Lumbar Dorsal Horn Neurons Research Articles

Astrocyte D-serine modulates the activation of neuronal NOS leading to the development of mechanical allodynia in peripheral neuropathy.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0–3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Lamina-specific population encoding of cutaneous signals in the spinal dorsal horn using multi-electrode arrays.

Key points Traditional, widely used in vivo electrophysiological techniques for the investigation of spinal processing of somatosensory information fail to account for the diverse functions of each lamina.To overcome this oversimplification, we have used multi‐electrode arrays, in vivo, to simultaneously record neuronal activity across all laminae of the spinal dorsal horn.Multi‐electrode arrays are sensitive enough to detect lamina‐ and region‐specific encoding of different subtypes of afferent fibres and to detect short‐lived changes in synaptic plasticity as measured by the application of cutaneous electrical stimulation of varying intensity and frequency.Differential encoding of innocuous and noxious thermal and mechanical stimuli were also detected across the laminae with the technique, as were the effects of the application of capsaicin.This new approach to the study of the dorsal spinal cord produces significantly more information per experiment, permitting accelerated research whilst also permitting the effects of pharmacological tools to modulate network responses. The dorsal horn (DH) of the spinal cord is a complex laminar structure integrating peripheral signals into the central nervous system. Spinal somatosensory processing is commonly measured electrophysiologically in vivo by recording the activity of individual wide‐dynamic‐range neurons in the deep DH and extrapolating their behaviour to all cells in every lamina. This fails to account for the specialized processes that occur in each lamina and the considerable heterogeneity in cellular phenotype within and between laminae. Here we overcome this oversimplification by employing linear multi‐electrode arrays (MEAs) in the DH of anaesthetized rats to simultaneously measure activity across all laminae. The MEAs, comprising 16 channels, were inserted into the lumbar dorsal horn and peripheral neurons activated electrically via transcutaneous electrodes and ethologically with von Frey hairs (vFHs) or an aluminium heating block. Ascending electrical stimuli showed fibre thresholds with distinct dorsoventral innervation profiles. Wind up was observed across the DH during the C‐fibre and post‐discharge latencies following 0.5 Hz stimulation. Intrathecal application of morphine (5 ng/50 μl) significantly reduced Aδ‐ and C‐fibre‐evoked activity in deep and superficial DH. Light vFHs (≤10 g) predominantly activated intermediate and deep laminae whereas noxious vFHs (26 g) also activated the superficial laminae. Noxious heat (55°C) induced significantly greater activity in the superficial and deep laminae than the innocuous control (30°C). The application of these arrays produced the first description of the processing of innocuous and noxious stimuli throughout the intact DH.

HYP-17, a novel voltage-gated sodium channel blocker, relieves inflammatory and neuropathic pain in rats

Clinical and experimental studies suggest that voltage-gated sodium channels (VGSCs) play a key role in the pathogenesis of neuropathic pain and that blocking agents against these channels can be potentially therapeutic. In the current study, we investigated whether a novel compound, (-)-2-Amino-1-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-propan-1-one(HYP-17), binds to VGSCs and evaluated its inhibitory effect on Na+ currents of the rat dorsal root ganglia (DRG) sensory neurons and its analgesic effect on inflammatory and neuropathic pain. HYP-17 (10μM) reduced both the tetrodotoxin-sensitive (TTX-S) and the TTX-resistant (TTX-R) currents in DRG sensory neurons. However, neither the voltage-dependent activation curves nor the steady-state inactivation curves for TTX-S and TTX-R currents were changed by HYP-17. In rats injected with 5% formalin under the plantar surface of the hind paw, HYP-17 (10μg) significantly reduced both the early and late phase spontaneous pain behaviors. Systemic injection with HYP-17 (60mg/kg, i.p.) also significantly relieved the mechanical, cold, and warm allodynia induced by rat tail nerve injury. Furthermore, HYP-17 (60mg/kg, i.p.) significantly relieved the central neuropathic pain induced by spinal cord injury (SCI), and inhibited c-Fos expression in lumbar (L) 4–L5 spinal segments. Electrophysiological study showed that HYP-17 significantly attenuated the hyper-responsiveness of lumbar dorsal horn neurons. In addition, HYP-17 significantly reduced the levels of pp38MAPK and p-JNK in microglia and astrocytes, respectively, in the L4-L5 spinal dorsal horn. Therefore, our results indicate that HYP-17 has potential analgesic activities against nociceptive, inflammatory and neuropathic pain.

Widespread pain sensitization after partial infraorbital nerve transection in MRL/MPJ mice.

Clinical studies show that chronic pain can spread to adjacent or even distant body regions in some patients. However, little is known about how this happens. In this study, we found that partial infraorbital nerve transection (p-IONX) in MRL/MPJ mice induced not only marked and long-lasting orofacial thermal hyperalgesia but also thermal hyperalgesia from day 3 postoperatively (PO) and tactile allodynia from day 7 PO in bilateral hind paws. Pain sensitization in the hind paw was negatively correlated with facial thermal hyperalgesia at early but not late stage after p-IONX. After a rapid activation of c-Fos, excitability and excitatory synaptic neurotransmission in lumbar dorsal horn neurons were elevated from day 3 and day 7 PO, respectively. In addition, microglial activation after p-IONX transmitted caudally from the Vc in the medulla to lumber dorsal horn in a time-dependent manner. Inhibition of microglial activation by minocycline at early but not late stage after p-IONX postponed and attenuated pain sensitization in the hind paw. These results indicate that neuropathic pain after p-IONX in MRL/MPJ mice spreads from the orofacial region to distant somatic regions and that a rostral-caudal transmission of central sensitization in the spinal cord is involved in the spreading process of pain hypersensitivity.

Intradermal endothelin-1 excites bombesin-responsive superficial dorsal horn neurons in the mouse.

Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 μg/μl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 μg·ml(-1)·min(-1)), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans.

Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlative responses of lumbar dorsal horn neurons in the mouse

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2h) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently described “calf” model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300mN, 2Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4–30mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

The modality‐specific contribution of peptidergic and non‐peptidergic nociceptors is manifest at the level of dorsal horn nociresponsive neurons

We previously demonstrated that genetic and/or pharmacological ablation of the TRPV1+/peptidergic or the MrgprD+/non-peptidergic subset of nociceptors produced selective, modality-specific deficits in the behavioural responses to heat and mechanical stimuli, respectively. To assess whether this modality-specific contribution is also manifest at the level of spinal cord neuron responsiveness, here we made extracellular recordings from lumbar dorsal horn neurons of the mouse in response to graded thermal and mechanical stimulation. We found that, following intrathecal injection of capsaicin to eliminate the central terminals of TRPV1+ nociceptors, neurons in the region of laminae I and V of the spinal cord lost responsiveness to noxious heat (whether generated by a contact heat probe or diode laser), with no change in their response to noxious mechanical stimulation. In contrast, ablation of MrgprD+ afferents did not alter the response to noxious heat, but reduced the firing of superficial dorsal horn nociceptive-specific neurons in response to graded mechanical stimulation and decreased the relative number of wide dynamic range neurons that were exclusively mechanosensitive. Neither ablation procedure reduced the number of dorsal horn neurons that responded to noxious cold. These findings support the conclusion that TRPV1+ nociceptors are necessary and probably sufficient for noxious heat activation of dorsal horn neurons and that, despite their polymodal properties, TRPV1+ and MrgprD+ nociceptors provide modality-specific contributions to the response properties of spinal cord neurons.

Topical hindpaw application of L-menthol decreases responsiveness to heat with biphasic effects on cold sensitivity of rat lumbar dorsal horn neurons

Menthol is used in pharmaceutical applications because of its desired cooling and analgesic properties. The neural mechanism by which topical application of menthol decreases heat pain is not fully understood. We investigated the effects of topical menthol application on lumbar dorsal horn wide dynamic range and nociceptive-specific neuronal responses to noxious heat and cooling of glabrous hindpaw cutaneous receptive fields. Menthol increased thresholds for responses to noxious heat in a concentration-dependent manner. Menthol had a biphasic effect on cold-evoked responses, reducing the threshold (to warmer temperatures) at a low (1%) concentration and increasing threshold and reducing response magnitude at high (10%, 40%) concentrations. Menthol had little effect on responses to innocuous or noxious mechanical stimuli, ruling out a local anesthetic action. Application of 40% menthol to the contralateral hindpaw tended to reduce responses to cooling and noxious heat, suggesting a weak heterosegmental inhibitory effect. These results indicate that menthol has an analgesic effect on heat sensitivity of nociceptive dorsal horn neurons, as well as biphasic effects on cold sensitivity, consistent with previous behavioral observations.

Blockade effects of BIBN4096BS on CGRP-induced inhibition on whole-cell K+ currents in spinal dorsal horn neuron of rats

Calcitonin gene-related peptide (CGRP) plays an important role in the transmission and modulation of nociceptive information in the spinal cord. BIBN4096BS, a nonpeptide CGRP receptor antagonist, has been shown to be efficiency in clinical migraine treatment. The present study was performed to investigate the effects of BIBN4096BS on the CGRP-induced inhibition to whole-cell K+ currents in spinal wide dynamic range (WDR) neuron of rats. Application of BIBN4096BS inhibited the neuronal activity of WDR neurons in lumbar dorsal horn of the spinal cord in rats tested by extracellular recording method. Furthermore, CGRP induced inhibition on whole-cell K+ currents in cultured dorsal horn neurons of rats tested by whole-cell patch-clamp recording, and the effect was significantly blocked by BIBN4096BS. The results indicate that BIBN4096BS may produce antinociceptive effects at the spinal level in rats.

Neuropeptide Y receptor-expressing dorsal horn neurons: Role in nocifensive reflex responses to heat and formalin

Lumbar intrathecal injection of neuropeptide Y (NPY) is antinociceptive, particularly in models of nerve injury and inflammation. Intrathecal NPY does not alter nociception in mice null for the Y1 neuropeptide Y receptor (Y1R) and these mice show enhanced nocifensive reflex responses to aversive thermal, mechanical, visceral and chemical stimuli. Y1R and NPY receptor type 2 (Y2R) are present in the spinal dorsal horn presynaptically on primary afferent, and possibly interneuron terminals, but only Y1R is found postsynaptically on dorsal horn neurons. In the present study, we sought to assess the anatomic effects of lumbar intrathecal disulfide conjugate of neuropeptide Y and saporin (NPY-sap) and to determine the role of Y1R-expressing dorsal horn neurons in nocifensive responses to aversive thermal and chemical stimulation. Lumbar intrathecal injection of NPY-sap was used to selectively destroy Y1R-expressing lumbar dorsal horn neurons followed by testing nocifensive reflex responses on the hotplate and after hind-paw formalin injection. NPY-saporin decreased superficial dorsal horn staining for Y1R, but not neurokinin-1 receptor, mu opiate receptor or NPY peptide, and had no effect on Y1R cell counts in fourth lumbar spinal segment dorsal root ganglia. Loss of Y1R-expressing dorsal horn neurons was associated with increased first response latencies on the 44 °C hotplate and reduced total time rats spent licking and guarding hind paws during 600 s trials at 44 °C or 200 s trials at 47 °C. First hind-paw response latencies to high intensity phasic stimulation at 52 °C were unaffected. NPY-sap also reduced formalin-induced nocifensive behaviors during both interphase and phase II. These data demonstrate that selective destruction of Y1R-expressing superficial dorsal horn neurons, probably excitatory interneurons and/or projection neurons, reduces nocifensive reflex responses, particularly to activation of C nociceptors, and suggest a possible role for Y1R-expressing dorsal horn neurons in pain.

Expression of Adenosine A2A Receptors in the Rat Lumbar Spinal Cord and Implications in the Modulation of N-Methyl-d-Aspartate Receptor Currents

The presence of A(2A) receptors in the dorsal horn of the spinal cord remains controversial. At this level, activation of N-methyl-d-aspartate (NMDA) receptors induces wind-up, which is clinically expressed as hyperalgesia. Inhibition of NMDA receptor currents after activation of A(2A) receptors has been shown in rat neostriatal neurons. In this study, we sought to establish the presence of adenosine A(2A) receptors in the lamina II of the rat lumbar dorsal horn neurons and investigated whether the activation of A(2A) receptors is able to modulate NMDA receptor currents. Experiments were conducted in the rat lumbar spinal cord. The presence of adenosine A(2A) receptor transcripts inside the lumbar spinal cord is assessed with the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Western blot experiments are performed at the same level. The RT-PCR technique is also performed specifically in the lamina II, and the presence of adenosine A(2A) receptor transcripts is assessed in neurons from the lamina II with the single-cell RT-PCR technique. The effect of adenosine A(2A) receptor activation on NMDA receptor currents is studied by the whole-cell configuration of the patch clamp technique. RT-PCR performed on the lumbar spinal cord revealed the presence of adenosine A(2A) receptor transcripts. Western blot experiments revealed the presence of A(2A) receptors in the lumbar spinal cord. RT-PCR performed on the substantia gelatinosa also revealed the presence of adenosine A(2A) receptor transcripts. Finally, single cell RT-PCR revealed the presence of adenosine A(2A) receptor transcripts in a sample of lamina II neurons. Patch clamp recordings showed an inhibition of NMDA currents during the application of a selective A(2A) agonist. These results demonstrate the presence of A(2A) receptor on neurons from the substantia gelatinosa of the rat lumbar dorsal horn and the inhibition of NMDA-induced currents by the application of a selective A(2A) receptor agonist. Therefore, A(2A) receptor ligands could modulate pain processing at the spinal cord level.

Immobilizing Doses of Halothane, Isoflurane or Propofol, Do Not Preferentially Depress Noxious Heat-Evoked Responses of Rat Lumbar Dorsal Horn Neurons with Ascending Projections

The spinal cord is an important site where volatile anesthetics decrease sensation and produce immobility. Beyond this knowledge, our understanding of a site of anesthetic action is limited. Previous evidence suggests that dorsal horn neurons with ascending projections may be more susceptible to depression by general anesthetics than local spinal interneurons. In this study we evaluated the effects of volatile and injectable general anesthetics on lumbar dorsal horn neurons with and without ascending projections. Thirty-seven adult male rats underwent laminectomies at C1, for placement of a stimulating electrode, and T13/L1, for extracellular recording from the spinal cord dorsal horn. Neuronal responses to heat were evaluated under two doses of halothane, isoflurane, or propofol anesthesia. Under both halothane and isoflurane anesthesia, increasing the dose from 0.8 to 1.2 minimum alveolar concentration (MAC) had no significant effect on heat-evoked responses in neurons that had ascending projections identified via antidromic stimulation (AD) or those without ascending projections (nAD). Heat responses in AD neurons 1 min after i.v. administration of 3 and 5 mg/kg of propofol were reduced to 60% +/- 18% (mean +/- SE) and 39% +/- 14% of control respectively. Similarly, in nAD neurons responses were reduced to 56% +/- 14% and 50% +/- 10% of control by 3 and 5 mg/kg propofol respectively. Our findings suggest, at peri-MAC concentrations, these general anesthetics do not preferentially depress lumbar dorsal horn neurons with ascending projections compared to those with no identifiable ascending projections.

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.

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.

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.

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.

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)

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.

Temporal plasticity of dorsal horn somatosensory neurons after acute and chronic spinal cord hemisection in rat

Unilateral T13 hemisection of the rat spinal cord produces a model of chronic spinal cord injury (SCI) that is characterized by bilateral hyperexcitability of lumbar dorsal horn neurons, and behavioral signs of central pain. While we have demonstrated that responsiveness of multireceptive (MR) dorsal horn neurons is dramatically increased at 28 days after injury, the effects of acute hemisection are unknown and predicted to be different than observed chronically. In the present study, the consequences of T13 hemisection are examined acutely at 45 min in MR neurons both ipsilateral and contralateral to the site of injury, and compared to the same class of cells at 28 days after injury ( n=20 cells total per group: 2–3 cells/side of the cord from n=5 animals). Acutely, ipsilateral to the hemisection, both spontaneous and evoked activity of MR neurons were significantly increased, whereas contralaterally, only evoked activity was significantly increased. In animals 28 days after hemisection, spontaneous activity of MR neurons was comparable to intact levels ipsilaterally, and cells exhibited hyperexcitability to evoked stimuli bilaterally. Expansion of cutaneous receptive fields was observed only in hindpaws ipsilateral to the lesion, acutely. These results demonstrate dynamic plasticity in properties of dorsal horn somatosensory neurons after SCI.