CGRP-ir did not change following the CCI [8]. Our RIA data support a reduction in CGRP-ir and SP-ir after the CCI but only at the later time (i.e. 60 days). Even at this time-point, however, the reduction in peptide levels is not as great as those seen 31 days after transection. At least two possibilities may explain this finding. Perhaps
K.C. Kajander, J. Xu I Neuroscience Letters 186 (1995) 184-188
peptide levels after CCI are never as low as after complete transection of the nerve, or perhaps peptide levels are returning toward normal by 60 days after the CCI. We believe either of these possibilities may explain our results, but further study is needed to resolve this issue. It is interesting to note that the error bars for CGRP-ir and SP-ir data f-com the 10-day CCI group (see Figs. 1 and 2) are the largest of any of the experimental groups. This result suggests to us that some rats in the 10-day CCI group exhibited a reduction in CGRP-ir and SP-ir, but that large variability in peptide levels existed between animais at this early time-point. Thus, although our data from the 10-day CCI group support the findings of Garrison et al. [8] by revealing no overall reduction in CGRPir, it is clear that a reduction in peptide levels occurs in some animais at this time-point. In general, our data suggest that reductions in levels of CGRP-ir and SP-ir after the CCI are not as great as after transection. It is not surprising that changes in CGRP-ir and SP-ir differ between sciatic nerve transection and the CCI. Anatomical evidence indicates that these two injuries differ dramatically. For example, nerve transection causes a complete peripheral denervation of ail axons [19]. The CCI, on the other hand, causes swelling of the nerve, which appears to result in a slow strangulation of the axons beneath the ligatures [6]. Massive degeneration of large myelinated fibers occurs, but there is only slight damage to thinly myel~nated and unmyelinated fibers [3]. It is these small diameter primary afferent axons that contain much of the CGRP-ir and SP-ir [13,16]. Thus, the CCI is only a partial peripheral denervation, and a smaller reduction in CGRP-ir and SP-ir following the CCI may be explainable primarily in terres of fewer injured smalldiameter primary afferent axons. However, Garrison et al. [8] observed a decrease only in SP-ir and not in CGRP-ir 14 days after the CCI. They hypothesized that the decrease in SP-ir may resuit from changes in SP-containing spinal interneurons and not necessarily from changes in primary afferent fibers (sec Discussion in Ref. [8] for details). They did not observe a change in CGRP-ir, which agrees with our data from the 10-day CCI group. They did not look beyond 14 days, however. Thus, the reduction in CGRP-ir (as primary afferent fibers are the only source of CGRP-ir in the dorsal spinal cord) and perhaps some of the reduction in SP-ir may be due to alterations in primary afferent fibers. It was somewhat surprising to us that there were significant bilateral decreases in SP-ir and CGRP-ir levels in the dorsal spinal cord at 31 days after sciatie nerve transection and in SP-ir at 60 days after the CCI. A significant decrease also occurred in CGRP-ir in the dorsal spinal cord contralateral to the CCI at 60 days. Although it generally has been assumed that unilaterally injuring a peripheral nerve causes pathological changes in the primary afferent fibers of that nerve and in the spinal cord ipsilateral to the injury, there is ample evidence for bilateral
187
changes after unilateral nerve injury [9,12,14,18]. In addition, Villar et al. [18] has reported bilateral changes in SP-ir in the dorsal spinal cord 14 days after transection of the sciatic nerve. The mechanism that underlies these bilateral changes in SP-ir and CGRP-ir remains unclear. It is possible that commissural pathways in the spinal cord regulate these changes [15]. It also is possible, however, that descending influences from supraspinal areas contribute to these bilateral peptide changes [2]. In conclusion, CGRP-ir and SP-ir are decreased in the dorsal spinal cord following transection injury and following CCI. Data from the current study demonstrate that the decrease is greater after nerve transection than after the CCI, and that the decreases are bilateral at later times (i.e. 31 and 60 days) after the injuries. The bilateral changes in peptide immunoreactivity may result from mechanisms present in the spinal cord or perhaps from a descending control system in the brain. W e hypothesize that changes in these peptides participate in other anatomical and biochemical changes that occur in the spinal cord during the development of neuropathic pain [7,10]. This work was supported by NIH Grant NS 29567. We thank C.M. DeLisle, G.J. Giesler, Y.-O. Lee, and A.K. Roche for their valuable comments on an earlier version of this manuscript. We also want to thank Dr. Kenneth M. Hargreaves for allowing us to use space in his laboratory. [1] Barbut, D., Polak, J.M. and Wall, P.D., Substance P in spinal cord dorsal hom decreases following peripheral nerve injury, Brain Res., 205 (1981) 289-298. [2] Basbaum, A.I., Clanton, C.H. and Fields, H., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. Comp. Neurol., 178 (1978) 209-224. [3] Basbaum, A.I., Gautron, M., Jazat, F., Mayes, M. and Guilbaud, G., The spectrum of fiber loss in a modei of neuropathic pain in the rat: an electron microscopic study, Pain, 47 (1991) 359-367. [4] 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. [5] Bennett, G.J., Kajander, K.C., Sahara, Y., ladarola, 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 P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Hom of tbe Spinal Cord, Plenum Press, New York, 1989, pp. 463-471. [6] Bennett, G.J., Experimental models of painful peripheral neuropathies, News Physiol. Soi., 5 (1990) 128-133. [7] 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 (1991) 249-252. [8] 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 (i 993) 205-214, [9] Greenman, M.J., Studies on the regeneration of the peroneai nerve of the albino rat: number and sectional areas of fibers: area relation of axis to sheath, J. Comp. Neurol., 23 (1913) 479-513. [10] Kajander. K,C., Sahara, Y,, ladarola, M.J. and Bennett, G.J.,
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[!1]
[12]
[13]
[14]
[15]
K.C. Kajander, J. Xu / Neuroscience Letters 186 (1995) 184-188
Dynorphin increases in the dorsal spinal cord in rats with a painfui peripheral neuropathy, Peptides, I 1 (1990) 719-728. Klein, C.M., Guillamondegui, O., Krenek, C.D., La Forte, R.A. and Coggeshall, R.E., Do neuropeptides in the dorsal horn change if the dorsal root ganglion oeil death that normally aecompanies peripheral nerve transection is prevented? Brain Res. 552 (1991) 273-282. Mao, J., Priee, D.D., Coghill, R.C., Mayer, D.J. and Hayes, R.L., Spatial patterns of spinal eord [14C]-2-deoxyglueose metabolie aetivity in a rat model of painful peripheral mononeuropathy, Pain, 50 (1992) 89-100. MeNeill, D.L., Coggeshall, R.E. and Carlton, S.M., A light and eleetron mieroseopie study of ealeitonin gene-related peptide in the spinal eord of the rat, Exp. Neurol., 99 (1988) 699-708. Nittono, K., On bilateral effects from the unilateral section of branches of the nervus trigeminus in the albino rat, J. Comp. Nenroi., 35 (1923) 133-161. Ram6n y Cajal, S., Histologie du Système Nerveux de l'Homme et des Vertébrés, Tome I, Consejo Supedor de Investigaciones Cientifieas, lnstituto Ramón y Cajal, Madrid, 1952, pp. 287-301.
[16] Ruda, M.A., Bennett, G.J. and Dubner, R., Neurochemistry and neural circuitry in the dorsal hom. In P.C. Emson, M.N. Rossor and M. Tonyama (Eds.), Progress in Brain Researeh, Vol. 66, EIsevier, Amsterdam, 1986, pp. 219-268. [17] Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal tord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422-430. [18] Villar, M.J., Cortés, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and H6kfelt, T., Neuropeptide expression in rat dorsal foot ganglion oeils and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587-604. [19] Weddell, G., Guttmann, L. and Gutmann, E., The local extension of nerve fibres into denervated areas of skin, J, Nenrol. Psyehiatry 4 (1941) 206-225. [20] Xu, J.-Y. and Kajander, K.C., Chromie gut suture reduees caleitonin gene-related peptide and substance P leveis in the spinal eord after chronie eonstriction injury, Pain, submitted.
NeuroscienceLetters186(1995)184-188
l~ll
Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury Keith C. Kajander a,b,*, Jiangyan Xu a aDepartment of Oral Science, 17-252 Moos Tower, 515 Delaware Street S.E., Universityof Minnesota, Minneapolis, MN 55455-0329, USA bDepartment of CeU Biology and Neuroanatomy, and Program in Neuroscience, Universityof Minnesota, Minneapolis, MN 55455-0329, USA
Received 14 October 1994; revisedversionreceived4 January 1995; accepted4 January 1995
Abstract
Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury. Keywords: Chronic constriction injury; Hyperalgesia; Nerve compression syndromes; Neuralgia; Neuroma; Neuropeptides; Radio-
immunoassay; Sciatic nerve
In the past few years, several animal models have been introduced for evaluating changes in the nervous system following peripheral nerve injury [6]. Of these models, the chronic constriction injury of the sciatic nerve (CCI) has become the focus of a great deal of recent research [4]. The CCI appears to injure selectively axons in the sciatic nerve; there is massive degeneration of large myelinated axons but only partial degeneration of thinly myelinated and unmyelinated axons [3,6]. As axonal degeneration occurs in the nerve, neurochemical alterations occur in the dorsal horn of the lumbar spinal cord in segments that receive intraspinal termination's of the sciatic nerve. For example~ calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir), as evaluated using immunocytochemical techniques, is decreased in the ipsilateral dorsal horn at 10, 20, and 28 days after the CCI [5,7]. Bennett et al. [5] confirmed these immunocytochemical findings using radioimmunoassay (RIA). However, a recent study reported that immunocytochemical * Corresponding author (to Departmentof Oral Science), Tel.: + 1 612 6260632; Fax: +1 612 6262651; E-mail: [email protected].
labeling for CGRP-ir was not decreased in the ipsilateral spinal cord at either 7 or 14 days after the CCI [8]. These inconsistencies suggest that further evaluation of alterations in CGRP-ir and SP-ir within the spinal cord is needed. In addition, previous studies have focused on short-term (7-28 days) alterations while changes at rimes later than these have not been evaluated. We decided to re-examine this issue using RIA, and we included additional groups not used in the previous studies. The major goal of this study was to determine levels of CGRP-ir and SP-ir in the dorsal spinal tord at a time-point long after induction of the CCI (60 days), but it also was desirable to re-evaluate peptide levels round shortly after the injury (10 days). Peptide levels at these two rimes were compared to each other, to peptide levels from unoperated control animals, and also to peptide levels seen after transection of the sciatic nerve. Thirty, adult, maie Sprague-Dawley rats (275-350 g) were randomly divided into rive groups. Rats in two groups had the left sciatic nerve transected and were sacrificed 10 (n = 6) and 31 (n = 6) days later. Rats in two other groups had the CCI induced on the left sciatic nerve and were sacrificed 10 (n = 5) and 60 (n = 7) days later.
0304-3940/95/$09.50 © 1995 E|sevier Science lreland Ltd. Ail rights reserved SSDI 0304-3940(95)1 1294-4
185
K.C. Kajander, J. Xu I Neuroscience Letters 186 (1995) 184-188
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Fig. 1. Histograms illustrating changes in CGRP-ir foliowing transection or CCI of the left sciatic nerve. Bars represent means + standard error of the means for CGRP-ir in the L4-L5 segments of the spinal dorsal quadrants. Contra, quadrant eontralateral to the injury; Ipsi, quadrant ipsilateral to the injury; C, unoperated eontrol group. Asterisk (*) indicates significant difference on the relevant side from the unoperated eonrol group. Triangle (A) indicates significant difference on relevant side between 31-day and 10-day transection groups or on relevant side between 60-day and 10-day CCI groups. Circle (O) indicates significant difference between relevant side of 10-day transection and 10-day CCI groups or between relevant side of 31-day transection and 60-day CCI groups. Diamond (~) indicates significant difference between sides in the same experimental group. *P < 0.05, **P < 0.01, ZXp< 0.05, zXZXp< 0.01, Op < 0.05, OOp < 0.01, ~ P < 0.05, <>~P < 0.01. Rats in the fifth group served as unoperated controls (n = 6); two of these rats were sacrificed at the same rime as rats in the two 10-day groups, and four were sacrificed at 60 days. Ail rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). For rats in the transection groups, the left sciatic nerve was exposed, two ligatures were tied tightly about it (5 mm apart), and a short length of the nerve ( 3 4 mm) was removed from between the ligatures. The CCI was produced according to the protocol of Bennett and Xie [4]. The left sciatic nerve was slightly constricted by tying four chromic gut ligatures. At the time of sacrifice, rats were deeply anesthetized (80 mg/kg i.p.) and decapitated. As described previously [10], the spinal cord was forcibly ejected, and the approximate lumbar 4-5 (L4-5) spinal segments were isolated and divided into left dorsal, left ventral, right dorsal, and right ventral quadrants by two perpendicular cuts through the central canal. Tissue samples were quickly frozen in multi-well plates and stored at - 8 0 ° C until assayed. The RIA procedures are described elsewhere [20]. Tissue samples from ail groups were blindly assayed simultaneously in duplicate. Total protein levels, as determined using the method of Lowry, were used to normalize pep-
tide levels between samples. Only results from the dorsal quadrants are reported here. After a significant two-way A N O V A (treatment x rime) for each peptide, Duncan's multiple range test was performed to examine for differences in means. We specified Duncan's test to evaluate six comparisons: (1) side-to-side (i.e. ipsilateral versus contralateral) differences within each group; (2) differences between the four treatment groups and the unoperated control group; (3) differences between the 10-day transection group and the 31-day transection group; (4) differences between the 10day CCI group and the 60-day CCI group ((3) and (4) represent a change over time); (5) differences between the 10-day transection group and the 10-day CCI group; and (6) differences between the 31-day transection group and the 60-day CCI group. Ail comparisons, except (1), were made between the same side (e.g. ipsilateral versus ipsilateral). Significance levels were set for at least P < 0.05 for ail tests. No side-to-side differences (comparison (1)) existed in either CGRP-ir or SP-ir in the unoperated control group (Figs. 1 and 2; data from the two rats sacrificed with the 10-day groups and from the four rats sacrificed with the 60-day group are combined). Significant side-to-side differences occurred in the two transection groups. Levels of CGRP-ir and SP-ir decreased ipsilateral to transection 10 days after injury (P < 0.01; Figs. 1 and 2). Ipsilateral CGRP-ir remained significantly lower 31 days after tran-
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Fig. 2. Histogramsillustrating changes in SP-ir following transeefion or CCI of the left seiatic nerve. Bars reprcsent means + standard error of the means for SP-ir in the L4-L5 segments of the spinal dorsal quadrants. Sec Fig. 1 for abbreviations. Asterisk (*) indicatcs significant diffcrencc on relevant side from thc unoperated control group. Triangle (A) indicatcs significant difference on relevant side betwoen 31-day and 10-day transection groups. Diamond (~) indicatcs significant differencc betwoen sides in the saine exp¢rimental group. *P < 0.05, **P < 0.01, AZ~p< 0.01, ~(>P < 0.01.
186
K.C. Kajander, J. Xu / Neuroscience Letters 186 (1995) 184-188
section (P < 0.05; Fig. 1). No side-to-side differences in either peptide occurred 10 or 60 days after the CCI. Peptide levels in the injury groups were compared to levels in the unoperated control group (comparison (2)). Levels of CGRP-ir (P < 0.01; Fig. 1) and SP-ir (P < 0.05; Fig. 2) decreased ipsilaterally 10 days after transection. Levels of CGRP-ir and SP-ir decreased bilaterally 31 days after transection ( P < 0.01; Figs. 1 and 2). Ten days after the CCI, peptide levels were not different from control values. However, 60 days after the CCI, levels of CGRP-ir were decreased contralaterally ( P < 0.05; Fig. 1), and levels of SP-ir were decreased bilaterally (P < 0.05; Fig. 2). Changes in peptide levels also were evaluated over time within the same injury group (comparisons (3) and (4)). Levels of CGRP-ir and SP-ir decreased bilaterally 31 days after nerve transection compared to levels at 10days (P<0.01; Figs. 1 and 2). Thus, both peptides decreased bilaterally over time after transection. At 60 days after the CCI, levels of CGRP-ir were decreased ipsilaterally (P < 0.05; Fig. 1), but levels of SP-ir were unchanged compared to levels at 10 days. Comparisons also were made between transection and CCI groups at 10 days (comparison (5)) and between the transection group at 31 days and the CCI group at 60 days (comparison (6)). After transection, ipsilateral levels of CGRP-ir at 10 and 31days were lower than 10 and 60 days after the CCI (P < 0.01; Fig. 1). However, contralateral levels of CGRP-ir 10 days after transection were higher than 10 days after the CCI (P < 0.05; Fig. 1), but contralateral levels of CGRP-ir at 31 days after transection were lower than 60 days after the CCI (P < 0.05; Fig. 1). No significant differences existed in SP-ir between the transection and CCI groups. The main purpose of this study was to evaluate levels of CGRP-ir and SP-ir in the dorsal spinal cord at a timepoint long after induction of the CCI. Sixty days was chosen for this purpose because in the original CCI report by Bennett and Xie [4] rats were still hyperalgesic at 60 days. In addition, CGRP-ir and SP-ir had not been evaluated this long after induction of the CCI. Two major findings emerged from this study. First, levels of CGRP-ir and SP-ir decreased in the dorsal quadrant of the spinal cord ipsilateral to either a transection injury or the CCI. This decrease was much more pronounced following the transection injury. In addition, levels of CGRP-ir and SPir decreased further with increasing time after the injury. Second, the decrease in peptides was bilateral 31 days after transection, and both peptides were reduced on the contralateral side of the dorsal spinal cord 60 days after the CCI. We included several groups of rats in the experimental design for the following reasons. A 10-day CCI group was included because we wanted to compare our peptide data with other data previously reported for the CCI [5,7,8]. The unoperated control group was included to
serve as a negative control in the assays (i.e. this group represents normal levels of peptides), while two transection groups were included to serve as positive controls (i.e. we expected these groups would represent the greatest change from normal peptide levels). Previous reports indicate that SP-ir is reduced in the spinal cord by 5 7 days after nerve transection, while peak reduction is reached at 15-31 days [1,11,17,18]. We included the 10day transection group to make a direct comparison with the levels seen 10 days after the CCI. The 31-day transection group was included because we believed this nerve injury at this time time-point would provide the maximal decrease in spinal peptide levels [1,17]. This group provided the standard by which we could assess extent of decrease seen 60 days after the CCI. Thus, inclusion of an unoperated control group and two different transection groups provided levels of CGRP-ir and SP-ir to compare with levels seen 10 and 60 days after the CCI. It is well documented that SP-ir decreases in the ipsilateral dorsal spinal cord following peripheral nerve transection, and our findings of SP-ir from ipsilateral cord confirm the results of others [1,11,17,18]. Whether CGRP-ir decreases in the ipsilateral dorsal spinal cord after nerve transection is not as clear [11,18]. Villar et al. [18] reported that CGRP-ir was not decreased at times up to 14 days after transection of the sciatic nerve (cf. their Fig. 7 for what appears to be a decrease, however). On the other hand, Klein et al. [11] reported that CGRP-ir was decreased at 60 days after transection of the sciatic nerve. They used an experimental paradigm that was more complex than a simple transection procedure, but nonetheless their data indicated that CGRP-ir decreased in the ipsilateral dorsal spinal cord following sciatic nerve transection. Our data demonstrating that CGRP-ir decreased following sciatic nerve transection are the first to show these changes occur at times as early as 10 and 31 days after the injury. It also is interesting to note that this decrease in CGRP-ir became bilateral by 31 days. Additional research is needed to substantiate and further evaluate these decreases in CGRP-ir following transection of a peripheral nerve. Changes in CGRP-ir and SP-ir following the CCI are not as well documented as those following transection. Three reports have been published on CGRP-ir and SP-ir after induction of the CCI [5,7,8]. Ail three indicated that SP-ir decreased, but this decrease was not very large, ranging from 15 to 21% [5]. Two of these reports indicated that CGRP-ir also decreased [5,7], but the most recent one, in which quantitative image-analysis techniques were used to evaluate immunocytochemical data, found that CGRP-ir did not change following the CCI [8]. Our RIA data support a reduction in CGRP-ir and SP-ir after the CCI but only at the later time (i.e. 60 days). Even at this time-point, however, the reduction in peptide levels is not as great as those seen 31 days after transection. At least two possibilities may explain this finding. Perhaps
K.C. Kajander, J. Xu I Neuroscience Letters 186 (1995) 184-188
peptide levels after CCI are never as low as after complete transection of the nerve, or perhaps peptide levels are returning toward normal by 60 days after the CCI. We believe either of these possibilities may explain our results, but further study is needed to resolve this issue. It is interesting to note that the error bars for CGRP-ir and SP-ir data f-com the 10-day CCI group (see Figs. 1 and 2) are the largest of any of the experimental groups. This result suggests to us that some rats in the 10-day CCI group exhibited a reduction in CGRP-ir and SP-ir, but that large variability in peptide levels existed between animais at this early time-point. Thus, although our data from the 10-day CCI group support the findings of Garrison et al. [8] by revealing no overall reduction in CGRPir, it is clear that a reduction in peptide levels occurs in some animais at this time-point. In general, our data suggest that reductions in levels of CGRP-ir and SP-ir after the CCI are not as great as after transection. It is not surprising that changes in CGRP-ir and SP-ir differ between sciatic nerve transection and the CCI. Anatomical evidence indicates that these two injuries differ dramatically. For example, nerve transection causes a complete peripheral denervation of ail axons [19]. The CCI, on the other hand, causes swelling of the nerve, which appears to result in a slow strangulation of the axons beneath the ligatures [6]. Massive degeneration of large myelinated fibers occurs, but there is only slight damage to thinly myel~nated and unmyelinated fibers [3]. It is these small diameter primary afferent axons that contain much of the CGRP-ir and SP-ir [13,16]. Thus, the CCI is only a partial peripheral denervation, and a smaller reduction in CGRP-ir and SP-ir following the CCI may be explainable primarily in terres of fewer injured smalldiameter primary afferent axons. However, Garrison et al. [8] observed a decrease only in SP-ir and not in CGRP-ir 14 days after the CCI. They hypothesized that the decrease in SP-ir may resuit from changes in SP-containing spinal interneurons and not necessarily from changes in primary afferent fibers (sec Discussion in Ref. [8] for details). They did not observe a change in CGRP-ir, which agrees with our data from the 10-day CCI group. They did not look beyond 14 days, however. Thus, the reduction in CGRP-ir (as primary afferent fibers are the only source of CGRP-ir in the dorsal spinal cord) and perhaps some of the reduction in SP-ir may be due to alterations in primary afferent fibers. It was somewhat surprising to us that there were significant bilateral decreases in SP-ir and CGRP-ir levels in the dorsal spinal cord at 31 days after sciatie nerve transection and in SP-ir at 60 days after the CCI. A significant decrease also occurred in CGRP-ir in the dorsal spinal cord contralateral to the CCI at 60 days. Although it generally has been assumed that unilaterally injuring a peripheral nerve causes pathological changes in the primary afferent fibers of that nerve and in the spinal cord ipsilateral to the injury, there is ample evidence for bilateral
187
changes after unilateral nerve injury [9,12,14,18]. In addition, Villar et al. [18] has reported bilateral changes in SP-ir in the dorsal spinal cord 14 days after transection of the sciatic nerve. The mechanism that underlies these bilateral changes in SP-ir and CGRP-ir remains unclear. It is possible that commissural pathways in the spinal cord regulate these changes [15]. It also is possible, however, that descending influences from supraspinal areas contribute to these bilateral peptide changes [2]. In conclusion, CGRP-ir and SP-ir are decreased in the dorsal spinal cord following transection injury and following CCI. Data from the current study demonstrate that the decrease is greater after nerve transection than after the CCI, and that the decreases are bilateral at later times (i.e. 31 and 60 days) after the injuries. The bilateral changes in peptide immunoreactivity may result from mechanisms present in the spinal cord or perhaps from a descending control system in the brain. W e hypothesize that changes in these peptides participate in other anatomical and biochemical changes that occur in the spinal cord during the development of neuropathic pain [7,10]. This work was supported by NIH Grant NS 29567. We thank C.M. DeLisle, G.J. Giesler, Y.-O. Lee, and A.K. Roche for their valuable comments on an earlier version of this manuscript. We also want to thank Dr. Kenneth M. Hargreaves for allowing us to use space in his laboratory. [1] Barbut, D., Polak, J.M. and Wall, P.D., Substance P in spinal cord dorsal hom decreases following peripheral nerve injury, Brain Res., 205 (1981) 289-298. [2] Basbaum, A.I., Clanton, C.H. and Fields, H., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain modulating systems, J. Comp. Neurol., 178 (1978) 209-224. [3] Basbaum, A.I., Gautron, M., Jazat, F., Mayes, M. and Guilbaud, G., The spectrum of fiber loss in a modei of neuropathic pain in the rat: an electron microscopic study, Pain, 47 (1991) 359-367. [4] 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. [5] Bennett, G.J., Kajander, K.C., Sahara, Y., ladarola, 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 P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Hom of tbe Spinal Cord, Plenum Press, New York, 1989, pp. 463-471. [6] Bennett, G.J., Experimental models of painful peripheral neuropathies, News Physiol. Soi., 5 (1990) 128-133. [7] 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 (1991) 249-252. [8] 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 (i 993) 205-214, [9] Greenman, M.J., Studies on the regeneration of the peroneai nerve of the albino rat: number and sectional areas of fibers: area relation of axis to sheath, J. Comp. Neurol., 23 (1913) 479-513. [10] Kajander. K,C., Sahara, Y,, ladarola, M.J. and Bennett, G.J.,
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[16] Ruda, M.A., Bennett, G.J. and Dubner, R., Neurochemistry and neural circuitry in the dorsal hom. In P.C. Emson, M.N. Rossor and M. Tonyama (Eds.), Progress in Brain Researeh, Vol. 66, EIsevier, Amsterdam, 1986, pp. 219-268. [17] Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal tord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422-430. [18] Villar, M.J., Cortés, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and H6kfelt, T., Neuropeptide expression in rat dorsal foot ganglion oeils and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587-604. [19] Weddell, G., Guttmann, L. and Gutmann, E., The local extension of nerve fibres into denervated areas of skin, J, Nenrol. Psyehiatry 4 (1941) 206-225. [20] Xu, J.-Y. and Kajander, K.C., Chromie gut suture reduees caleitonin gene-related peptide and substance P leveis in the spinal eord after chronie eonstriction injury, Pain, submitted.