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Plasticity in spinal nociception after peripheral nerve section: reduced effectiveness of the NMDA receptor antagonist MK-801 in blocking wind-up and central sensitization of the flexor reflex
BRAIN RESEARCH ELSEVIER
Brain Research 670 (1995) 342-346
Plasticity in spinal nociception after peripheral nerve section: reduced effectiveness of the NMDA receptor antagonist MK-801 in blocking wind-up and central sensitization of the flexor reflex Xiao-Jun Xu a, XU Zhang b, Tomas H6kfelt b, Zsuzsanna Wiesenfeld-Hallin a,. a
Department of Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden b Department ofNeuroscience, Karolinska Institute, Stockholm, Sweden Accepted 16 November 1994
Abstract
We have examined and compared the effects of systemically applied MK-801, an NMDA receptor/channel blocker, on the wind-up and facilitation of the flexor reflex during and after conditioning stimulation (CS) of C-afferents in rats with intact sciatic nerves or 13-16 days after axotomy. In rats with intact sciatic nerves, intravenous MK-801 (0.5 mg/kg) partially reduced wind-up and totally blocked reflex facilitation following C-fiber CS to the sural nerve. In contrast, 13-16 days after unilateral section of the sciatic nerve, the same dose of MK-801 failed to reduce the wind-up and reflex facilitation following C-fiber CS to the axotomized sural nerve, although the duration of reflex facilitation was significantly shortened. These findings indicate that the involvement of NMDA receptors in mediating activity-dependent spinal hyperexcitability is substantially reduced after peripheral nerve section, possibly reflecting a reduced release of glutamate by primary sensory afferents.
Keywords: MK-801; NMDA; Nociception; Sensitization; Flexor reflex; Plasticity
The release of excitatory amino acid glutamate and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in mediating activity-dependent ioazrease in spinal cord excitability after conditioning repetitive stimulation of high threshold afferents. Thus, N M D A receptor antagonists reduced a n d / o r blocked the successive increase in response to repetitive stimuli, termed wind-up [13], in dorsal and ventral horn neurons [3-5,18], as well as in the flexor reflex preparation [29,30] during conditioning stimulation (CS) activating C afferents. Furthermore, N M D A antagonists also abolished the heterosynaptic facilitation of the flexor reflex (central sensitization) [21] following the CS of C-afferents in both skin and muscle nerves [29,30]. Peripheral nerve injury leads to profound and complex morphological and functional changes in spinal nociceptive systems, including changes in neurotrans-
* Corresponding author. Fax: (46) (8) 774-8856. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 3 6 0 - 8
mitter expression. For example, substance P (SP) and calcitonin gene-related peptide (CGRP) are downregulated, whereas the levels of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and galanin (GAL) increase dramatically in small dorsal root ganglion ( D R G ) neurons (see [9] for refs.). We have recently studied the fine structure of peptide-containing glomeruli of primary afferents in lamina II of the rat dorsal horn 14 days after peripheral axotomy [34,35]. The results show that many GAL-, VIP- and PHI-immunoreactive large dense core vesicles (LDCVs) are present close to the nerve cell membrane of the glomeruli, but outside the synaptic zone. In addition there was often a loss of synaptic vesicles, especially close to the synapse. This is in agreement with several earlier studies showing that peripheral axotomy induces transganglionic changes, either termed 'transganglionic degeneration' [1,6,7] or 'degenerative atrophy' [2,11]. Taken together these findings suggest that transmission via peptides may be retained following peripheral axotomy, albeit that V | P / P H I and GAL have replaced C G R P and SP, whereas the release of
X.-J. Xu et aL / B r a i n Research 670 (1995) 342-346
classical transmitters, e.g. glutamate [15], may be reduced, leading to an attenuation of glutamatergic synaptic transmission. The present experiments were conducted to examine the effect of systemically applied MK-801, an N M D A r e c e p t o r / c h a n n e l blocker, on the activity-dependent increase in spinal cord excitability in rats after peripheral axotomy in order to verify this hypothesis. The experiments were performed on 15 female S p r a g u e - D a w l e y rats weighing 200-250 g (B and K Universal, Stockholm, Sweden). In eight rats the sciatic nerve was intact and in the other 7 rats the left sciatic nerve was transected 13-16 days prior to the acute physiological experiments. The sciatic nerve section was performed under methohexital anesthesia (Brietal, Lilly, Indianapolis, USA, 70 m g / k g , i.p.). The tibial and peroneal branches of the sciatic nerve were unilaterally ligated and sectioned distal to the ligation. The tibial nerve was transected distally to the branch point of the sural nerve. The sural nerve was ligated and sectioned as distally as possible in order that the sural nerve by itself could be electrically stimulated proximal to the site of section. In the acute experiments, the animals were briefly anesthetized with methohexital, ventilated and decerebrated by aspiration of the forebrain and midbrain. The spinal cord was exposed by a laminectomy at mid-thoracic level and sectioned at Th8-9. An i.t. catheter (PE 10) was implanted caudally to the transection with its tip on the lumbar spinal cord (L4-5). The flexor reflex was elicited by supramaximal electric shocks (0.5 ms, 10 mA, 1 / m i n ) to the sural nerve above the level of nerve section in axotomized rats and at a similar level in rats with intact nerves. A CS train (1 Hz, 20 stimuli) of the same strength as the test stimuli was administered to evoke a facilitation of the reflex magnitude. The flexor reflex was recorded as E M G activity via stainless steel needle electrodes inserted into the ipsilateral posterior biceps f e m o r i s / s e m i tendinosus muscles. The number of action potentials elicited during the reflex was integrated over 2 s and recorded on a chart recorder. During the CS, the E M G was integrated in 1 s intervals to evaluate wind-up. During the experiments the heart rate and rectal temperature of the rat were monitored. MK-801 (Merck, Sharp and Dohme) was dissolved in 0.9% saline and injected through an intravenous cannula in the left jugular vein in a volume of 0.2 ml. In rats with intact sciatic nerves, electrical stimulation of high threshold afferents in the sural nerve evoked a flexor reflex, which was usually highly stable in the course of the experiments. When the same stimulus was used as a conditioning train, the reflex magnitude was gradually increased during the CS (Fig. 1). The flexor reflex in response to the test stimuli was briefly facilitated after the termination of CS, which
343
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Fig. 1. Summary of the effects of 0.5 m g / k g i.v. MK-801 on the wind-up of the flexor reflex during a 0.9 Hz, 20 shock C-fiber conditioning stimulus train (CS) in rats with intact (n = 8) and sectioned (n = 7) sciatic nerves. Note that wind-up developed similarly in rats with intact (open columns) and sectioned (hatched columns) sciatic nerves. MK-801 partially reduced the extent of wind-up in normal rats (dotted columns), but not in axotomized rats (filled columns). *P < 0.05, * *P < 0.01 and * * *P < 0.005 compared to normal controls with Wilcoxon matched-pairs test. The wind-up is expressed as average increase in reflex magnitude over baseline response prior to or following MK-801 administration in response to five consecutive stimuli. Variability is expressed as standard error here.
was maximal at 0.5 min (Fig. 2A). MK-801 consistently depressed the baseline flexor reflex (Fig. 2A), reduced the development of wind-up by about 50% (Fig. l) and blocked the facilitation of the flexor reflex induced by the CS by 93 + 3% (n = 8). In rats in which the sciatic nerve had been transected 13-16 days previously, the flexor reflex elicited by electrical stimulation of the sural nerve proximal to the transection was indistinguishable from the reflex recorded in rats with intact sciatic nerves (see also [20,22]). The wind-up of the reflex during the conditioning train and the subsequent reflex facilitation were also similar to normal rats [22]. The baseline flexor reflex was depressed by MK-801 in a manner which was similar to normal rats. But in contrast to normal rats, MK-801 had no effect on wind-up (Fig. 1). The maximal reflex facilitation evoked by CS of the sural nerve was blocked by MK-801 only by 18.7 + 6.3% (n = 7, Fig. 2B), which was significantly less than in rats with intact sciatic nerves (unpaired t-test, t = 11.0, P < 0.0001). However, the duration of reflex facilitation was shorter after the administration of MK-801 compared to the control response in axotomized rats (reduction from 2.7 _+ 0.2 min to 0.8 _+ 0.3 rain, P < 0.05). While being very effective in reducing wind-up of the neuronal response to repetitive C-fiber stimulation [3-5], numerous authors have reported that N M D A antagonists did not influence the response of dorsal horn neurons to the initial C-fiber input [3-5,8]. Both we [30] and Woolf and Thompson [29] reported that in addition to suppressing reflex wind-up and facilitation,
X.-J. Xu et al. /Brain Research 670 (1995) 342-346
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Time (rain) Fig. 2. Illustration of the effects of 0.5 m g / k g i.v. MK-801 on the facilitation of the flexor reflex induced by conditioning stimulation of the sural nerve in a rat with intact sciatic nerves (A) and in another rat 14 days after ipsilateral sciatic nerve section (B). Note that MK-801 produced similar depression of the baseline reflex in both preparations. However, while MK-801 totally blocked the reflex facilitation by the sural nerve CS in the rat with intact sciatic nerve (A), it failed to do so in the axotomized rats (B).
N M D A antagonists were also effective in reducing the baseline flexor reflex. It is suggestive from these results that certain site(s) in the flexor reflex pathway, possibly within the motoneuron pool, are sensitive to blockade of the N M D A receptor and are responsible for the reflex depressive effect of MK-801. On the other hand, the reflex wind-up and facilitation during and following the C-fiber afferent CS possibly reflects similar changes in dorsal horn neurons involved in nociceptive transmission. In the present study, we observed that i.v. MK-801 depressed the baseline flexor reflex in a similar manner in rats with intact and sectioned sciatic nerves, indicating that the sites responsible for this effect of MK-801 were not altered by peripheral axotomy. In contrast, unlike in rats with intact sciatic nerves [30], MK-801 totally lost its effectiveness in reducing wind-up and in blocking maximal reflex facilitation following the CS of C-afferents in axotomized
rats. However, MK-801 reduced the duration of facilitation, indicating that it still had some residual effect. These results imply that activation of the N M D A receptor may be less critically involved in the generation of spinal cord hyperexcitability following C-fiber input after peripheral nerve section. Considering the morphological observation of substantial loss of synaptic vesicles in peptide-containing glomeruli in primary afferent terminals in axotomized rats [34,35], it is most likely that our results reflect the inability of C-afferent terminals to release glutamate upon repetitive stimulation. It is also possible that there is a down-regulation of the N M D A receptor or a reduced coupling between the released glutamate and the postsynaptic N M D A receptors after nerve injury. Dysfunction in glutamatergic transmission may also underly the observation that repetitive stimulation of muscle afferents in axotomized rats failed to elicit prolonged increase in spinal cord excitability [22]. We have previously reported that the facilitatory effect of C-fiber conditioning stimulation can also be blocked by antagonists of tachykinin receptors in rats with intact sciatic nerves [25,26],. We and others have shown that there is a synergistic interaction between activation of N M D A receptors and the NK~ tachykinin receptor in eliciting wind-up and central sensitization [14,19,30]. After peripheral nerve section, the level of substance P was depleted in primary sensory afferents [10] and this was reflected by a gradual reduction in the effectiveness of tachykinin antagonists in blocking flexor reflex facilitation by conditioning C-fiber stimulation [24]. In contrast, vasoactive intestinal peptide (VIP), which normally does not mediate spinal hyperexcitability [32], becomes an important mediator after nerve injury [23,24]. It is unclear, however, whether VIP is solely responsible for spinal hyperexcitability or whether it interacts with compounds other than glutamate. Moreover, it is also possible that other neuroactive substances, such as purines [16], may have a more important role after axotomy, which needs to be examined. N M D A receptor antagonists have been shown to relieve neuropathic pain-related behavior in rats associated with partial nerve constriction [17,33], leading to the suggestion that such drugs may be useful in treating human neuropathic pain. The present results, however, indicate that this may not be the case when pain arises after complete nerve transection. In a clinical report no analgesic effect following i.t. administration of the N M D A receptor antagonist CPP was observed on the spontaneous pain and allodynia in the territory of the injured nerve [12]. These are symptoms which may result from sensitization of dorsal horn neurons receiving input from injured primary afferents [28]. As the inputs of at least two major excitatory sensory transmitters, glutamate and SP, are impaired after
X.-J. Xu et aL /Brain Research 670 (19951 342-346
nerve section, it is interesting to suggest that drugs which interfere with neurotransmission in axotomized sensory neurons, such as VIP antagonists [23,24], CCK-B antagonists [31] or galanin agonists [27], may have more relevance in managing neuropathic pain.
This work is supported by the Swedish Medical Research Council (07913, 2887), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, Marianne and Marcus Wallenbergs Stiftelse, King Gustav V's and Queen Victoria's Stiftelse, Stiftelsen Lars Hiertas Minne and research funds of the Karolinska Institute.
[1] Aldskogius, H., Arvidsson, J. and Grant, G., Axotomy induced changes in primary sensory neurons. In S,A. Scotts (Ed.), Sensory Neurons - - Diversity, Development, and Plasticity, Oxford University Press, New York, 1992, pp. 363-383. [2] Csillik, B. and Knyihar-Csillik, E., The Protean Gate: Structure and Plasticity of the Primary Nociceptive Analyzer, Akademiai Kiado, Budapest, 1986. [3] Davies, S.N. and Lodge, D., Evidence for involvement of Nmethylaspartate receptors in 'wind up' of class 2 neurones in the dorsal horn of the rat, Brain Res., 424 (1987) 402-406. [4] Diekenson, A.H. and Sullivan, A.F., Evidence for a role of the NMDA receptor in the frequency-dependent potentiation of deep rat dorsal horn nociceptive neurones following C-fiber stimulation, Neuropharmacology, 26 (19871 1235-1238. [5] Dickenson, A.H. and Sullivan, A.F., Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat, Brain Res., 506 (1990) 31-39. [6] Grant, G., Neuronal changes central to the site of axon transection. A method for the identification of retrograde changes in perikarya, dentrites and axons by silver impregnation. In W.J.H. Nauta and S.O.E. Eddesson (Eds.), Comtemporary Research Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 173-185. [7] Grant, G. and Arvidsson, J., Transganglionic degeneration in trigeminal primary sensory neurons, Brain Res., 95 (1975) 265279. [8] Headley, P.M., Parsons, C.G. and West D.C., The role of N-methylaspartate receptors in mediating responses of rat and cat spinal neurones to defined sensory stimuli, J. Physiol., 385 (1987) 169-180. [9] H6kfelt, T,, Zhang, X. and Wiesenfeld-Hallin, Z., Messenger plasticity in primary sensory neurons following axotomy and its functional implications, Trends Neurosci., 17 (1994) 22-29. [10] Jessell, T.M., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P depletion in the dorsal horn of the rat spinal cord after section of the peripheral processes of primary sensory neurons, Brain Res., 168 (19791 247--259. [11] Knyihar-Csillik, E. and Csillik, B., FRAP: Histochemistry of the Primary Nociceptit~e Neuron, Progress in Histochemistry and Cytochemistry, Vol. 14, Gustav Fischer, Stuttgart, pp. 1-137. [12] Kristensen, J.D., Svensson, B. and Gorth, Jr., T., The NMDAreceptor antagonist CPP abolishes neurogenic 'wind-up pain' after intrathecal administration in humans, Pain, 51 (1992) 249-253. [13] Mendell, L.M., Physiological properties of unmyelinated fiber projection to the spinal cord, Exp. Neurol., 16 (1966) 316-332. [14] Randic, M., Hecimovic, H. and Ryu, P.D., Substance P modulates glutamate-induced currents in acutely isolated spinal dorsal horn neurones, Neurosci. Lett., 117 (1990) 74-80.
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[15] Salt, T.E. and Hill, R.C., Neurotransmitter candidates of somatosensory primary afferent fibers, Neuroscience, 10 (1983) 1083-1103. [16] Salter, M.E., De Koninck, Y. and Henry, J.L., Physiological roles for adenosine and ATP in synaptic transmission in the spinal dorsal horn, Prog. Neurobiol., 41 (1993) 125-156. [17] Tal, M. and Bennett, G.J., Dextrorphan relieves neuropathic heat-evoked hyperalgesia in the rat, Neurosci. Lett., 151 (1993) 107-111. [18] Thompson, S.W.N., King, A.E. and Woolf, C.J., Activity-dependent changes in rat ventral horn neurons in vitro: summation of prolonged afferent evoked postsynaptic depolarizations produce a D-2-amino-5-phosphonovaleric acid sensitive windup, Eur. J. Neurosci., 2 (1990) 638-649. [19] Thompson, S.W.N., Urban, L. and Dray, A., Contribution of NK1 and NK2 receptor activation to high threshold fibre evoked ventral root responses in the rat spinal cord in vitro, Brain Res., 625 (1993) 100-108. [20] Wall, P.D., Fitzgerald, M. and Gibson, S.J., The response of rat spinal cord cells to unmyelinated afferents after peripheral nerve section and after changes in substance P levels, Neuroscience, 6 (1981) 2205-2215. [21] Wall, P.D. and Woolf, C.J., Muscle but not cutaneous C-afferent input produces prolonged increases in the excitability of the flexion reflex in the rat, J. Physiol., 356 (1984) 443-458. [22] Wall, P.D. and Woolf, C.J., The brief and the prolonged facilitatory effects of unmyelinated afferent input on the rat spinal cord are independently influenced by peripheral nerve section, Neuroscience, 17 (1986) 1199-1205. [23] Wiesenfeld-Hallin, Z., Nerve injury alters the interaction between C-fibre activity and intrathecal neuropeptides on the flexor reflex in rat, Brain Res., 486 (1989) 205-.213. [24] Wiesenfeld-Hallin, Z., Xu, X.-J., H~kanson, R., Feng, D.-M. and Folkers, K., Plasticity of the peptidergic mediation of spinal reflex facilitation after peripheral nerve section in the rat, Neurosci. Lett., 116 (1990) 293-298. [25] Wiesenfeld-Hallin, Z., Xu, X.-J., H~kanson, R., Feng, D,-M and Folkers, K., The specific antagonistic effect of intrathecal spantide I1 on susbtance P- and C-fiber conditioning stimulation-induced facilitation of the nociceptive flexor reflex in rat, Brain Res., 526 (1990) 284-29(I. [26] Wiesenfeld-Hallin, Z., Xu, X.-J., H~kanson, R., Feng, D.-M. and Folkers, K., Tachykinins mediate changes in spinal reflexes after activation of unmyelinated muscle afferents in the rat, Acta Physiol. Scand., 141 (1991) 57-61. [27] Wiesenfeld-Hallin, Z., Xu, X.-J., Langel, IJ., Bedecs, K., H6kfelt, T. and Bartfai, T., Galanin mediated control of pain: enhanced role after nerve injury, Proc. Natl. Acad. Sci USA, 89 (1992) 3334-3337. [28] Woolf, C.J., Central mechanisms of acute pain. In M.R. Bond, J.R. Charlton and C.J. Woolf (Eds.), Proceedings of the VI World Congress on Pain, Pain Research and Clinical Management, Vol. 4, Elsevier, Amsterdam, 1991, pp. 25-34. [29] Woolf, C.J. and Thompson, S.W.N., The induction and maintenance of central sensitization is dependent on N-methyl-Daspartic acid receptor activation: implications for the treatment of post-injury pain hypersensitivity states, Pain, 44 (1991) 293300. [30] Xu, X.-J., Dalsgaard, C.-J. and Wiesenfeld-ltallin, Z., Spinal substance P and N-methyl-D-aspartate receptors are coactived in central sensitization of nociceptive flexor reflex. Neuroscience, 51 (1992) 641-648. [31] Xu, X.-J., Puke, M.J.C., Verge, V.M.K., Wiesenfeld-Hallin, Z., Hughes, J. and H6kfelt T., Up-regulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain, Neurosci. Lett., 152 (19931 129-132.
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[32] Xu, X.-J. and Wiesenfeld-Hallin, Z., An analogue of growth hormone releasing factor (GRF), (Ac-Tyr 1, D-PheZ)-GRF-(1 29), specifically antagonizes the facilitation of the flexor reflex induced by intrathecal vasoactive intestinal peptide in rat spinal cord, Neuropeptides, 18 (1991) 129-135. [33] Yamamoto, T. and Yaksh, T.L., Spinal pharmacology of thermal hyperalgesia induced by constriction injury of sciatic nerve. Excitatory amino acid antagonists, Pain, 49 (1992) 121-128.
[34] Zhang, X., Bean, A.J., Wiesenfeld-Hallin, Z., Xu, X.-J. and H6kfelt, T., Ultrastructural studies on peptides in the dorsal horn of the spinal cord. III. Effects of peripheral axotomy with special reference to galanin, Neuroscience, in press. [35] Zhang, X., Bean, A.J., Wiesenfeld-Hallin, Z. and H6kfelt, T., Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord. IV. Effects of peripheral axotomy with special reference to NPY and VIP/PHI, Neuroscience, in press.
Brain Research 670 (1995) 342-346
Plasticity in spinal nociception after peripheral nerve section: reduced effectiveness of the NMDA receptor antagonist MK-801 in blocking wind-up and central sensitization of the flexor reflex Xiao-Jun Xu a, XU Zhang b, Tomas H6kfelt b, Zsuzsanna Wiesenfeld-Hallin a,. a
Department of Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden b Department ofNeuroscience, Karolinska Institute, Stockholm, Sweden Accepted 16 November 1994
Abstract
We have examined and compared the effects of systemically applied MK-801, an NMDA receptor/channel blocker, on the wind-up and facilitation of the flexor reflex during and after conditioning stimulation (CS) of C-afferents in rats with intact sciatic nerves or 13-16 days after axotomy. In rats with intact sciatic nerves, intravenous MK-801 (0.5 mg/kg) partially reduced wind-up and totally blocked reflex facilitation following C-fiber CS to the sural nerve. In contrast, 13-16 days after unilateral section of the sciatic nerve, the same dose of MK-801 failed to reduce the wind-up and reflex facilitation following C-fiber CS to the axotomized sural nerve, although the duration of reflex facilitation was significantly shortened. These findings indicate that the involvement of NMDA receptors in mediating activity-dependent spinal hyperexcitability is substantially reduced after peripheral nerve section, possibly reflecting a reduced release of glutamate by primary sensory afferents.
Keywords: MK-801; NMDA; Nociception; Sensitization; Flexor reflex; Plasticity
The release of excitatory amino acid glutamate and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in mediating activity-dependent ioazrease in spinal cord excitability after conditioning repetitive stimulation of high threshold afferents. Thus, N M D A receptor antagonists reduced a n d / o r blocked the successive increase in response to repetitive stimuli, termed wind-up [13], in dorsal and ventral horn neurons [3-5,18], as well as in the flexor reflex preparation [29,30] during conditioning stimulation (CS) activating C afferents. Furthermore, N M D A antagonists also abolished the heterosynaptic facilitation of the flexor reflex (central sensitization) [21] following the CS of C-afferents in both skin and muscle nerves [29,30]. Peripheral nerve injury leads to profound and complex morphological and functional changes in spinal nociceptive systems, including changes in neurotrans-
* Corresponding author. Fax: (46) (8) 774-8856. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 3 6 0 - 8
mitter expression. For example, substance P (SP) and calcitonin gene-related peptide (CGRP) are downregulated, whereas the levels of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and galanin (GAL) increase dramatically in small dorsal root ganglion ( D R G ) neurons (see [9] for refs.). We have recently studied the fine structure of peptide-containing glomeruli of primary afferents in lamina II of the rat dorsal horn 14 days after peripheral axotomy [34,35]. The results show that many GAL-, VIP- and PHI-immunoreactive large dense core vesicles (LDCVs) are present close to the nerve cell membrane of the glomeruli, but outside the synaptic zone. In addition there was often a loss of synaptic vesicles, especially close to the synapse. This is in agreement with several earlier studies showing that peripheral axotomy induces transganglionic changes, either termed 'transganglionic degeneration' [1,6,7] or 'degenerative atrophy' [2,11]. Taken together these findings suggest that transmission via peptides may be retained following peripheral axotomy, albeit that V | P / P H I and GAL have replaced C G R P and SP, whereas the release of
X.-J. Xu et aL / B r a i n Research 670 (1995) 342-346
classical transmitters, e.g. glutamate [15], may be reduced, leading to an attenuation of glutamatergic synaptic transmission. The present experiments were conducted to examine the effect of systemically applied MK-801, an N M D A r e c e p t o r / c h a n n e l blocker, on the activity-dependent increase in spinal cord excitability in rats after peripheral axotomy in order to verify this hypothesis. The experiments were performed on 15 female S p r a g u e - D a w l e y rats weighing 200-250 g (B and K Universal, Stockholm, Sweden). In eight rats the sciatic nerve was intact and in the other 7 rats the left sciatic nerve was transected 13-16 days prior to the acute physiological experiments. The sciatic nerve section was performed under methohexital anesthesia (Brietal, Lilly, Indianapolis, USA, 70 m g / k g , i.p.). The tibial and peroneal branches of the sciatic nerve were unilaterally ligated and sectioned distal to the ligation. The tibial nerve was transected distally to the branch point of the sural nerve. The sural nerve was ligated and sectioned as distally as possible in order that the sural nerve by itself could be electrically stimulated proximal to the site of section. In the acute experiments, the animals were briefly anesthetized with methohexital, ventilated and decerebrated by aspiration of the forebrain and midbrain. The spinal cord was exposed by a laminectomy at mid-thoracic level and sectioned at Th8-9. An i.t. catheter (PE 10) was implanted caudally to the transection with its tip on the lumbar spinal cord (L4-5). The flexor reflex was elicited by supramaximal electric shocks (0.5 ms, 10 mA, 1 / m i n ) to the sural nerve above the level of nerve section in axotomized rats and at a similar level in rats with intact nerves. A CS train (1 Hz, 20 stimuli) of the same strength as the test stimuli was administered to evoke a facilitation of the reflex magnitude. The flexor reflex was recorded as E M G activity via stainless steel needle electrodes inserted into the ipsilateral posterior biceps f e m o r i s / s e m i tendinosus muscles. The number of action potentials elicited during the reflex was integrated over 2 s and recorded on a chart recorder. During the CS, the E M G was integrated in 1 s intervals to evaluate wind-up. During the experiments the heart rate and rectal temperature of the rat were monitored. MK-801 (Merck, Sharp and Dohme) was dissolved in 0.9% saline and injected through an intravenous cannula in the left jugular vein in a volume of 0.2 ml. In rats with intact sciatic nerves, electrical stimulation of high threshold afferents in the sural nerve evoked a flexor reflex, which was usually highly stable in the course of the experiments. When the same stimulus was used as a conditioning train, the reflex magnitude was gradually increased during the CS (Fig. 1). The flexor reflex in response to the test stimuli was briefly facilitated after the termination of CS, which
343
500-
'~
400 -
E 300 c
==
-
200
100-
o~ 0
-
1-5
6-10 11-15 Stimulus number
16-20
Fig. 1. Summary of the effects of 0.5 m g / k g i.v. MK-801 on the wind-up of the flexor reflex during a 0.9 Hz, 20 shock C-fiber conditioning stimulus train (CS) in rats with intact (n = 8) and sectioned (n = 7) sciatic nerves. Note that wind-up developed similarly in rats with intact (open columns) and sectioned (hatched columns) sciatic nerves. MK-801 partially reduced the extent of wind-up in normal rats (dotted columns), but not in axotomized rats (filled columns). *P < 0.05, * *P < 0.01 and * * *P < 0.005 compared to normal controls with Wilcoxon matched-pairs test. The wind-up is expressed as average increase in reflex magnitude over baseline response prior to or following MK-801 administration in response to five consecutive stimuli. Variability is expressed as standard error here.
was maximal at 0.5 min (Fig. 2A). MK-801 consistently depressed the baseline flexor reflex (Fig. 2A), reduced the development of wind-up by about 50% (Fig. l) and blocked the facilitation of the flexor reflex induced by the CS by 93 + 3% (n = 8). In rats in which the sciatic nerve had been transected 13-16 days previously, the flexor reflex elicited by electrical stimulation of the sural nerve proximal to the transection was indistinguishable from the reflex recorded in rats with intact sciatic nerves (see also [20,22]). The wind-up of the reflex during the conditioning train and the subsequent reflex facilitation were also similar to normal rats [22]. The baseline flexor reflex was depressed by MK-801 in a manner which was similar to normal rats. But in contrast to normal rats, MK-801 had no effect on wind-up (Fig. 1). The maximal reflex facilitation evoked by CS of the sural nerve was blocked by MK-801 only by 18.7 + 6.3% (n = 7, Fig. 2B), which was significantly less than in rats with intact sciatic nerves (unpaired t-test, t = 11.0, P < 0.0001). However, the duration of reflex facilitation was shorter after the administration of MK-801 compared to the control response in axotomized rats (reduction from 2.7 _+ 0.2 min to 0.8 _+ 0.3 rain, P < 0.05). While being very effective in reducing wind-up of the neuronal response to repetitive C-fiber stimulation [3-5], numerous authors have reported that N M D A antagonists did not influence the response of dorsal horn neurons to the initial C-fiber input [3-5,8]. Both we [30] and Woolf and Thompson [29] reported that in addition to suppressing reflex wind-up and facilitation,
X.-J. Xu et al. /Brain Research 670 (1995) 342-346
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Time (rain) Fig. 2. Illustration of the effects of 0.5 m g / k g i.v. MK-801 on the facilitation of the flexor reflex induced by conditioning stimulation of the sural nerve in a rat with intact sciatic nerves (A) and in another rat 14 days after ipsilateral sciatic nerve section (B). Note that MK-801 produced similar depression of the baseline reflex in both preparations. However, while MK-801 totally blocked the reflex facilitation by the sural nerve CS in the rat with intact sciatic nerve (A), it failed to do so in the axotomized rats (B).
N M D A antagonists were also effective in reducing the baseline flexor reflex. It is suggestive from these results that certain site(s) in the flexor reflex pathway, possibly within the motoneuron pool, are sensitive to blockade of the N M D A receptor and are responsible for the reflex depressive effect of MK-801. On the other hand, the reflex wind-up and facilitation during and following the C-fiber afferent CS possibly reflects similar changes in dorsal horn neurons involved in nociceptive transmission. In the present study, we observed that i.v. MK-801 depressed the baseline flexor reflex in a similar manner in rats with intact and sectioned sciatic nerves, indicating that the sites responsible for this effect of MK-801 were not altered by peripheral axotomy. In contrast, unlike in rats with intact sciatic nerves [30], MK-801 totally lost its effectiveness in reducing wind-up and in blocking maximal reflex facilitation following the CS of C-afferents in axotomized
rats. However, MK-801 reduced the duration of facilitation, indicating that it still had some residual effect. These results imply that activation of the N M D A receptor may be less critically involved in the generation of spinal cord hyperexcitability following C-fiber input after peripheral nerve section. Considering the morphological observation of substantial loss of synaptic vesicles in peptide-containing glomeruli in primary afferent terminals in axotomized rats [34,35], it is most likely that our results reflect the inability of C-afferent terminals to release glutamate upon repetitive stimulation. It is also possible that there is a down-regulation of the N M D A receptor or a reduced coupling between the released glutamate and the postsynaptic N M D A receptors after nerve injury. Dysfunction in glutamatergic transmission may also underly the observation that repetitive stimulation of muscle afferents in axotomized rats failed to elicit prolonged increase in spinal cord excitability [22]. We have previously reported that the facilitatory effect of C-fiber conditioning stimulation can also be blocked by antagonists of tachykinin receptors in rats with intact sciatic nerves [25,26],. We and others have shown that there is a synergistic interaction between activation of N M D A receptors and the NK~ tachykinin receptor in eliciting wind-up and central sensitization [14,19,30]. After peripheral nerve section, the level of substance P was depleted in primary sensory afferents [10] and this was reflected by a gradual reduction in the effectiveness of tachykinin antagonists in blocking flexor reflex facilitation by conditioning C-fiber stimulation [24]. In contrast, vasoactive intestinal peptide (VIP), which normally does not mediate spinal hyperexcitability [32], becomes an important mediator after nerve injury [23,24]. It is unclear, however, whether VIP is solely responsible for spinal hyperexcitability or whether it interacts with compounds other than glutamate. Moreover, it is also possible that other neuroactive substances, such as purines [16], may have a more important role after axotomy, which needs to be examined. N M D A receptor antagonists have been shown to relieve neuropathic pain-related behavior in rats associated with partial nerve constriction [17,33], leading to the suggestion that such drugs may be useful in treating human neuropathic pain. The present results, however, indicate that this may not be the case when pain arises after complete nerve transection. In a clinical report no analgesic effect following i.t. administration of the N M D A receptor antagonist CPP was observed on the spontaneous pain and allodynia in the territory of the injured nerve [12]. These are symptoms which may result from sensitization of dorsal horn neurons receiving input from injured primary afferents [28]. As the inputs of at least two major excitatory sensory transmitters, glutamate and SP, are impaired after
X.-J. Xu et aL /Brain Research 670 (19951 342-346
nerve section, it is interesting to suggest that drugs which interfere with neurotransmission in axotomized sensory neurons, such as VIP antagonists [23,24], CCK-B antagonists [31] or galanin agonists [27], may have more relevance in managing neuropathic pain.
This work is supported by the Swedish Medical Research Council (07913, 2887), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, Marianne and Marcus Wallenbergs Stiftelse, King Gustav V's and Queen Victoria's Stiftelse, Stiftelsen Lars Hiertas Minne and research funds of the Karolinska Institute.
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