Effects of spinal administration of muscimol on C- and A-fibre evoked neuronal responses of spinal dorsal horn neurones in control and nerve injured rats

Neuropathic pain is a common clinical problem with complex aetiology, mechanisms and symptoms. Alterations in spinal gamma-aminobutyric acid (GABA) receptors may contribute to persistent pain states. The aim of the present study is to investigate potential changes of spinal GABA(A)-receptor function following peripheral nerve injury. Effects of spinal administration of the GABA(A)-receptor agonist muscimol (0.1-30 microg/50 microl) on electrically-evoked responses of spinal neurones in control, spinal nerve ligated and sham operated halothane-anaesthetised rats were studied. Spinal muscimol significantly (10 microg/50 microl) reduced evoked Abeta-, Adelta- and C-fibre responses of spinal neurones in control rats (58+/-22% of control, P<0.05; 3+/-2% of control, P<0.001; and 8+/-7% of control, P<0.001; respectively). Muscimol produced significantly greater inhibition of Adelta- and C-fibre evoked neuronal responses compared to Abeta-fibre evoked neuronal responses in control rats (P<0.001). C-fibre mediated post-discharge responses and the non-potentiated C-fibre evoked responses were significantly inhibited by muscimol in control rats. Inhibitory effects of muscimol (10 microg/50 microl) were blocked by pre-application of spinal bicuculline (10 microg/50 microl). Following either sham surgery, or spinal nerve ligation, spinal muscimol inhibited Abeta-, Adelta- and C-fibre evoked responses of spinal neurones to a similar extent, however significant inhibitory effects on the post-discharge response were not observed in nerve injured rats. Our data demonstrate that GABA(A)-receptor control of Abeta- and Adelta-fibre evoked responses are not altered in nerve injured or sham operated rats, compared to control. However, following nerve injury we report a reduction in GABA(A)-receptor control of C-fibre responses, in particular in relation to post-discharge responses.     

Spinal cord injury triggers sensitization of wide dynamic range dorsal horn neurons in segments rostral to the injury

A spinal cord injury (SCI) was produced in adult rats by complete spinal cord transection at L6-S1. Neuropathic pain behaviors similar to the chronic central pain (CCP) syndrome in human, such as thermal hyperalgesia, mechanical allodynia and autotomy, were present in these rats after spinal cord injury. Meanwhile, wide dynamic range (WDR) neurons recorded in the spinal dorsal horn rostral to the lesion responded as high frequency of spontaneous activities, long duration of after-discharges to noxious electrical stimuli and an augmented wind-up to 0.5 Hz stimuli. By using bupivacaine powder, a sodium channel blocker, at the locus of transection immediate after nerve injury, the chronic pain behaviors were prevented; the hyperexcitability of WDR neurons was also substantially reduced. It is suggested that spinal cord transection induces the CCP syndromes, which may be evoked and maintained by the hyperexcitability in WDR neurons rostrally. Reducing the neuronal activity at the site of lesion following injury may prevent the development of CCP after SCI.     

Oxytocin actions on afferent evoked spinal cord neuronal activities in neuropathic but not in normal rats

A hypothalamic oxytocinergic-descending pathway that reaches the dorsal horn of the spinal cord has been well documented and recently related to states of pain and analgesia. In order to study the action of the neuropeptide oxytocin (OT) on pain-related responses, we compared dorsal horn neuronal responses to electrical and mechanical stimulation of receptive fields in normal and neuropathic rats. Spinal nerve (L5 and L6) ligation (Chung rats) was used to produce experimental neuropathy. Single unit activity was recorded at the L4-L5 level from neurons identified as wide dynamic range presenting latency responses corresponding to A-beta, A-delta, C fibers and also exhibiting post-discharge, and wind-up. We tested intrathecally applied doses of 0.05, 0.1, 1, 2, 5, 10 I.U. of OT. Minor effects on responses to electrical stimulation were present in normal rats. Mechanical responses evoked by von Frey filaments were slightly reduced in normal animals. In neuropathic rats a dose of 1 I.U. produced a significant reduction in C-fibers and post-discharge activities, and doses of 2 I.U. caused a further, pronounced reduction in post-discharge, wind-up, and input values. However, the most marked change was the post-discharge reduction at 10 and 20 min after OT administration. Mechanical responses were significantly reduced in terms of their discharge rate response in neuropathic rats. The contrasting results obtained in normal and neuropathic rats revealed an important distinction between these animals and indicate that plastic changes occur as a consequence of nerve damage. In neuropathic rats, mechanisms involving ascending noxious information to the paraventricular nuclei and descending OT activities could be altered so sensitizing the OT receptors of the spinal dorsal horn cells and could explain our observations. Our results point out an anti-algesic OT effect in neuropathic rats.     

Inhibition of spinal nociceptive responses after intramuscular injection of capsaicin involves activation of noradrenergic and opioid systems

Extracellular recordings of wide dynamic range neurones in the dorsal horn driven by electrical stimulation of the sciatic nerve were performed in intact urethane-anaesthetized Sprague–Dawley rats. The electrically evoked neuronal responses were defined as A- and C-fibres responses according to latencies, and the effect of a deep nociceptive conditioning stimulus induced by 200 μg capsaicin (8-methyl-N-vanillyl-6-noneamide) injected into the contralateral gastrocnemius–soleus muscle was studied for at least 30 min. Independent of the size and location of the receptive field of the neurone under study, a clear inhibition of the neuronal responses was observed. The electrically evoked C-fibre responses were inhibited to 53% of baseline 15–30 min after injection of capsaicin. This inhibition was only slightly attenuated by 125 nmol of the α-adrenoceptor antagonist phentolamine or 250 nmol of the opioid receptor antagonist naloxone applied directly onto the spinal cord when the two compounds were administered separately 5 min before capsaicin. In contrast, when a mixture of the two compounds was given 5 min before capsaicin, the effect of capsaicin was completely abolished. These results indicate that activation of the capsaicin-sensitive afferents in the gastrocnemius–soleus muscle inhibits the electrically evoked C-fibre responses in the dorsal horn by activating noradrenergic and opioidergic inhibitory systems. Moreover, our data indicate that the activation of these two systems following injection of capsaicin has a sub-additive inhibitory effect on the wide dynamic range neurones in the spinal cord. We conclude that only one of these systems is sufficient for the inhibition to occur.     

The influence of temporal summation on a C-fibre reflex in the rat: effects of lesions in the rostral ventromedial medulla (RVM)

In intact rats, an inhibitory mechanism counteracts the increase in excitability of a flexor reflex seen in spinal animals following high-intensity, repetitive stimulation of C-fibres. We tested the hypothesis that the rostral ventromedial medulla (RVM) is involved in these processes. Electromyographic responses elicited by electrical stimulation of the sural nerve, were recorded from the ipsilateral biceps femoris in halothane-anaesthetised, sham-operated or RVM-lesioned rats. There were no significant differences between the C-fibre reflexes in the two groups in terms of their thresholds, latencies, durations or mean recruitment curves. The excitability of the C-fibre reflex was tested following 20 s of high-intensity homotopic electrical conditioning stimuli at 1 Hz. During the conditioning period, the EMG responses first increased in both groups (the wind-up phenomenon), but then decreased in the sham-operated rats and plateaued in the RVM-lesioned rats. These effects were followed by inhibitions that were very much smaller in the RVM-lesioned rats, both in terms of their magnitudes and their durations. It is concluded that the RVM is involved in inhibitory feedback mechanisms elicited by temporal summation of C-fibre afferents that both counteract the wind-up phenomenon and trigger long periods of inhibition.     

Spinal GABA(B)-receptor antagonism increases nociceptive transmission in vivo.

GABA(B) receptors modulate primary afferent fibre evoked responses of spinal neurones. Here effects of the selective GABA(B) receptor antagonist, CGP-35348, on electrically-evoked responses of spinal neurones in control and carrageenan-inflamed rats were studied. Spinal CGP-35348 (0.1-10 microg/50 microl) did not alter Abeta- or Adelta-fibre evoked neuronal responses in control rats, although C-fibre evoked responses and post discharge responses of spinal neurones were significantly facilitated by 3.0 and 10.0 microg/50 microl CGP-35348 (p < 0.05). In carrageenan-treated animals, spinal CGP-35348 did not alter electrically evoked responses of spinal neurones at any dose. Our data suggest that following acute peripheral inflammation there is loss of endogenous GABA(B) receptor mediated inhibition of C-fibre transmission at the level of the spinal cord.     

Differential pharmacological modulation of the spontaneous stimulus-independent activity in the rat spinal cord following peripheral nerve injury

Peripheral nerve injury is a significant clinical problem that is often difficult to treat. The major clinical symptoms are numbness, tactile and cooling allodynia, hyperalgesias as well as ongoing pain. In animal models of neuropathy, abnormal responses to applied (or evoked) stimuli can be gauged, but spontaneous pain, a major clinical issue, has proved very difficult to assess. In neuropathic animals, spinal neuronal hyperexcitability indicative of peripheral and central changes with high levels of spontaneous neuronal firing has been reported. This latter stimulus-independent firing of sensory neurones may be a measure related to ongoing pain. Two weeks after L5/6 spinal nerve ligation, deep dorsal horn neurones were recorded in halothane-anesthetized rats. The majority of neurones in neuropathic rats showed increased levels of spontaneous firing with irregular firing patterns. We examined and compared the effects of 5 centrally acting pharmacological agents: morphine (i.t. or i.v.), gabapentin, ketamine, memantine and mepyramine on stimulus-independent neuronal firing. This ongoing activity showed high sensitivity to gabapentin (s.c.) and morphine (i.t.) administration, being significantly reduced in a dose-dependent manner. Morphine administered via the systemic route produced modest but non-significant reductions of spontaneous activity. The two NMDA receptor antagonists, ketamine and memantine, and the histamine H1 receptor antagonist, mepyramine, produced minor effects at doses known to be effective on stimulus evoked measures of deep dorsal horn neurones. This may form an electrophysiological basis for the efficacy of gabapentin and spinal morphine on ongoing pain in patients with peripheral neuropathy.     

Effects of peripheral nerve injury on functional spinal VR1 receptors

VR1 receptors, present on Adelta- and C-fibres and post-synaptic sites within the spinal cord dorsal horn, is an integrator of noxious stimuli. Here, the contribution of spinal VR1 receptors to spinal nociceptive processing in nerve injured (selective spinal nerve ligated SNL) and sham anaesthetised rats was studied. Spinal capsazepine (0.5-30 microM), a competitive VR1 antagonist, reduced noxious evoked responses of spinal neurones to a greater extent in sham operated rats, compared to SNL rats. Significant differences between the effect of spinal capsazepine on the non-potentiated component of the C-fibre evoked response of SNL and sham operated rats are reported (p< 0.01, two-way ANOVA). Our data suggest there is a functional plasticity of the spinal VR1 receptor following nerve injury.     

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.     

Chronic Spinal Nerve Ligation Induces Changes in Response Characteristics of Nociceptive Spinal Dorsal Horn Neurons and in Their Descending Regulation Originating in the Periaqueductal Gray in the Rat

We studied whether a chronic neuropathy induced by unilateral spinal nerve ligation changes the response characteristics of spinal dorsal horn wide-dynamic range (WDR) neurons or their periaqueductal gray (PAG)-induced descending modulation. Experiments were performed in rats with behaviorally demonstrated allodynia induced by spinal nerve ligation and in a group of nonneuropathic control rats. The stimulus–response functions of WDR neurons for mechanical and thermal stimuli and the modulation of their peripherally evoked responses by electrical stimulation of the PAG were determined under pentobarbital anesthesia. The results showed that neuropathy caused a significant leftward shift in stimulus–response functions for mechanical stimuli. In contrast, stimulus–response functions for noxious heat stimuli in the neuropathic limb were, if anything, shifted rightward, although this shift was short of statistical significance. In neuropathic rats, PAG stimulation produced a significantly stronger attenuation of spinal neuronal responses induced by noxious heat in the unoperated than in the operated side. At the intensity that produced attenuation of noxious heat stimuli, PAG stimulation did not produce any significant change in spinal neuronal responses evoked by mechanical stimuli either from the operated or the nonoperated hindlimb of the neuropathic rats. Spontaneous activity of WDR neurons was higher in the operated side of neuropathic rats than in control rats. Afterdischarges evoked by peripheral stimuli were observed in 1/16 of the WDR neurons ipsilateral to spinal nerve ligation and not at all in other experimental groups. The WDR neurons studied were not activated by innocuous or noxious cold stimuli. The results indicate that spinal nerve ligation induces increased spontaneous activity and enhanced responses to mechanical stimuli in the spinal dorsal horn WDR neurons, whereas noxious heat-evoked responses are not significantly changed or if anything, attenuated. Moreover, the inhibition of noxious heat stimuli by PAG stimulation is attenuated in the neuropathic side. It is proposed that the observed changes in the response characteristics of the spinal dorsal horn WDR neurons and in their descending modulation may contribute to the neuropathic symptoms in these animals.     

Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.     

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague-Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH-133, and the CB1 receptor agonist, arachidonyl-2-chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham-operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH-133 (3 microm) or ACEA (1 microm) significantly (P<0.001) attenuated capsaicin-evoked calcium responses in DRG neurons in neuropathic and sham-operated rats. The CB2 receptor antagonist, SR144528, (1 microm) significantly inhibited the effects of JWH-133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 microm). In vivo experiments evaluated the effects of spinal administration of JWH-133 (8-486 ng/50 microL) and ACEA (0.005-500 ng/50 microL) on mechanically evoked responses of neuropathic and sham-operated rats. Spinal JWH-133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham-operated rats. These inhibitory effects were blocked by SR144528 (0.001 microg/50 microL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham-operated rats, these effects were blocked by SR141716A (0.01 microg/50 microL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham-operated rats suggesting that spinal CB2 may be an important analgesic target.     

Activation of spinal cannabinoid 1 receptors inhibits C-fibre driven hyperexcitable neuronal responses and increases [35S]GTPgammaS binding in the dorsal horn of the spinal cord of noninflamed and inflamed rats

The analgesic potential of cannabinoid (CB) receptor agonists is of clinical interest. Improved understanding of the mechanisms of action of cannabinoids at sites involved in the modulation of acute and sustained inflammatory nociceptive transmission, such as the spinal cord, is essential. In vivo electrophysiology was used to compare the effect of the synthetic CB agonist, HU210, on acute transcutaneous electrical-evoked responses of dorsal horn neurons of noninflamed anaesthetized rats and anaesthetized rats with a peripheral carrageenin inflammation. CB receptor G-protein coupling in lumbar spinal cord sections of noninflamed and carrageenin-inflamed rats was studied with in vitro autoradiography of guanylyl 5'-[gamma-[35S]thio]triphosphate ([35S]GTPgammaS) binding. Spinal HU210 significantly inhibited the C-fibre-mediated late (300-800 ms) postdischarge response of dorsal horn neurons of noninflamed and carrageenin-inflamed rats; the CB1 receptor antagonist SR141716A blocked the effect of HU210. HU210 had limited effects on A-fibre-evoked dorsal horn neuronal responses of both groups of rats. HU210 significantly increased [35S]GTPgammaS binding in the dorsal horn of the spinal cord of both groups of rats compared with basal [35S]GTPgammaS binding; SR141716A blocked these effects. The predominant effect of spinal HU210, via CB1 receptor activation, was on the C-fibre driven postdischarge responses, a measure of neuronal hyperexcitability following repetitive C-fibre stimulation. Sustained, but not enhanced, antinociceptive effects of HU210 following carrageenin inflammation are reported; CB receptor G-protein coupling was not altered by inflammation. These results strengthen the body of evidence suggesting CB agonists may be an important novel analgesic approach for the treatment of sustained pain states.     

Spinal administration of capsazepine inhibits noxious evoked responses of dorsal horn neurons in non-inflamed and carrageenan inflamed rats

Vanilloid VR1 receptors are located in the dorsal horn of the spinal cord. The aim of the present study was to determine the role of spinal vanilloid receptors (VR1) during nociceptive processing in control and inflamed rats. Effects of spinal administration of capsazepine (0.5-30 microM/50 microl), a competitive VR1 antagonist, on innocuous and noxious evoked responses of spinal neurones were studied in halothane anaesthetised rats. Transcutaneous electrical-evoked neuronal responses of spinal neurones were recorded in control and carrageenan (2%, 3 h) inflamed rats. Spinal application of capsazepine did not significantly alter Abeta-fibre evoked responses of neurones, however Adelta-fibre evoked responses were significantly inhibited by capsazepine in both non-inflamed and carrageenan inflamed rats (30 microM: non-inflamed 31+/-8% of control, P<0.01: carrageenan-inflamed 43+/-6% of control, P<0.01). Similarly, the evoked C-fibre mediated post-discharge responses of spinal neurones in non-inflamed and carrageenan inflamed rats were reduced by capsazepine (30 microM: non-inflamed 41+/-14% of control, P<0.01: carrageenan-inflamed 31+/-9% of control, P<0.01). These results demonstrate a role of spinal VR1 receptors during noxious, but not innocuous transmission, at the level of the spinal cord. The degree of effect of capsazepine on evoked neuronal responses was similar in control and inflamed rats, suggesting that the role of spinal VR1 receptors is not altered following short-term peripheral inflammation. Our data suggest that following noxious peripheral stimulation, spinal VR1 receptors are activated, but the endogenous ligands mediating this effect remain to be elucidated.     

Cortical hyperexcitability in response to preserved spinothalamic inputs immediately after spinal cord hemisection

Chronic injury of the main somatosensory pathways ascending along the spinal cord - the dorsal columns and the spinothalamic tract - can produce both changes in the organization of cortical somatotopic maps and neuropathic pain. Little is known, however, about the early neurophysiological changes occurring immediately after injury. We bilaterally recorded the neural activity of the hindpaw representation of the primary somatosensory cortex evoked by stimuli delivered to the hindpaws before and immediately after a thoracic spinal cord hemisection in anesthetized rats. This unilateral spinal cord injury allowed us to separately investigate the cortical effects of deafferenting the dorsal column (stimuli ipsilateral to the hemisection) or the spinothalamic tract (stimuli contralateral to the hemisection). The hemisection produced immediate bilateral changes in the cortical responses evoked by stimuli delivered to the hindpaw ipsilateral to the hemisection (deafferented dorsal column): an expected loss of classical short-latency cortical responses, accompanied by an unexpected appearance of long-latency activations. At the population level, these activations reflected a progressive stimulus-induced transition of the hindpaw somatosensory cortex from up-and-down states to a sustained activated state. At the single-cell level, these cortical activations resembled the “wind-up” typically observed – with the same type of stimuli – in the dorsal horn cells originating the spinothalamic tract. Virtually no changes were observed in the responses evoked by stimuli delivered to the hindpaw contralateral to the hemisection (deafferented spinothalamic tract). These results suggest that spinal cord hemisection immediately produces an abnormal hyperexcitability of the primary somatosensory cortex in response to preserved spinothalamic inputs from the hindpaw. This immediate cortical hyperexcitability could be important to understand the long-term development of cortical reorganization and neuropathic pain after incomplete spinal cord lesions.     

Enlargement of the receptive field size to low intensity mechanical stimulation in the rat spinal nerve ligation model of neuropathy

One characteristic of plasticity after peripheral tissue or nerve damage is receptive field reorganization, and enlargement of receptive field size has been suggested to occur in certain models of neuropathic pain. The aim of the present study was to explore whether enlargement of neuronal receptive fields could contribute to the mechanical allodynia found on the ipsilateral paw in the spinal nerve ligation model of neuropathy. After ligation of L(5)-L(6) spinal nerves, all rats developed behavioral signs of mechanical allodynia, while the sham-operated control group displayed no such changes. The characteristics of the evoked responses of the neurones recorded in the dorsal horn of the rats were similar between the spinal nerve ligation, the sham operated control group, and the nonoperated control group, except for spontaneous activity, which was significantly increased in the spinal nerve ligation group. The mean size of the receptive field on the ipsilateral hindpaw, mapped using low-intensity stimulation with 9-g von Frey hair, was significantly increased in the spinal nerve ligation group, as compared to the sham-operated group. No significant difference was seen with 15- or 75-g von Frey hairs. The distribution of the receptive fields over the plantar surface of the paw was similar between the study groups. The enlargement of receptive field for non-noxious touch could be an indication of central sensitization in this model.     

The Cav2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch-evoked pain). Evidence exists for nerve damage-induced plasticity in both transmission and modulatory systems, including changes in voltage-dependent calcium channel (VDCC) expression and function; however, the role of Ca(v)2.3 calcium channels has not clearly been defined. Here, the effects of SNX-482, a selective Ca(v)2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [Kim & Chung, (1992)Pain, 50, 355-363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham-operated animals. Spinal SNX-482 (0.5-4 microg/50 microL) exerted dose-related inhibitions of noxious C-fibre- and Adelta-fibre-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX-482. In contrast, non-noxious Abeta-mediated responses were not affected by SNX-482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX-482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX-482-sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in Ca(V)2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.     

Intraspinal transplantation of GABAergic neural progenitors attenuates neuropathic pain in rats: a pharmacologic and neurophysiological evaluation

Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABA(A) receptor antagonist bicuculline (BIC), GABA(B) receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1-3week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals.     

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV–V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.     

Ionotropic glutamate receptors contribute to maintained neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 µg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.5 and 1 µg) significantly attenuated neuronal activity in all three phenotypes of neurons (*p < 0.05). However, MCPG (group I/II metabotropic glutamate receptor antagonist, 100 µg) had no effect. The present study, in the context of previous work, suggests that ionotropic glutamate receptor activation play critical roles in the maintenance of neuronal hyperexcitability and neuropathic “below-level” pain behavior following spinal hemisection injury.