Library

Notes: Abstract Electrophysiological experiments in anesthetized cats and rats were performed in order to study the effects of dipyrone on single afferent fibers from the knee joint and on spinal cord neurons with knee joint input. The neurons were activated and/or rendered hyperexcitable by an acute inflammation in the knee joint. In the joint nerve in cats, intravenous dipyrone (25–100 mg/kg) reduced ongoing activity in 10/12 thinly myelinated afferents but only in 1/10 unmyelinated afferents; the responses to movements of the inflamed knee were reduced in 8/10 thinly myelinated but only in 3/10 unmyelinated units. The reduction of activity was significant 20–30 min after application and was maximal at 60–180 min. In the spinal cord of spinalized cats, intravenous dipyrone (25–100 mg/kg) reduced ongoing activity and/or responses to pressure onto the inflamed knee in 14/16 neurons and in non-spinalized rats similar effects were seen in 10/11 neurons. Effects on spinal cord neurons started 5–10 min after application and were maximal after 20–40 min. These data show pronounced suppression of inflammation-induced nociception by dipyrone and they suggest that the spinal cord is a major site of action of this compound.

Notes: Abstract  Administration of the N-type calcium channel antagonist ω-conotoxin GVIA to the spinal cord reduces spinal neuronal responses to innocuous and noxious pressure applied to the knee, both in rats with normal knees and in rats in which a knee inflammation has induced a state of hyperexcitability in spinal neurons (Neugebauer et al. 1996, J Neurophysiol 76: 3740–3749). In the present experiments we studied whether the development of hyperexcitability of spinal neurons induced by intra-articular injection of mustard oil, an excitant of C-fibres, can be influenced by spinal pretreatment with ω-conotoxin GVIA. In anaesthetized rats, responses of wide-dynamic-range neurons were recorded in the spinal dorsal horn when standardized stimulation with innocuous and noxious pressure was applied to the knee and ankle joints. Injection of mustard oil into the knee joint cavity caused an initial neuronal discharge followed by an early (peaking at about 15 min) and a late (after 60 min) facilitation of responses to innocuous and noxious stimulation of the knee. Responses to ankle stimulation showed only the late facilitation. When ω-conotoxin GVIA (20 μl, 1 μM) was applied into a small trough onto the spinal cord above the recording site the responses to articular stimulation were reduced. Furthermore, when mustard oil was injected while ω-conotoxin GVIA was on the spinal cord, the early increase in the neuronal responses to innocuous pressure on the knee and the late increase in responses to noxious pressure on the ankle were significantly smaller than those observed in rats not treated with ω-conotoxin GVIA; the drop in the responses to noxious pressure on the knee was not significant. Thus the spinal application of ω-conotoxin GVIA reduced but did not completely prevent the fast and slow development of neuronal hyperexcitability of spinal cord neurons produced by a prompt and strong excitation of afferent C-fibres. This suggests that N-type calcium channels are important for the development of spinal cord hyperexcitability.

Notes: Summary 1. Recordings were made from 16 ascending tract cells in the spinal cords of anaesthetized, spinalized cats before and after an acute arthritis was produced by injection of kaolin and carrageenan into the knee joint. 2. The responses tested routinely were to passive flexion of the knee, an innocuous movement. In some cases, responses to other movements were also tested, and changes in background discharge rates were monitored. 3. Control recordings for a period of 1 h or in 3 cases of 3 h indicated that the responses to flexion were reasonably stationary. 4. Four tract cells that initially showed little or no response to flexion of the knee joint developed large responses within 1 to 2 h after inflammation of the joint. 5. Another 9 cells were tested that had responses to flexion of the knee joint prior to inflammation. In 6 cases, inflammation produced enhanced static or transient responses. In 2 cases, the effect of flexion was initially inhibitory or variable, but after inflammation these cells showed large excitatory responses. In the other case, inflammation had no effect. Background discharges were increased by inflammation in 6 of these 9 cells. 6. The effect of inflammation of the knee joint was tested on 3 tract cells that had no clearly defined receptive field in the knee. In 1 case, a response developed to knee flexion after acute inflammation was produced. In the other 2 cases, there were initially responses to knee flexion, but these were unchanged by inflammation. 7. Two of the cells tested had bilateral receptive fields in or around the knee joints. Inflammation of one knee joint enhanced the responses to flexion of the same but not of the contralateral knee in one case but greatly increased the responses to flexion of both knees in the other case. 8. Injections of prostaglandin (PGE2) caused an enhancement of the responses to knee flexion beyond that caused by inflammation in 5 of 7 cases. One cell whose responses to flexion of the knee were unaffected by inflammation showed inhibitory responses to prostaglandin injections into the inflamed knee joint. 9. The effects of inflammation on the responses of ascending tract cells of the spinal cord appear to serve as a useful neural model of the events responsible for the development of arthritic pain.

Notes: Summary 1.Responses were recorded from 160 ascending tract cells in segments L4 to L6 of the spinal cord in chloralose anaesthetized, spinalized cats. The tract cells were identified by antidromic activation following stimulation of pathways in the lateral and ventral funiculi at the level of the spinal cord transection at the thoracolumbar junction. Axonal conduction velocities ranged from 9 to 114 m/s. 2. A sample of 152 of the neurones examined could be subdivided according to the distribution of their receptive fields into 49 cells activated just from receptors located in skin (“s” cells), 17 neurones excited by receptors in deep tissues (“d” cells), 15 units with a convergent input from receptors in skin and deep tissues (“sd” cells), and 25 neurones with a convergent input from the knee joint and either skin (“sj” cells), deep tissues (“dj” cells) or both (“sdj” cells). No receptive fields could be demonstrated for the remaining 46 neurones. 3. “S” and “sj” cells were found almost exclusively in the dorsal horn, whereas many “d”, “sd”, “sdj” and “dj” units were in the ventral horn. Almost all of the cells that lacked receptive fields were in the ventral horn or intermediate grey. 4. Ninety-one of 158 cells (56%) demonstrated no background activity. Of these, 43 cells (27%) lacked receptive fields. Many of the silent neurones were in the ventral horn, but some were in the dorsal horn. Of 25 cells having knee joint input, 18 (72%) had background activity. 5. All of the neurones that had a receptive field in the knee joint also had a convergent input from receptors in other tissues. In 3 cases, there was a receptive field in the skin over the foot (“sj” cells). For 16 cells, receptive fields included not only the knee joint but also skin and deep tissue (“sdj” cells). Usually, the cutaneous receptive field was near the knee joint, but sometimes it was remote, such as on the foot. The deep receptive fields were chiefly in the muscles of the thigh and/or leg. For 6 “dj” cells, the receptive fields included not only the knee joint but also deep fields like those of “sdj” cells. 6. Cutaneous receptive fields were classified as “low threshold” (cells excited best by innocuous intensities of mechanical stimulation), “wide dynamic range” (cells activated by weak mechanical stimuli, but the best responses were to noxious stimuli) or high threshold (innocuous stimuli had little effect, but noxious mechanical stimuli produced a vigorous discharge). Similarly, stimulation of the knee joint with weak mechanical stimuli could excite some neurones, while others could be activated by weak or strong articular stimuli but were excited best by noxious stimuli, and still other neurones were activated by knee joint stimuli only if the intensity was noxious. 7. In several instances, contralateral receptive fields were noted. These were generally in deep tissue or in the knee joint. 8. It was concluded that many of the responses to articular stimulation of the spinal cord ascending tract cells examined in this study could have been mediated by the fine afferent fibres that supply the knee joint. Although further work will be required to determine which particular ascending tracts transmit nociceptive information concerning the knee joint, it can be proposed that many of the responses demonstrated here were likely to play a role in either joint pain of in triggering responses associated with joint pain.

Notes: Experiments were performed in barbiturate-anaesthetized, spinalized cats to investigate the effect of calcitonin gene-related peptide (CGRP) on the spatial distribution of immunoreactive substance P (ir-SP) in the spinal cord released by electrical nerve stimulation and noxious mechanical stimuli. The presence of ir-SP was assessed with microprobes bearing C-terminus-directed antibodies to SP. CGRP was microinjected into the grey matter of the spinal cord near microprobe insertion sites at depths of 2500, 2000, 1500 and 1000 μm using minute amounts (in total 0.2–0.5 μl) of Ringer solution containing CGRP at a concentration of 10−5 or 10−3 M. In the untreated cord electrical stimulation of the tibial nerve (suprathreshold for all C fibres) elicited release of ir-SP which was centred in and around the lamina II. After microinjection of CGRP, stimulation-associated ir-SP was detected in a region extending from the cord surface down to the ventral horn. This pattern was similar to that observed after the microinjection of synthetic peptidase inhibitors (Duggan et al., Brain Res., 579, 261–269, 1992). The large expansion of sites accessed by ir-SP was time-dependent, reaching a maximal effect within 10–40 min after microinjection of CGRP, and reversal was observed in subsequent probes. A similar expansion of the regions accessed by ir-SP after microinjection of CGRP was also observed when release of ir-SP was evoked by noxious mechanical stimulation of the toes. These results indicate that one important function of CGRP in the spinal cord may be the control of the intraspinal sites and neuronal circuits accessed by released substance P, possibly by inhibition of endopeptidases responsible for peptide degradation.

Notes: In spinal cord neurons in anesthetized rats, the role of neurokinin A and neurokinin-2 receptors in the processing of nociceptive information from the knee joint was studied. The specific non-peptide antagonist at the neurokinin-2 receptor, SR48968, its inactive R-enantiomer, SR48965, neurokinin A, substance P and (R, S)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), were administered ionophoretically close to neurons with input from the knee joint. SR48968 reduced the effects of exogenous neurokinin A, but not those of exogenous substance P and AMPA, indicating selective blockade of neurokinin-2 receptors. In most neurons with input from the normal knee joint, SR48968 reduced dose-dependently the responses to noxious pressure applied to the knee, and in ˜50% of the neurons the responses to innocuous pressure. The administration of SR48968 during the induction of an experimental joint inflammation markedly attenuated the development of inflammation-evoked hyperexcitability. In hyperexcitable neurons with input from the inflamed joint, SR48968 reduced the responses to noxious and innocuous pressure. The relative reduction of the responses was more pronounced than in neurons with input from the normal joint. None of the effects of SR48968 was mimicked by SR48965. These data show that neurokinin-2 receptors are involved in the spinal processing of nociceptive information from the normal joint. Furthermore, neurokinin-2 receptors must be coactivated at an early stage of inflammation, to allow the generation of hyperexcitability. Finally, neurokinin-2 receptors are involved in the maintenance of hyperexcitability during inflammation. In summary, spinal neurokinin-2 receptors are important in the generation of pain in the normal and inflamed joint.

Notes: The role of the endogenous kappa opioid system in the control of neuronal activity has been studied in the spinal cord of normal rats and in rats with Freund's adjuvant induced unilateral inflammation of the ankle under barbiturate anaesthesia. During recordings from neurons with ankle input the kappa receptor agonist U50,488H and/or the kappa antagonist nor-binaltorphamine were administered ionophoreticaliy using multibarrel electrodes. In most neurons tested U50, 488H reduced the responses evoked by pressure applied across the ankle whereas smaller proportions of neurons showed increased activity or were not affected. The kappa opioid antagonist nor-binaltorphamine affected more neurons in rats with inflammation than in control rats. Ongoing activity was increased in 7 of 19 (37%) neurons in control rats, in 16 of 24 (67%) neurons in the acute phase of inflammation (2 days post inoculation) and in 15 of 23 (65%) neurons in the chronic phase of inflammation (16–20 days post inoculation). During application of nor-binaltorphamine in control rats, the responses to pressure were increased in 9 cells (36%), reduced in 7 cells (28%) and unaffected in 9 cells (36%). In the acute phase of inflammation significantly more neurons (11 of 15, 73%) showed enhanced responses to pressure during ionophoresis of nor-binaltorphamine but not in the chronic phase. These results show that spinal cord neurons with ankle input are influenced by the endogenous kappa opioid system particularly under inflammatory conditions. The upregulation of this system under inflammatory conditions may serve to counteract inflammation-induced hyperexcitability.

Notes: High threshold voltage-dependent P- and Q-type calcium channels are involved in neurotransmitter release. In order to investigate the role of P- and Q-type calcium channels in the mechanosensory (nociceptive) processing in the spinal cord, their participation in the responses of spinal wide-dynamic-range neurons to innocuous and noxious mechanical stimulation of the knee and ankle joints was studied in 30 anaesthetized rats. The knee was either normal or acutely inflamed by kaolin/carrageenan. During the topical application of ω-agatoxin IVA (P-type channel antagonist, 0.1 μM) onto the dorsal surface of the spinal cord, the responses to innocuous and noxious pressure applied to the normal knee were increased to respectively 124 ± 42% and 114 ± 23% of predrug values (mean ± SD, P 〈 0.05, 14 neurons). By contrast, in rats with an inflamed knee, the responses to innocuous and noxious pressure applied to the knee were reduced to respectively 72 ± 19 and 73 ± 22% of baseline (mean ± SD, P 〈 0.01, 13 neurons). In the same neurons, ω-agatoxin IVA slightly increased the responses to pressure on the non-inflamed ankle whether the knee was normal or inflamed. Thus P-type calcium channels seem to acquire a predominant importance in the excitation of spinal cord neurons by mechanosensory input from inflamed tissue and hence in the generation of inflammatory pain. By contrast, the Q-type channel antagonist, ω-conotoxin MVllC (1 or 100 μM), had no significant effect upon responses to innocuous or noxious pressure applied to either normal or inflamed knees (25 neurons).