Neuroscience Letters, l 15 (1990) 24-28 Elsevier Scientific Publishers Ireland Ltd.
24
NSL 06972
MK-801-induced sprouting by CGRP immunoreactive primary afferent fibers in the dorsal spinal cord of the rat Clifford H. Harris, Elizabeth L. Fagan, Ronald L. Shew, Thad C. Kammerlocher and Daniel L. McNeill Department q[' Anatomical Sciences, University q] Oklahoma. Oklahoma City, OK 73190 (U.S.A.) (Received 18 December 1989; Revised version received 13 February 1990; Accepted 20 February 1990)
Key word~': MK-801: Sprouting; Primary afferent fiber; Spinal cord: Rat In the present study, rats received daily injections of the N-methyl-t)-aspartate (NMDA) receptor antagonist, MK-801, over 30 consecutive days. The effects of MK-801 on the distribution of calcitonin generelated peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were subsequently examined. In addition to the normal immunostaining pattern in laminae I, II and lateral V, a dense network of CGRP-immunoreactive fibers was observed along the medial border of the dorsal horn and within the dorsal grey commissure. This marked increase in immunoreactivity was virtually eliminated following dorsal rhizotomy. These observations suggest that MK-801 induces intraspinal sprouting by CGRP immunoreactive primary afferent fibers in vivo.
MK-801 has been characterized as a non-competitive antagonist of the N-methylD-aspartate subtype of glutamate receptor [4, 16]. In recent in vivo studies, MK-801 has been observed to reduce the extent of tissue damage following contusive injury to the spinal cord [3, 7]. MK-801 has also been demonstrated to exert a neuroprotective effect in the CNS following ischemic injury [6, 8, 9, 10, 12, 13, 15]. While these studies are significant, the effects of MK-801 on fibers in the uninjured cord have not been assessed. In the present study, alterations in the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were examined following chronic treatment with MK-801. The rational for selecting CGRP as an appropriate marker was due to its localization within a specific subpopulation of primary afferent fibers and terminals within the rat dorsal spinal cord [5, 11]. By confining the effects of MK-801 to one fiber population, data interpreta-
Correspondence: D.L. McNeill, Department of Anatomical Sciences, University of Oklahoma, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. 0304-3940/90/$ 03.50 (c) 1990 Elsevier Scientific Publishers Ireland Ltd.
25
tion is simplified. An increase in the distribution of CGRP-immunoreactive fibers would suggest that MK-801 may not only serve as a neuroprotective agent, but may induce intraspinal sprouting as well. Twelve male Sprague-Dawley rats (30 days old, Sasco, Omaha, NE) were divided into 3 groups. Group 1 rats (n=4) received no MK-801 and served as controls. Group 2 rats (n = 4) received 1.0 mg/kg, of MK-801 (i.p., 1% in dH20, Merck, Sharp and Dohme Research Labs., Rahway, NJ) over 30 consecutive days. Group 3 animals (n=4) received an identical series of MK-801 injections. However, on day 23, each group 3 rat was anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg, i.p.) and a lumbar hemi-laminectomy performed. The L1-S4 dorsal roots on one side were identified and transected. The wound was closed, the animals allowed to recover and the MK-801 injections resumed until day 30. On the afternoon following the last MK-801 injection, all animals were anesthetized with sodium pentobarbital and perfused transcardially with 300 ml of 0.9% saline followed by 300 mls of 4% paraformaldehyde in 0.1 M, pH 7.2, phosphate buffer. A complete lumbar laminectomy was then performed and the 5th lumbar (Ls) spinal cord segment dissected and placed in fresh fixative overnight. The following morning, the tissue was rinsed in phosphate buffer then embedded in O.C.T. compound (Miles, Inc., Elkhart, IN). Cryostat sections (20/zm) of the entire segment were cut and placed in phosphate buffer. The free-floating sections were then incubated with an antiserum to human CGRP (1:1, 600, Peninsula Labs., Inc., Belmont, CA) then processed for FITC fluorescence using a modification of the indirect immunofluorescence technique of Coons et al. [2]. Following the immunocytochemistry, the sections were placed on glass slides. Five sections from each segment were then randomly selected and photographed using an Olympus Vanox-S microscope equipped with an epifluorescent illuminator. As an immunocytochemical control, additional sections from control animals were incubated with an antiserum that had been preadsorbed for 24 h with 10/zg/ml of human CGRP (Peninsula Labs., Inc., Belmont, CA). The CGRP immunostaining pattern in the dorsal spinal cords of control rats from this study was identical to that previously reported by Gibson et al. [5] and McNeill et al. [11]. Briefly, dense regions of immunoreactivity included: (1) a broad band in laminae I and II outer, (2) a band of fibers extending along the lateral margin of the dorsal horn into laminae V and (3) 2 small bundles of fibers located dorsal and ventral to the central canal. In addition, a small, variable number of CGRP-immunoreactive fibers were observed extending into the deeper laminae of the dorsal horn, along the medial aspect of the dorsal horn and within the dorsal grey commissure (Fig. I A). No immunoreactivity was observed in sections incubated with antiserum that had been preadsorbed with CGRP. In all sections from rats receiving MK-801, a marked difference in the CGRP immunostaining pattern was observed. In addition to the dense regions of immunoreactivity described for control animals, MK-801-treated animals had a marked increase in the density of CGRP-immunoreactive fibers within the deeper laminae of the dorsal horn (Fig. 1B). More striking, was the presence of a dense bundle of fibers coursing along the medial borders of the dorsal horns (Fig. 1B). These medial bun-
26
Fig. 1. C G R P immunoreactivity in sections from the L5 spinal cord of a control rat (A), an MK-801treated rat (B) and a rat receiving MK-801 and a unilateral dorsal rhizotomy (C). The photographed areas of the sections are represented by the box on the schematic. Note that in the MK-801-treated spinal cord (B), there is a marked increase in C G R P immunoreactivity in the deeper laminae of the dorsal horn (large arrowheads), along the medial border of the dorsal horns (small arrowheads) and in the dorsal grey commissure (open arrow). Following unilateral dorsal rhizotomy, the increase in C G R P immunoreactivity is virtually eliminated ipsilateral to the lesion, cc, central canal; DH, dorsal horn: DF, dorsal funiculus: DG. dorsal grey commissure. Bar ~ 200 l~m.
27
dies extended into the dorsal grey commissure where they appeared to form a 'plexus' dorsal to the central canal (Fig. 1B). In the MK-801-treated rats that had received a unilateral dorsal rhizotomy 7 days prior to sacrifice, virtually all CGRP immunostaining was eliminated in the dorsal horn and dorsal grey commissure unilateral to the rhizotomies. However, on the contralateral side, the marked increase in CGRP immunoreactivity was still present (Fig. 1C). In the present study, a marked increase in the density and distribution of C G R P immunoreactive fibers in the medial dorsal horn and in the dorsal grey commissure was observed following treatment with MK-801. The elimination of CGRP immunoreactivity following dorsal rhizotomy suggests that these fibers are primary afferent in origin. In studies from other laboratories, the pattern of C G R P immunostaining in the normal dorsal spinal cord appeared to be similar to the pattern we observed following MK-801 treatment [5, 14]. Factors contributing to this similarity include variations in the segmental level examined [5], species and strain variation [5] and differences in C G R P antibody characteristics [14]. Nonetheless, in this study, there was a marked difference in the C G R P immunostaining pattern between control and MK801-treated rats. While MK-801 has been observed to increase the mean neurite length of dentate granule cells in vitro [1], these data suggest that MK-801 is also capable of altering fibers in vivo. Additional studies are necessary to determine the mechanism by which MK-801 induces intraspinal sprouting by primary afferent fibers and whether this phenomenon is ubiquitous among other fiber tracts of the spinal cord. We wish to thank Ms. Cassandra G. Howerton for her excellent technical assistance. This work was supported by the Kent Waldrep National Paralysis Foundation, the Presbyterian/Harris Foundation and a Biomedical Research Support Grant.
I Brewer, G.J. and Cotman, C.W., NMDA promotes branching, MK801 stimulates elongation of dentate granule neurons, Soc. Neurosci. Abstr., 14 (1988) 115. 2 Coons, A.H., Leduc, F.H. and Conolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to the study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 49~50. 3 Faden, A.I., Lemke, M., Simon, R.P. and Noble, L.J., N-methyl-D-aspartate antagonist MK-801 improves outcome following traumatic spinal cord injury in rats: Behavioral, anatomic, and neurochemical studies, J. Neurotrauma, 5 (1988) 33~45. 4 Foster, A.C. and Wong, E.H.F., The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403~410. 5 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulberry, P.M., Ghatel, M., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species, J. Neurosci., 4 (1984) 3101-3111. 6 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 7 Gomez-Pinilla, F., Tram, H., Cotman, C.W. and Nieto-Sampedro, M., Neuroprotective effect of MK801 and U-50488H after contusive spinal cord injury, Exp. Neurol., 104 (1989) 118-124. 8 Hattori, H., Morin, A.M., Schwartz, P.H., Fujikawa, D.G. and Wasterlain, C.G., Posthypoxic treat-
28 ment with MK-801 reduces hypoxic-ischemic damage in the neonatal rat, Neurology, 39 (1989) 713 718.
9 Kochhar, A., Zivin., A. Lyden, P.D. and Mazzarella, V., Glutamate antagonist therapy reduces neurologic deficits produced by local central nervous system ischemia, Arch. Neurol., 45 (1988) 148 153. 10 McDonald, J.W., Silverstein, F.S. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359 361. I 1 McNeill, D.L., Coggeshall. R.E. and Carlton, S.M., A light and electron microscopic study ofcalcitonin gene-related peptide in the spinal cord of the rat, Exp. Neurol., 99 (1988) 699 708. 12 Ozyurt, E., Graham, D.I., Woodruff', G.N. and McCuttoch, J., Protective effect of the glutamate antagonist, MK-801 in focal cerebral ischemia in the cat, J. Cereb. Blood Flow Metab., 8 (1988) 138 143. 13 Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. and McCulloch, J., The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat, Ann. Neurol., 24 (1988) 543 551. 14 Popper, P. and Micevych, P.E., The effect of castration on calcitonin gene-related peptide in spinal motor neurons, Neuroendocrinology, 50 (1989) 338 343. 15 Simon, R.P., Swan, J.H., Griffiths, T. and Melbrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850 852. 16 Wong, E.H.F,, Kemp, J.A., Priestley, T., Knight, A.R.. Woodruff, G.N. and Iversen, L.L., The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7104 7108.
24
NSL 06972
MK-801-induced sprouting by CGRP immunoreactive primary afferent fibers in the dorsal spinal cord of the rat Clifford H. Harris, Elizabeth L. Fagan, Ronald L. Shew, Thad C. Kammerlocher and Daniel L. McNeill Department q[' Anatomical Sciences, University q] Oklahoma. Oklahoma City, OK 73190 (U.S.A.) (Received 18 December 1989; Revised version received 13 February 1990; Accepted 20 February 1990)
Key word~': MK-801: Sprouting; Primary afferent fiber; Spinal cord: Rat In the present study, rats received daily injections of the N-methyl-t)-aspartate (NMDA) receptor antagonist, MK-801, over 30 consecutive days. The effects of MK-801 on the distribution of calcitonin generelated peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were subsequently examined. In addition to the normal immunostaining pattern in laminae I, II and lateral V, a dense network of CGRP-immunoreactive fibers was observed along the medial border of the dorsal horn and within the dorsal grey commissure. This marked increase in immunoreactivity was virtually eliminated following dorsal rhizotomy. These observations suggest that MK-801 induces intraspinal sprouting by CGRP immunoreactive primary afferent fibers in vivo.
MK-801 has been characterized as a non-competitive antagonist of the N-methylD-aspartate subtype of glutamate receptor [4, 16]. In recent in vivo studies, MK-801 has been observed to reduce the extent of tissue damage following contusive injury to the spinal cord [3, 7]. MK-801 has also been demonstrated to exert a neuroprotective effect in the CNS following ischemic injury [6, 8, 9, 10, 12, 13, 15]. While these studies are significant, the effects of MK-801 on fibers in the uninjured cord have not been assessed. In the present study, alterations in the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were examined following chronic treatment with MK-801. The rational for selecting CGRP as an appropriate marker was due to its localization within a specific subpopulation of primary afferent fibers and terminals within the rat dorsal spinal cord [5, 11]. By confining the effects of MK-801 to one fiber population, data interpreta-
Correspondence: D.L. McNeill, Department of Anatomical Sciences, University of Oklahoma, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. 0304-3940/90/$ 03.50 (c) 1990 Elsevier Scientific Publishers Ireland Ltd.
25
tion is simplified. An increase in the distribution of CGRP-immunoreactive fibers would suggest that MK-801 may not only serve as a neuroprotective agent, but may induce intraspinal sprouting as well. Twelve male Sprague-Dawley rats (30 days old, Sasco, Omaha, NE) were divided into 3 groups. Group 1 rats (n=4) received no MK-801 and served as controls. Group 2 rats (n = 4) received 1.0 mg/kg, of MK-801 (i.p., 1% in dH20, Merck, Sharp and Dohme Research Labs., Rahway, NJ) over 30 consecutive days. Group 3 animals (n=4) received an identical series of MK-801 injections. However, on day 23, each group 3 rat was anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg, i.p.) and a lumbar hemi-laminectomy performed. The L1-S4 dorsal roots on one side were identified and transected. The wound was closed, the animals allowed to recover and the MK-801 injections resumed until day 30. On the afternoon following the last MK-801 injection, all animals were anesthetized with sodium pentobarbital and perfused transcardially with 300 ml of 0.9% saline followed by 300 mls of 4% paraformaldehyde in 0.1 M, pH 7.2, phosphate buffer. A complete lumbar laminectomy was then performed and the 5th lumbar (Ls) spinal cord segment dissected and placed in fresh fixative overnight. The following morning, the tissue was rinsed in phosphate buffer then embedded in O.C.T. compound (Miles, Inc., Elkhart, IN). Cryostat sections (20/zm) of the entire segment were cut and placed in phosphate buffer. The free-floating sections were then incubated with an antiserum to human CGRP (1:1, 600, Peninsula Labs., Inc., Belmont, CA) then processed for FITC fluorescence using a modification of the indirect immunofluorescence technique of Coons et al. [2]. Following the immunocytochemistry, the sections were placed on glass slides. Five sections from each segment were then randomly selected and photographed using an Olympus Vanox-S microscope equipped with an epifluorescent illuminator. As an immunocytochemical control, additional sections from control animals were incubated with an antiserum that had been preadsorbed for 24 h with 10/zg/ml of human CGRP (Peninsula Labs., Inc., Belmont, CA). The CGRP immunostaining pattern in the dorsal spinal cords of control rats from this study was identical to that previously reported by Gibson et al. [5] and McNeill et al. [11]. Briefly, dense regions of immunoreactivity included: (1) a broad band in laminae I and II outer, (2) a band of fibers extending along the lateral margin of the dorsal horn into laminae V and (3) 2 small bundles of fibers located dorsal and ventral to the central canal. In addition, a small, variable number of CGRP-immunoreactive fibers were observed extending into the deeper laminae of the dorsal horn, along the medial aspect of the dorsal horn and within the dorsal grey commissure (Fig. I A). No immunoreactivity was observed in sections incubated with antiserum that had been preadsorbed with CGRP. In all sections from rats receiving MK-801, a marked difference in the CGRP immunostaining pattern was observed. In addition to the dense regions of immunoreactivity described for control animals, MK-801-treated animals had a marked increase in the density of CGRP-immunoreactive fibers within the deeper laminae of the dorsal horn (Fig. 1B). More striking, was the presence of a dense bundle of fibers coursing along the medial borders of the dorsal horns (Fig. 1B). These medial bun-
26
Fig. 1. C G R P immunoreactivity in sections from the L5 spinal cord of a control rat (A), an MK-801treated rat (B) and a rat receiving MK-801 and a unilateral dorsal rhizotomy (C). The photographed areas of the sections are represented by the box on the schematic. Note that in the MK-801-treated spinal cord (B), there is a marked increase in C G R P immunoreactivity in the deeper laminae of the dorsal horn (large arrowheads), along the medial border of the dorsal horns (small arrowheads) and in the dorsal grey commissure (open arrow). Following unilateral dorsal rhizotomy, the increase in C G R P immunoreactivity is virtually eliminated ipsilateral to the lesion, cc, central canal; DH, dorsal horn: DF, dorsal funiculus: DG. dorsal grey commissure. Bar ~ 200 l~m.
27
dies extended into the dorsal grey commissure where they appeared to form a 'plexus' dorsal to the central canal (Fig. 1B). In the MK-801-treated rats that had received a unilateral dorsal rhizotomy 7 days prior to sacrifice, virtually all CGRP immunostaining was eliminated in the dorsal horn and dorsal grey commissure unilateral to the rhizotomies. However, on the contralateral side, the marked increase in CGRP immunoreactivity was still present (Fig. 1C). In the present study, a marked increase in the density and distribution of C G R P immunoreactive fibers in the medial dorsal horn and in the dorsal grey commissure was observed following treatment with MK-801. The elimination of CGRP immunoreactivity following dorsal rhizotomy suggests that these fibers are primary afferent in origin. In studies from other laboratories, the pattern of C G R P immunostaining in the normal dorsal spinal cord appeared to be similar to the pattern we observed following MK-801 treatment [5, 14]. Factors contributing to this similarity include variations in the segmental level examined [5], species and strain variation [5] and differences in C G R P antibody characteristics [14]. Nonetheless, in this study, there was a marked difference in the C G R P immunostaining pattern between control and MK801-treated rats. While MK-801 has been observed to increase the mean neurite length of dentate granule cells in vitro [1], these data suggest that MK-801 is also capable of altering fibers in vivo. Additional studies are necessary to determine the mechanism by which MK-801 induces intraspinal sprouting by primary afferent fibers and whether this phenomenon is ubiquitous among other fiber tracts of the spinal cord. We wish to thank Ms. Cassandra G. Howerton for her excellent technical assistance. This work was supported by the Kent Waldrep National Paralysis Foundation, the Presbyterian/Harris Foundation and a Biomedical Research Support Grant.
I Brewer, G.J. and Cotman, C.W., NMDA promotes branching, MK801 stimulates elongation of dentate granule neurons, Soc. Neurosci. Abstr., 14 (1988) 115. 2 Coons, A.H., Leduc, F.H. and Conolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to the study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 49~50. 3 Faden, A.I., Lemke, M., Simon, R.P. and Noble, L.J., N-methyl-D-aspartate antagonist MK-801 improves outcome following traumatic spinal cord injury in rats: Behavioral, anatomic, and neurochemical studies, J. Neurotrauma, 5 (1988) 33~45. 4 Foster, A.C. and Wong, E.H.F., The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403~410. 5 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulberry, P.M., Ghatel, M., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species, J. Neurosci., 4 (1984) 3101-3111. 6 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 7 Gomez-Pinilla, F., Tram, H., Cotman, C.W. and Nieto-Sampedro, M., Neuroprotective effect of MK801 and U-50488H after contusive spinal cord injury, Exp. Neurol., 104 (1989) 118-124. 8 Hattori, H., Morin, A.M., Schwartz, P.H., Fujikawa, D.G. and Wasterlain, C.G., Posthypoxic treat-
28 ment with MK-801 reduces hypoxic-ischemic damage in the neonatal rat, Neurology, 39 (1989) 713 718.
9 Kochhar, A., Zivin., A. Lyden, P.D. and Mazzarella, V., Glutamate antagonist therapy reduces neurologic deficits produced by local central nervous system ischemia, Arch. Neurol., 45 (1988) 148 153. 10 McDonald, J.W., Silverstein, F.S. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359 361. I 1 McNeill, D.L., Coggeshall. R.E. and Carlton, S.M., A light and electron microscopic study ofcalcitonin gene-related peptide in the spinal cord of the rat, Exp. Neurol., 99 (1988) 699 708. 12 Ozyurt, E., Graham, D.I., Woodruff', G.N. and McCuttoch, J., Protective effect of the glutamate antagonist, MK-801 in focal cerebral ischemia in the cat, J. Cereb. Blood Flow Metab., 8 (1988) 138 143. 13 Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. and McCulloch, J., The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat, Ann. Neurol., 24 (1988) 543 551. 14 Popper, P. and Micevych, P.E., The effect of castration on calcitonin gene-related peptide in spinal motor neurons, Neuroendocrinology, 50 (1989) 338 343. 15 Simon, R.P., Swan, J.H., Griffiths, T. and Melbrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850 852. 16 Wong, E.H.F,, Kemp, J.A., Priestley, T., Knight, A.R.. Woodruff, G.N. and Iversen, L.L., The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7104 7108.
