Lumbar Dorsal Horn Research Articles

Interferon-γ induced disruption of GABAergic inhibition in the spinal dorsal horn in vivo

The proinflammatory cytokine interferon-γ (IFN-γ), which can be present in elevated levels in the central nervous system during pathological conditions, may be involved in the generation of persistent pain states by inducing neuronal hyperexcitability. The aim of the present study was to examine whether loss of dorsal horn GABAergic inhibition may underlie this IFN-γ-mediated neuronal hyperexcitability. Repetitive intrathecal injections of recombinant rat IFN-γ (1000 U) or control buffer were administered to rats every second day for eight days. Electrophysiological recordings from lumbar dorsal horn neurons (n=46) were performed under halothane anaesthesia. Cellular responses were recorded before, during and after microiontophoretic application of the GABA antagonist bicuculline. In control animals, all cellular responses studied were significantly enhanced in the presence of bicuculline, including increased spontaneous activity, enhanced responses to innocuous and noxious mechanical stimulation and reduced paired-pulse depression. In contrast, in IFN-γ-treated animals, bicuculline ejection had little or no facilitating effect on neuronal responses and instead a significant proportion of neurons displayed reduced responses. Seventy-four percent of cells from IFN-γ treated animals showed a reduction in the response to noxious stimulation and 47% of the cells showed increased rather than reduced paired-pulse depression in the presence of bicuculline, thus suggesting IFN-γ-induced excitatory actions by GABA. These findings show that the prolonged presence of increased levels of IFN-γ in the central nervous system may contribute to the generation of central sensitization and persistent pain by reducing inhibitory tone in the dorsal horn. This implies a potential link between disinhibition and cytokine action in the spinal cord.

Spinal Prostaglandins Facilitate Exaggerated A- and C-fiber-mediated Reflex Responses and Are Critical to the Development of Allodynia Early after L5-L6 Spinal Nerve Ligation

Spinal prostaglandins are important in the early pathogenesis of spinal nerve ligation (SNL)-induced allodynia. This study examined the effect of SNL on the expression of cyclooxygenase (COX)-1, COX-2, and prostaglandin E2 receptors in the rat lumbar spinal cord, and the temporal and pharmacologic relation of these changes to the exaggerated A- and C-fiber-mediated reflex responses and allodynia, 24 h after injury. Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery. Paw withdrawal threshold, electromyographic analysis of the biceps femoris flexor reflex, and immunoblotting of the spinal cord were used. Both allodynia (paw withdrawal threshold of < or = 4 g) and exaggerated A- and C-fiber-mediated reflex responses (i.e., decrease in activation threshold, increase in evoked activity, including windup; P < 0.05) were evident 24 h after SNL but not sham surgery. Allodynic animals exhibited significant increases in prostaglandin E2 receptor (subtypes 1-3) and COX-1 (but not COX-2) expression in the ipsilateral lumbar dorsal horn. The corresponding ventral horns and contralateral dorsal horn were unchanged from sham controls. Exaggerated A- and C-fiber-mediated reflex responses were significantly attenuated by intrathecal SC-560 or SC-51322, but not SC-236, given 24 h after SNL. These results provide further evidence that spinal prostaglandins, derived primarily from COX-1, are critical in the exaggeration of A- and C-fiber input and allodynia, 24 h after SNL.

(642): Spinal nerve ligation-induced allodynia is dependent on the NFκ-B-mediated induction of spinal COX-2 and prostaglandin synthesis in the rat

L5/L6 spinal nerve ligation (SNL) induces early, spinal prostaglandin (PG)-dependent allodynia (<10 days after injury), including the increased expression of COX-2 in the ipsilateral lumbar dorsal horn 3 days after SNL. This study investigated the spinal expression of nuclear factor κ-B (NFκ-B; a modulator of COX-2 transcription), its relationship to the induction of COX-2, and the respective contributions of NFκ-B and COX-2 in the development of SNL-induced allodynia. Male, Sprague-Dawley rats (100-140g), fitted with intrathecal (i.t.) catheters, underwent SNL- or sham-surgery. Allodynia was defined as a paw withdrawal threshold (PWT) of ≤4g (baseline ≥15g). The biceps femoris flexor reflex was measured electromyographically, and lumbar segments were analyzed for NFκ-B and COX-2 protein expression. Allodynia and A- and C-fibre hyperexcitability (i.e. decreased activation threshold, increased evoked activity, including windup; p<0.05) were evident 3 days after SNL- but not sham-surgery. NFκ-B expression was increased in the ipsilateral, but not contralateral, dorsal horn of allodynic animals. This was apparent 12h after SNL and remained elevated at 3 days (p<0.05). Sham-surgery had no effect on NFκ-B (p>0.05). The NFκ-B inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 100μg i.t.), given immediately after SNL, decreased A- and C-fibre hyperexcitability 3 days later (p<0.05), and prevented the development of allodynia. Hyperexcitability was dose-dependently attenuated by i.t. SC-236 (COX-2 inhibitor), but not SC-560 (COX-1 inhibitor), given 3 days after SNL. Treatment with i.t. COX-inhibitors, immediately after SNL, also prevented the development of allodynia with no apparent effect on NFκ-B activation. These results indicate that: 1) the SNL-induced expression of spinal COX-2 is modulated by NFκ-B; 2) the hyperexcitability/allodynia observed 3 days after SNL is spinal PG-dependent; and 3) spinal COX-2 is primarily responsible for PG synthesis at this time point. While NFκ-B activation is a critical intermediate step, its influence on SNL-induced allodynia reflects the consequent actions of spinal PGs. L5/L6 spinal nerve ligation (SNL) induces early, spinal prostaglandin (PG)-dependent allodynia (<10 days after injury), including the increased expression of COX-2 in the ipsilateral lumbar dorsal horn 3 days after SNL. This study investigated the spinal expression of nuclear factor κ-B (NFκ-B; a modulator of COX-2 transcription), its relationship to the induction of COX-2, and the respective contributions of NFκ-B and COX-2 in the development of SNL-induced allodynia. Male, Sprague-Dawley rats (100-140g), fitted with intrathecal (i.t.) catheters, underwent SNL- or sham-surgery. Allodynia was defined as a paw withdrawal threshold (PWT) of ≤4g (baseline ≥15g). The biceps femoris flexor reflex was measured electromyographically, and lumbar segments were analyzed for NFκ-B and COX-2 protein expression. Allodynia and A- and C-fibre hyperexcitability (i.e. decreased activation threshold, increased evoked activity, including windup; p<0.05) were evident 3 days after SNL- but not sham-surgery. NFκ-B expression was increased in the ipsilateral, but not contralateral, dorsal horn of allodynic animals. This was apparent 12h after SNL and remained elevated at 3 days (p<0.05). Sham-surgery had no effect on NFκ-B (p>0.05). The NFκ-B inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 100μg i.t.), given immediately after SNL, decreased A- and C-fibre hyperexcitability 3 days later (p<0.05), and prevented the development of allodynia. Hyperexcitability was dose-dependently attenuated by i.t. SC-236 (COX-2 inhibitor), but not SC-560 (COX-1 inhibitor), given 3 days after SNL. Treatment with i.t. COX-inhibitors, immediately after SNL, also prevented the development of allodynia with no apparent effect on NFκ-B activation. These results indicate that: 1) the SNL-induced expression of spinal COX-2 is modulated by NFκ-B; 2) the hyperexcitability/allodynia observed 3 days after SNL is spinal PG-dependent; and 3) spinal COX-2 is primarily responsible for PG synthesis at this time point. While NFκ-B activation is a critical intermediate step, its influence on SNL-induced allodynia reflects the consequent actions of spinal PGs.

Extracellular Signal-Regulated Kinase-Regulated Microglia–Neuron Signaling by Prostaglandin E2Contributes to Pain after Spinal Cord Injury

Many patients with traumatic spinal cord injury (SCI) report pain that persists indefinitely and is resistant to available therapeutic approaches. We recently showed that microglia become activated after experimental SCI and dynamically maintain hyperresponsiveness of spinal cord nociceptive neurons and pain-related behaviors. Mechanisms of signaling between microglia and neurons that help to maintain abnormal pain processing are unknown. In this study, adult male Sprague Dawley rats underwent T9 spinal cord contusion injury. Four weeks after injury when lumbar dorsal horn multireceptive neurons became hyperresponsive and when behavioral nociceptive thresholds to mechanical and thermal stimuli were decreased, we tested the hypothesis that prostaglandin E2 (PGE2) contributes to signaling between microglia and neurons. Immunohistochemical data showed specific localization of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), an upstream regulator of PGE2 release, to microglial cells and a neuronal localization of the PGE2 receptor E-prostanoid 2 (EP2). Enzyme immunoassay analysis showed that PGE2 release was dependent on microglial activation and ERK1/2 phosphorylation. Pharmacological antagonism of PGE2 release was achieved with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and the microglial inhibitor minocycline. Cyclooxygenase-2 expression in microglia was similarly reduced by MEK1/2 inhibition. PD98059 and EP2 receptor blockade with AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) resulted in a decrease in hyperresponsiveness of dorsal horn neurons and partial restoration of behavioral nociceptive thresholds. Selective targeting of dorsal horn microglia with the Mac-1-SAP immunotoxin, a chemical conjugate of mouse monoclonal antibody to CD11b and the ribosome-inactivating protein saporin, resulted in reduced microglia staining, reduction in PGE2 levels, and reversed pain-related behaviors [corrected]. On the basis of these observations, we propose a PGE2-dependent, ERK1/2-regulated microglia-neuron signaling pathway that mediates the microglial component of pain maintenance after injury to the spinal cord.

Specific down-regulation of spinal μ-opioid receptor and reduced analgesic effects of morphine in mice with postherpetic pain

The analgesic effects of opioid agonists and the expression of mu- and kappa-opioid receptors were compared between mice with herpetic pain and those with postherpetic pain induced by herpetic virus inoculation. Morphine inhibited herpetic pain more effectively than postherpetic pain. Intrathecal injection reduced the analgesic effects of morphine on postherpetic pain, but intracerebroventricular injection did not. The kappa-opioid receptor agonist nalfurafine suppressed herpetic and postherpetic pain to similar degrees. mu-Opioid receptor-like immunoreactivities in the lumbar dorsal horn were markedly decreased at the postherpetic, but not herpetic, stage of pain. In the dorsal root ganglia, the expression of mu-opioid receptor mRNA was significantly decreased in mice with postherpetic pain, whereas the kappa-opioid receptor mRNA level was not altered. These results suggest that specific down-regulation of the mu-opioid receptor in the primary sensory neurons is responsible for the reduced analgesic action of morphine on postherpetic pain. The kappa-opioid receptor may be a useful target for the analgesic treatment of postherpetic neuralgia.

Cyclo-oxygenase-2-immunoreactive neurons in the lumbar dorsal horn in a chicken acute inflammation model

Acute and chronic peripheral inflammation is known to induce the expression of cyclo-oxygenase (COX)-2 in spinal cord neurons and increase the synthesis and release of prostaglandins (PG). Although these PG are presumed to cause inflammatory pain or hyperalgesia, the relationship between PG-producing cells in the dorsal horn and substance P (SP)-containing, pain-transmittimg nerve fibers remains unknown. In the present study we investigated immunohistochemically changes in the number of COX-2-containing neurons using the avidin-biotinylated peroxidase complex method in dorsal horn superficial laminae in chicken lumbosacral enlargement (L4, L5) under inflammatory conditions induced by unilateral intraplantar injection of complete Freund's adjuvant. After 12-24 h, a significant increase in the number of small COX-2-containing neurons was observed in lamina II on the injected side compared with the contralateral side. Furthermore, using fluorescent double-labeling for COX-2 and SP, an increase in the number of small COX-2-containing neurons in contact with SP-containing elements was observed ipsilaterally (1.4-1.6-fold compared with the contralateral side) in lamina II. Fluorescence triple-labeling of COX-2, SP and calcitonin gene-related peptide (CGRP) confirmed that the majority of these SP-containing elements coexisted with CGRP, indicating that these elements originated from primary afferent neurons. Using electron microscopy, two types of SP-containing axon terminals were found to form synapses with COX-2-containing neurons in lamina II. These results indicate that the number of COX-2-containing neurons increases concomitantly with an increase in the number of contacts of these neurons with SP-containing primary afferent fibers and suggest that this phenomenon is associated with PG production and the persistence of inflammatory pain.

Increased formalin-induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infant rats

When pregnant dams are stressed, there is a resultant alteration in brain development and behavior in their offspring. Prior work has shown increased nociceptive responses in adolescent or adult rats born of stressed dams. However, the age at which those changes first occur is not known. The aim of the present study was to evaluate the effect of prenatal stress on pain sensitivity in the formalin test in 7-day-old rats, behaviorally and by fos-like immunoreactivity (Fos-LI) in the lumbar spinal cord dorsal horn. The behavioral response to intraplantar injection of formalin is represented by two nociceptive phases separated by an interphase during which nociceptive responses decrease; the interphase is not seen until the start of the third postnatal week and appears as descending inhibitory monoaminergic systems develop. Prenatally stressed infants showed increased nociceptive responses in the second, tonic phase and a large increase in the number of formalin-induced Fos-LI neurons in the lumbar dorsal horn, a result consistent with the behavioral data. The increased nociception in prenatally stressed 7-day-old pups may be associated with the decrease in the intensity of serotonin-like immunoreactivity and density of serotonergic cells in the lumbar spinal cord dorsal horn and the dorsal raphe nucleus reported earlier.

Upregulation of persistent and ramp sodium current in dorsal horn neurons after spinal cord injury

Traumatic spinal cord injury (SCI) results not only in motor impairment, but also in chronic central neuropathic pain, which often is refractory to conventional treatment approaches. Upregulated expression of sodium channel Nav1.3 has been observed within the spinal dorsal horn neurons after SCI, and appears to contribute to neuronal hyperresponsiveness and pain-related behaviors. In this study we characterized the changes in sodium current properties within dorsal horn neurons after contusive SCI. Four weeks after adult male Sprague-Dawley rats underwent T9 spinal cord contusion injury, when behavioral nociceptive thresholds were decreased to both mechanical and thermal stimuli, whole-cell patch-clamp recordings were performed on acutely dissociated lumbar dorsal horn neurons. The cells demonstrated characteristic fast-activating and fast-inactivating sodium currents. SCI led to a shift of the steady-state activation and inactivation of the sodium current towards more depolarized potentials. The shifted steady-state inactivation shows similarities to that obtained from axotomized dorsal root ganglions, which were shown to upregulate Nav1.3. Small slow depolarizations below action potential threshold produced ramp currents, which were markedly enhanced by SCI (from 182 +/- 41 to 338 +/- 55 pA). The density of the noninactivating persistent sodium current was also significantly enhanced in neurons from SCI animals (from 17.4 +/- 3.2 to 27.7 +/- 4.4 pA/pF at 50-70 ms of depolarization). The increased persistent sodium current and ramp current, which are consistent with upregulation of Nav1.3 within dorsal horn neurons, suggest a basis for the hyperresponsiveness of these neurons following SCI.

Functional GABAA-Receptor–Mediated Inhibition in the Neonatal Dorsal Horn

Neonatal nociceptive circuits and dorsal horn cells are characterized by an apparent lack of inhibitory control: receptive fields are large and thresholds low in the first weeks of life. It has been suggested that this may reflect immature GABA(A)-receptor (GABA(A)R) signaling whereby an early developmental shift in transmembrane anion gradient is followed by a longer period of low Cl- extrusion capacity. To investigate whether functional GABA(A)R-mediated inhibition does indeed undergo postnatal regulation at the level of dorsal horn circuits, we applied the selective GABA(A)R antagonist gabazine to the spinal cord in anesthetized rat pups [postnatal day (P) 3 or 21] while recording spike activity in single lumbar dorsal horn cells in vivo. At both ages, blockade of GABA(A)R activity resulted in enlarged hind paw receptive field areas and increased activity evoked by low- and high-intensity cutaneous stimulation, revealing comparable inhibition of dorsal horn cell firing by spinal GABA(A)Rs at P3 and P21. This inhibition did not require descending pathways to the spinal cord because perforated patch-clamp recordings of deep dorsal horn neurons in P3 spinal cord slices also showed an increase in evoked spike activity after application of gabazine. We conclude that spinal GABAergic inhibitory transmission onto single dorsal horn cells "in vivo" is functional at P3 and that low Cl- extrusion capacity does not restrict GABAergic function over the normal range of evoked sensory activity. The excitability of neonatal spinal sensory circuits could reflect immaturity in other intrinsic or descending inhibitory networks rather than weak spinal GABAergic inhibition.

Activated Microglia Contribute to the Maintenance of Chronic Pain after Spinal Cord Injury

Traumatic spinal cord injury (SCI) results not only in motor impairment but also in chronic central pain, which can be refractory to conventional treatment approaches. It has been shown recently that in models of peripheral nerve injury, spinal cord microglia can become activated and contribute to development of pain. Considering their role in pain after peripheral injury, and because microglia are known to become activated after SCI, we tested the hypothesis that activated microglia contribute to chronic pain after SCI. In this study, adult male Sprague Dawley rats underwent T9 spinal cord contusion injury. Four weeks after injury, when lumbar dorsal horn multireceptive neurons became hyperresponsive and when behavioral nociceptive thresholds were decreased to both mechanical and thermal stimuli, intrathecal infusions of the microglial inhibitor minocycline were initiated. Electrophysiological experiments showed that minocycline rapidly attenuated hyperresponsiveness of lumbar dorsal horn neurons. Behavioral data showed that minocycline restored nociceptive thresholds, at which time spinal microglial cells assumed a quiescent morphological phenotype. Levels of phosphorylated-p38 were decreased in SCI animals receiving minocycline. Cessation of delivery of minocycline resulted in an immediate return of pain-related phenomena. These results suggest an important role for activated microglia in the maintenance of chronic central below-level pain after SCI and support the newly emerging role of non-neuronal immune cells as a contributing factor in post-SCI pain.

Licking and C-Fos Expression in the Dorsal Horn of the Spinal Cord After the Formalin Test

We investigated whether c-fos expression in the dorsal horn is affected by licking in the formalin test. Thirty adult Sprague-Dawley rats were divided into 5 groups of 6 rats each: a free condition control (Free Cont) group, formalin test under free condition (Free F-test) group, scrub stimulation under free condition (Free Scrub) group, restrained condition control (Restricted Cont) group, and formalin test under restrained condition (Restricted F-test) group. Animals in the three free condition groups and two restricted groups were put in a clear plastic chamber and a restraining chamber, respectively. Ten percent formalin was injected into the left rear paw in the Free and Restricted F-test groups. Animals in the Free Scrub group were scrubbed on the left rear paw with a wet cotton swab. The Free Cont, Restricted Cont, and Free Scrub groups showed little c-fos expression. The number of c-fos positive cells in the ipsilateral surface dorsal horn of the Restricted F-test group was significantly less than that of the Free F-test group (P < 0.05). The results indicated that the licking action increased c-fos expression of the lumbar dorsal horn in the formalin test.

Safety Evaluation of Intrathecal Substance P-Saporin, a Targeted Neurotoxin, in Dogs

Intrathecal (IT) substance P-Saporin (SP-SAP), a 33-kDa-targeted neurotoxin, produces selective destruction of superficial neurokinin 1 receptor (NK1r)-bearing cells in the spinal dorsal horn. In rats, SP-SAP prevents the formation of hyperalgesia and can reverse established neuropathic pain behavior in rodents. To determine the safety of this therapeutic modality in a large animal model, beagles received bolus IT lumbar injections of vehicle, SP-SAP (1.5, 15, 45, or 150 microg), or a nontargeted preparation of saporin (SAP, 150 microg) for immunohistological analysis of spinal cords. Doses of 15 microg SP-SAP and above produced a significant and equivalent loss of NK1r-bearing cells and dendrites in lumbar laminae II and I compared to vehicle- or SAP-treated animals. Cervical regions in all animals displayed no loss of NK1r immunoreactivity as compared to controls. Total numbers of neurons in the lumbar dorsal horn or alpha-motor neurons in the ventral horn demonstrated no significant changes. No increases in the astrocytic marker glial fibrillary acidic protein were noted following treatment with SP-SAP, suggesting a lack of generalized neurotoxicity. Additional dogs received doses of 1.5-150 microg SP-SAP or SAP and were sacrificed after 28 or 90 days to assess behavioral and physiological parameters. Although some acute motor signs were observed with both SP-SAP and SAP, no long-lasting significant events were noted in any of these animals. These data indicate no adverse toxicity at doses up to 10 times those necessary for producing loss of superficial NK1r-bearing neurons in a large animal model.

Increased Expression of Cyclooxygenase and Nitric Oxide Isoforms, and Exaggerated Sensitivity to Prostaglandin E2, in the Rat Lumbar Spinal Cord 3 Days after L5–L6 Spinal Nerve Ligation

Spinal prostaglandins seem to be important in the early pathogenesis of experimental neuropathic pain. Here, the authors investigated changes in the expression of cyclooxygenase and nitric oxide synthase (NOS) isoforms in the lumbar, thoracic, and cervical spinal cord and the pharmacologic sensitivity to spinal prostaglandin E2 (PGE2) after L5-L6 spinal nerve ligation (SNL). Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery 3 days before experimentation. Paw withdrawal threshold was monitored for up to 20 days. Immunoblotting, spinal glutamate release, and behavioral testing were examined 3 days after SNL. Allodynia (paw withdrawal threshold < or = 4 g) was evident 1 day after SNL and remained stable for 20 days. Paw withdrawal threshold was unchanged (P > 0.05) from baseline (> 15 g) after sham surgery except for a small but significant decrease on day 20. Cyclooxygenase 2, neuronal NOS, and inducible NOS were significantly increased in the ipsilateral lumbar dorsal horn after SNL. Expression in the contralateral dorsal horn and ventral horns (lumbar segments) or bilaterally (thoracic and cervical segments) was unchanged from sham controls. This was accompanied by a significant decrease in both the EC50 of PGE2-evoked glutamate release and the ED50 of PGE2 on brush-evoked allodynia. Enhanced sensitivity to PGE2 was localized to lumbar segments of SNL animals and attenuated by SC-51322 or S(+)-ibuprofen, but not R(-)-ibuprofen (100 mum). The increased expression of cyclooxygense-2, neuronal NOS, and inducible NOS and the enhanced sensitivity to PGE2 in spinal segments affected by SNL support the hypothesis that spinal prostanoids play an early pathogenic role in experimental neuropathic pain.

IS PROTEIN KINASE C (PKC) INVOLVED IN NOCICEPTION?

The study was designed to determine whether the protein kinase C (PKC) is involved in nociceptive c-Fos expression and the concomitant signaling processes of endogenous opioid-like substances (OLS) that modulate c-Fos expression in the spinal dorsal horn following formalin injection into the unilateral hindpaw in rats by using immunocytochemical techniques. In the first part of experiments in which rats were pretreated with intrathecal (i.t.) chelerythrine (Chel), an inhibitor of PKC, the nociceptive c-Fos-like immunoreactive (Fos-LI) neurons in the lumbar dorsal horn ipsilateral to the formalin injection were significantly suppressed with a reduction rate of 60.3% (p < .001) as compared to that in the control group with i.t. saline. In the second part of experiments in which rats were pretreated with i.t. naloxone (Nal), the nociceptive Fos-LI neurons were significantly increased by 53.2% (p < .01) as compared to that in the control group; however, when rats were pretreated with combined i.t. Nal + Chel, the nociceptive Fos-LI neurons exhibited a percentage reduction similar to that in group with i.t. Chel alone, although the real number of Fos-LI neurons in group with i.t. Nal + Chel still significantly surpassed that in group with i.t. Chen only. These results suggest that: (1) PKC may play an important role in the induction of nociceptive c-Fos expression; (2) nociceptive c-Fos expression is subject to the modulation of endogenous OLS that suppress the nociceptive responses of the dorsal horn neurons; and (3) PKC may not be involved in the signaling processes by which the endogenous OLS modulate the nociceptive c-Fos expression in the spinal level.

Spinal and Peripheral GABA-A and B Receptor Agonists for the Alleviation of Mechanical Hypersensitivity following Compressive Nerve Injury in the Rat

Background: This study was conducted to investigate the roles of the spinal and peripheral γ-aminobutyric acid (GABA)ergic systems for the mechanical hypersensitivity produced by chronic compression of the dorsal root ganglion (CCD). Methods: CCD was performed at the left 5th lumbar dorsal root ganglion. The paw withdrawal threshold (PWT) to von Frey stimuli was measured. The mechanical responsiveness of the lumbar dorsal horn neurons was examined. GABAergic drugs were delivered with intrathecal (i.t.) or intraplantar (i.pl.) injection or by topical application onto the spinal cord. Results: CCD produced mechanical hypersensitivity, which was evidenced by the decrease of the PWT, and it lasting for 10 weeks. For the rats showing mechanical hypersensitivity, the mechanical responsiveness of the lumbar dorsal horn neurons was enhanced. A similar increase was observed with the normal lumbar dorsal horn neurons when the GABA-A receptor antagonist bicuculline was topically applied. An i.t. injection of GABA-A or GABA-B receptor agonist, muscimol or baclofen, alleviated the CCD-induced hypersensitivity. Topical application of same drugs attenuated the CCD-induced enhanced mechanical responsiveness of the lumbar dorsal horn neurons. CCD-induced hypersensitivity was also improved by low-dose muscimol applied (i.pl.) into the affected hind paw, whereas no effects could be observed with high-dose muscimol or baclofen. Conclusions: The results suggest that the neuropathic pain associated with compression of the dorsal root ganglion is caused by hyperexcitability of the dorsal horn neurons due to a loss of spinal GABAergic inhibition. Peripheral application of low-dose GABA-A receptor agonist can be useful to treat this pain. (Korean J Pain 2006; 19: 22-32)

Persistent monoarthritis of the temporomandibular joint region enhances nocifensive behavior and lumbar spinal Fos expression after noxious stimulation to the hindpaw in rats

Effects of persistent temporomandibular joint (TMJ) inflammation on nociceptive responses of remote bodily areas of the rat were investigated. Monoarthritis of the TMJ region was evoked by the injection of complete Freund's adjuvant (CFA) into the left TMJ region. Rats without injection of CFA into the TMJ region served as controls (non-CFA group). Time spent on licking behavior evoked by the injection of formalin into the left hindpaw and withdrawal thresholds of mechanical stimulation to both sides of the hindpaw were measured during TMJ inflammation for 3 weeks. Furthermore, expression of Fos protein in the lumbar dorsal horn was immunohistochemically investigated following the injection of formalin into the hindpaw during TMJ inflammation. Formalin-evoked nocifensive behavioral activities were significantly enhanced at 10 and 14 days after CFA injection in the late phase, while the withdrawal threshold to mechanical stimulation was significantly decreased bilaterally at 8, 10 and 14 days after CFA injection. Both formalin-evoked licking behavior and mechanical withdrawal thresholds to bilateral hindpaw at 21 days after CFA injection were similar to those in the non-CFA group. The number of Fos-positive neurons in the lumbar dorsal horn ipsilateral to the formalin injection at 1 and 7 days after CFA injection into the TMJ were similar to those in the non-CFA group; however, those were significantly increased in the laminae I-II and V-VI of the lumbar dorsal horn at 14 days after CFA injection. TMJ inflammation for 7 and 14 days alone produced a small number of Fos-expressing neurons in the lumbar dorsal horn. These results provide evidence that persistent unilateral inflammation of the TMJ region causes an increase in behavioral hyperalgesia of the hindpaw, which is attributed to the modulation of neural activities, in part, in the lumbar dorsal horn, likely mediated by supraspinal neural mechanisms.

The possible role of the NO-cGMP pathway in nociception: Different spinal and supraspinal action of enzyme blockers on rat dorsal horn neurones

In the literature, the pro- or antinociceptive effects of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) are discussed controversially. Our laboratory and others have reported that in the spinal cord a local lack of NO has an excitatory action on the ongoing (background) activity of dorsal horn neurones. Here, we tested the hypothesis that this effect of NO is mediated by cGMP and that part of the controversy is due to differences in the spinal and supraspinal actions of both compounds. In anaesthetised rats, impulse activity of lumbar dorsal horn neurones was recorded, and blockers of NO- and cGMP-synthesis, as well as the phosphodiesterase 5 (PDE5) inhibitor sildenafil (which increases the cGMP level), or 8-Bromo-cGMP (a membrane permeable cGMP analogue) were administered spinally or supraspinally. Topical superfusion of the spinal cord with a blocker of the guanylyl cyclase (ODQ) to reduce the cGMP level led to an increase in background activity of nociceptive lumbar dorsal horn neurones similar to that caused by l-NAME, a blocker of the NO synthase. Spinal superfusion with sildenafil or 8-Bromo-cGMP had no excitatory effect. In contrast, injections of sildenafil or 8-Bromo-cGMP into the third cerebral ventricle caused an increased background activity in lumbar dorsal horn neurones, while l-NAME and ODQ were ineffective. The results show that at the spinal level, a lack of cGMP and NO has an excitatory action on dorsal horn neurones, whereas supraspinally an elevated level of cGMP is excitatory.

Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury

Spinal cord contusion injury (SCI) is known to induce pain-related behaviour, as well as hyperresponsiveness in lumbar dorsal horn nociceptive neurons associated with the aberrant expression of Na(v)1.3, a rapidly repriming voltage-gated sodium channel. Many of these second-order dorsal horn neurons project to third-order neurons in the ventrobasal complex of the thalamus. In this study we hypothesized that, following SCI, neurons in the thalamus undergo electrophysiological changes linked to aberrant expression of Na(v)1.3. Adult male Sprague-Dawley rats underwent contusion SCI at the T9 thoracic level. Four weeks post-SCI, Na(v)1.3 protein was upregulated within thalamic neurons in ventroposterior lateral (VPL) and ventroposterior medial nuclei, where extracellular unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, and expansion of peripheral receptive fields. Altered electrophysiological properties of VPL neurons persisted after interruption of ascending spinal barrage by spinal cord transection above the level of the injury. Lumbar intrathecal administration of specific antisense oligodeoxynucleotides generated against Na(v)1.3 caused a significant reduction in Na(v)1.3 expression in thalamic neurons and reversed electrophysiological alterations. These results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after SCI.

Differential effects of halothane and isoflurane on lumbar dorsal horn neuronal windup and excitability

Windup of spinal nociceptive neurones may underlie temporal summation of pain, influencing the minimum alveolar concentration (MAC) of anaesthetics required to prevent movement to supramaximal stimuli. We hypothesized that halothane and isoflurane would differentially affect windup of dorsal horn neurones. We recorded 18 nociceptive dorsal horn neurones exhibiting windup to 1 Hz electrical hindpaw stimuli in rats. Effects of 0.8 and 1.2 MAC isoflurane and halothane were recorded in the same neurones (counterbalanced, crossover design). Windup was calculated as the total number of C-fibre (100-400 ms latency) plus afterdischarge (400-1000 ms latency) spikes/20 stimuli (area under curve, AUC) or absolute windup (C-fibre plus afterdischarge-20 x initial response). Increasing isoflurane from 0.8 to 1.2 MAC did not affect AUC, but increased absolute windup from 429 (62) to 618 (84) impulses/20 stimuli (P<0.05) and depressed the initial C-fibre response from 14 (3) to 8 (2) impulses (P<0.05). Increasing halothane from 0.8 to 1.2 MAC depressed AUC from 690 (79) to 537 (65) impulses/20 stimuli (P<0.05) and the initial response from 18 (2) to 13 (2) impulses (P<0.05), but absolute windup was not affected. Absolute windup was 117% greater during 1.2 MAC isoflurane compared with 1.2 MAC halothane. Windup was significantly greater under isoflurane than halothane anaesthesia at 1.2 MAC, whereas the initial C-fibre response was suppressed more by isoflurane. These findings suggest that these two anaesthetics have mechanistically distinct effects on neuronal windup and excitability.

The central termination of sensory fibers from nerves to the gastrocnemius muscle of the rat

Peripheral nerves innervating muscles have sensory fibers that relay information into the CNS information about proprioception, pain, and the metabolic state of the muscle. The present study shows the primary afferent projections into the spinal cord of the nerves innervating the gastrocnemius muscle of the rat using the transganglionic transport of a cocktail of horseradish peroxidase (HRP) conjugated to cholera toxin and wheat germ agglutinin; these markers have been shown to label large and small fibers, respectively. A dense projection into lamina I of the lumbar dorsal horn and a more moderate projection into lamina V were seen. Moreover, dense reaction product was found in the most medial aspect of lamina II, especially lamina II inner part, and less in lamina III and IV of levels L3–L5. Lamina VI had dense reaction product from the rostral sacral levels of the spinal cord that continued into Clarke’s column at rostral lumbar levels. The nucleus gracilis also was labeled. Other nerves emerging from the popliteal fossa, including the tibial, peroneal, and sural nerves, also were injected with the HRP cocktail and their projections compared with those from the gastrocnemius muscle. Projections from the gastrocnemius muscle only partially overlapped with those from the tibial nerve, from which the nerves to the gastrocnemius muscle branch. However, the topology of projections from these nerves to laminae II-IV of the dorsal horn differed from that of the nerves of the gastrocnemius muscle, suggesting there was little spread to other nerves in the popliteal fossa. It was also noted that large labeled processes, presumably dendrites of retrogradely labeled motoneurons, entered the dorsal horn. These data provide information on the central projections of both the large and small fibers innervating the gastrocnemius muscle, and may aid in determining the circuitry utilized in the exercise pressor reflex as well as muscle pain.