Spinal Nociceptive Processing Research Articles

An Open-Source Tool for Investigation of Differential RNA Expression Between Spinal Cord Cells of Male and Female Mice.

Chronic pain is a highly debilitating condition that differs by type, prevalence, and severity between men and women. To uncover the molecular underpinnings of these differences, it is critical to analyze the transcriptomes of spinal cord pain-processing networks for both sexes. Despite several recently published single-nucleus RNA-sequencing (snRNA-seq) studies on the function and composition of the mouse spinal cord, a gene expression analysis investigating the differences between males and females has yet to be performed. Here, we combined data from three different large-scale snRNA-seq studies, which used sex-identified adult mice. Using SeqSeek, we classified more than 37,000 unique viable cells within predicted cell types with the use of machine learning. We then utilized DESeq2 to identify significant differentially expressed genes (DEGs) between males and females in a variety of cell populations, including superficial dorsal horn (SDH) neurons. We found a large number of DEGs between males and females in all cells, in neurons, and in SDH neurons of the mouse spinal cord, with a greater level of differential expression in inhibitory SDH neurons compared to excitatory SDH neurons. The results of these analyses are available on an open-source web-app: https://justinbellavance.shinyapps.io/snRNA_Visualization/. Lastly, we used gene set enrichment analysis to identify sex-enriched pathways from our previously identified DEGs. Through this, we have identified specific genetic players within the rodent spinal cord that diverge between males and females, which may underlie reported sex differences in spinal nociceptive mechanisms and pain processing.

Mode of delivery alters sensitivity to thermal and chemical stimuli in adult rats: An experimental study.

The mode of delivery might prompt a long-lasting alteration in physiological and behavioral responsiveness in offspring. This study was intended to evaluate if the mode of delivery could alter sensitivity to thermal and chemical stimuli in female rats. 56 adult female Wistar rats (200-220 gr) that were born by vaginal or cesarean section (C-section) were used (n = 28/each). Inflammatory pain was induced by subcutaneous injection of formalin into the hind paw. The thermal nociceptive threshold was determined by tail-flick and hot plate tests. Besides, the Western blot test was used to evaluate the spinal cord levels of c-Fos and c-Jun proteins. Formalin-induced inflammation was significantly decreased in C-section group as compared to vaginally born rats (p 0.001). The baseline nociceptive threshed and morphine-induced analgesia were significantly increased in C-section groups in comparison to vaginally born rats. In addition, the levels of c-Fos and c-Jun proteins were significantly decreased in the spinal cord of C-section rats as compared to vaginally born animals (p 0.01). Morphine treatment could decrease the expression of c-Fos and c-Jun in the C-section group (p 0.05). Overall, C-section rats showed lower spinal nociceptive processing and neuronal activity later in life, compared to the vaginal born rats.

AKAP150 and its Palmitoylation Contributed to Pain Hypersensitivity Via Facilitating Synaptic Incorporation of GluA1-Containing AMPA Receptor in Spinal Dorsal Horn.

The A-kinase anchoring protein 150 (AKAP150) organizes kinases and phosphatases to regulate AMPA receptors (AMPARs) that are pivotal for synaptic plasticity. AKAP150 itself undergoes S-palmitoylation. However, the roles of AKAP150 and its palmitoylation in spinal nociceptive processing remain unknown. In this study, we found that intraplantar injection of complete Freund's adjuvant (CFA) significantly increased the synaptic expression of AKAP150 and caused a reorganization of AKAP150 signaling complex in spinal dorsal horn. Knockdown of AKAP150 or interruption of its interactions with kinases effectively suppressed the CFA-induced synaptic expression of GluA1 subunit of AMPARs. Our data also showed that an upregulation of AKAP150 palmitoylation was involved in the synaptic redistribution of AKAP150. Inhibition of AKAP150 palmitoylation by expression of palmitoylation-defective mutant AKAP150 (C36, 123S) effectively repressed the CFA-induced phosphorylation and synaptic expression of GluA1 subunit, meanwhile, attenuated the development of mechanical allodynia and thermal hyperalgesia. Furthermore, we found that an increased expression of palmitoyl acyltransferase ZDHHC2 contributed to the upregulation of AKAP150 palmitoylation and GluA1 accumulation in inflamed mouse. These data indicated that AKAP150 and its palmitoylation were involved in AMPA receptor-dependent modification of nociceptive transmission, and the manipulations of AKAP150 signaling complex and palmitoylation might serve as potential therapeutic strategies for persistent pain after inflammation.

Calcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain.

AimsCalcitonin gene‐related peptide (CGRP) as a regulator of astrocyte activation may facilitate spinal nociceptive processing. Histone H3 lysine 9 acetylation (H3K9ac) is considered an important regulator of cytokine and chemokine gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K9ac in the activation of astrocytes, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain.MethodsAstroglial cells (C6) were treated with CGRP and differentially enrichments of H3K9ac on gene promoters were examined using ChIP‐seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on astrocyte activation and H3K9ac signaling in CCI‐induced neuropathic pain. Specific inhibitors were employed to delineate the involved signaling.ResultsIntrathecal injection of CGRP and CCI increased the number of astrocytes displaying H3K9ac in the spinal dorsal horn of rats. Treatment of CGRP was able to up‐regulate H3K9ac and glial fibrillary acidic protein (GFAP) expression in astroglial cells. ChIP‐seq data indicated that CGRP significantly altered H3K9ac enrichments on gene promoters in astroglial cells following CGRP treatment, including 151 gaining H3K9ac and 111 losing this mark, which mostly enriched in proliferation, autophagy, and macrophage chemotaxis processes. qRT‐PCR verified expressions of representative candidate genes (ATG12, ATG4C, CX3CR1, MMP28, MTMR14, HMOX1, RET) and RTCA verified astrocyte proliferation. Additionally, CGRP treatment increased the expression of H3K9ac, CX3CR1, and IL‐1β in the spinal dorsal horn. CGRP antagonist and HAT inhibitor attenuated mechanical and thermal hyperalgesia in CCI rats. Such analgesic effects were concurrently associated with the reduced levels of H3K9ac, CX3CR1, and IL‐1β in the spinal dorsal horn of CCI rats.ConclusionOur findings highly indicate that CGRP is associated with the development of neuropathic pain through astrocytes‐mediated neuroinflammatory responses via H3K9ac in spinal dorsa horn following nerve injury.This study found that CGRP act on their astrocytic receptors and lead to H3K9 acetylation (H3K9ac), which are mainly associated with proliferation‐, autophagy‐, and inflammation‐related gene expression. The number of astrocytes with H3K9ac expression is increased after nerve injury. Inhibition of CGRP attenuates the development of neuropathic pain, which was accompanied by the suppression of H3K9ac, CX3CR1, and IL‐1β expression in CCI rats.

Optogenetic Manipulations of Amygdala Neurons Modulate Spinal Nociceptive Processing and Behavior Under Normal Conditions and in an Arthritis Pain Model.

The amygdala is an important neural substrate for the emotional–affective dimension of pain and modulation of pain. The central nucleus (CeA) serves major amygdala output functions and receives nociceptive and affected–related information from the spino-parabrachial and lateral–basolateral amygdala (LA–BLA) networks. The CeA is a major site of extra–hypothalamic expression of corticotropin releasing factor (CRF, also known as corticotropin releasing hormone, CRH), and amygdala CRF neurons form widespread projections to target regions involved in behavioral and descending pain modulation. Here we explored the effects of modulating amygdala neurons on nociceptive processing in the spinal cord and on pain-like behaviors, using optogenetic activation or silencing of BLA to CeA projections and CeA–CRF neurons under normal conditions and in an acute pain model. Extracellular single unit recordings were made from spinal dorsal horn wide dynamic range (WDR) neurons, which respond more strongly to noxious than innocuous mechanical stimuli, in normal and arthritic adult rats (5–6 h postinduction of a kaolin/carrageenan–monoarthritis in the left knee). For optogenetic activation or silencing of CRF neurons, a Cre–inducible viral vector (DIO–AAV) encoding channelrhodopsin 2 (ChR2) or enhanced Natronomonas pharaonis halorhodopsin (eNpHR3.0) was injected stereotaxically into the right CeA of transgenic Crh–Cre rats. For optogenetic activation or silencing of BLA axon terminals in the CeA, a viral vector (AAV) encoding ChR2 or eNpHR3.0 under the control of the CaMKII promoter was injected stereotaxically into the right BLA of Sprague–Dawley rats. For wireless optical stimulation of ChR2 or eNpHR3.0 expressing CeA–CRF neurons or BLA–CeA axon terminals, an LED optic fiber was stereotaxically implanted into the right CeA. Optical activation of CeA–CRF neurons or of BLA axon terminals in the CeA increased the evoked responses of spinal WDR neurons and induced pain-like behaviors (hypersensitivity and vocalizations) under normal condition. Conversely, optical silencing of CeA–CRF neurons or of BLA axon terminals in the CeA decreased the evoked responses of spinal WDR neurons and vocalizations, but not hypersensitivity, in the arthritis pain model. These findings suggest that the amygdala can drive the activity of spinal cord neurons and pain-like behaviors under normal conditions and in a pain model.

Anti-nociceptive effects of oxytocin receptor modulation in healthy volunteers-A randomized, double-blinded, placebo-controlled study.

There is increasing evidence for oxytocin as a neurotransmitter in spinal nociceptive processes. Hypothalamic oxytocinergic neurons project to the spinal dorsal horn, where they activate GABA-ergic inhibitory interneurons. The present study tested whether the long-acting oxytocin-analogue carbetocin has anti-nociceptive effects in multi-modal experimental pain in humans. Twenty-five male volunteers received carbetocin 100 mcg and placebo (0.9% NaCl) on two different sessions in a randomized, double-blinded, cross-over design. Multi-modal quantitative sensory testing (QST) including a model of capsaicin-induced hyperalgesia and allodynia were performed at baseline and at 10, 60 and 120min after drug administration. QST data were analysed using mixed linear and logistic regression models. Carbetocin plasma concentrations and oxytocin receptor genotypes were quantified and assessed in an exploratory fashion. An anti-nociceptive effect of carbetocin was observed on intramuscular electrical temporal summation (estimated difference: 1.26mA, 95% CI 1.01 to 1.56mA, p=.04) and single-stimulus electrical pain thresholds (estimated difference: 1.21mA, 95% CI 1.0 to 1.47mA, p=.05). Furthermore, the area of capsaicin-induced allodynia was reduced after carbetocin compared to placebo (estimated difference: -6.5 cm2 , 95% CI -9.8 to -3.2 cm2 , p<.001). This study provides evidence of an anti-nociceptive effect of carbetocin on experimental pain in humans. This study provides evidence of the anti-nociceptive effect of intravenous administration of the oxytocin agonist carbetocin in healthy male volunteers.

Spinal Excitatory Dynorphinergic Interneurons Contribute to Burn Injury-Induced Nociception Mediated by Phosphorylated Histone 3 at Serine 10 in Rodents

The phosphorylation of serine 10 in histone 3 (p-S10H3) has recently been demonstrated to participate in spinal nociceptive processing. However, superficial dorsal horn (SDH) neurons involved in p-S10H3-mediated nociception have not been fully characterized. In the present work, we combined immunohistochemistry, in situ hybridization with the retrograde labeling of projection neurons to reveal the subset of dorsal horn neurons presenting an elevated level of p-S10H3 in response to noxious heat (60 °C), causing burn injury. Projection neurons only represented a small percentage (5%) of p-S10H3-positive cells, while the greater part of them belonged to excitatory SDH interneurons. The combined immunolabeling of p-S10H3 with markers of already established interneuronal classes of the SDH revealed that the largest subset of neurons with burn injury-induced p-S10H3 expression was dynorphin immunopositive in mice. Furthermore, the majority of p-S10H3-expressing dynorphinergic neurons proved to be excitatory, as they lacked Pax-2 and showed Lmx1b-immunopositivity. Thus, we showed that neurochemically heterogeneous SDH neurons exhibit the upregulation of p-S10H3 shortly after noxious heat-induced burn injury and consequential tissue damage, and that a dedicated subset of excitatory dynorphinergic neurons is likely a key player in the development of central sensitization via the p-S10H3 mediated pathway.

Loss of cortical control over the descending pain modulatory system determines the development of the neuropathic pain state in rats.

The loss of descending inhibitory control is thought critical to the development of chronic pain but what causes this loss in function is not well understood. We have investigated the dynamic contribution of prelimbic cortical neuronal projections to the periaqueductal grey (PrL-P) to the development of neuropathic pain in rats using combined opto- and chemogenetic approaches. We found PrL-P neurons to exert a tonic inhibitory control on thermal withdrawal thresholds in uninjured animals. Following nerve injury, ongoing activity in PrL-P neurons masked latent hypersensitivity and improved affective state. However, this function is lost as the development of sensory hypersensitivity emerges. Despite this loss of tonic control, opto-activation of PrL-P neurons at late post-injury timepoints could restore the anti-allodynic effects by inhibition of spinal nociceptive processing. We suggest that the loss of cortical drive to the descending pain modulatory system underpins the expression of neuropathic sensitisation after nerve injury.

The serotonin receptor 2A (HTR2A) rs6313 variant is associated with higher ongoing pain and signs of central sensitization in neuropathic pain patients.

The serotonin receptor 2A (HTR2A) has been described as an important facilitation mediator of spinal nociceptive processing leading to central sensitization (CS) in animal models of chronic pain. However, whether HTR2A single nucleotide variants (SNVs) modulate neuropathic pain states in patients has not been investigated so far. The aim of this study was to elucidate the potential association of HTR2A variants with sensory abnormalities or ongoing pain in neuropathic pain patients. At total of 240 neuropathic pain patients and 253 healthy volunteers were included. Patients were phenotypically characterized using standardized quantitative sensory testing (QST). Patients and controls were genotyped for HTR2A g.-1438G>A (rs6311) and c.102C>T (rs6313). Genotype-related differences in QST parameters were assessed considering QST profile clusters, principal somatosensory components and sex. There was an equal distribution of rs6313 and linked rs6311 between patients and controls. However, the rs6313 variant was significantly associated with a principal component of pinprick hyperalgesia and dynamic mechanical allodynia, indicating enhanced CS in patients with sensory loss (-0.34±0.15 vs. +0.31±0.11 vs., p<.001). In this cluster, the variant allele was also associated with single QST parameters of pinprick hyperalgesia (MPT, +0.64±0.18 vs. -0.34±0.23 p=.002; MPS, +0.66±0.17 vs. -0.09±0.23, p=.009) and ongoing pain was increased by 30%. The specific association of the rs6313 variant with pinprick hyperalgesia and increased levels of ongoing pain suggests that the HTR2A receptor might be an important modulator in the development of CS in neuropathic pain. This article presents new insights into serotonin receptor 2A-mediating mechanisms of central sensitization in neuropathic pain patients. The rs6313 variant allele was associated with increased mechanical pinprick sensitivity and increased levels of ongoing pain supporting a contribution of central sensitization in the genesis of ongoing pain providing a possible route for mechanism-based therapies.

Spinal spatial integration of nociception and its functional role assessed via the nociceptive withdrawal reflex and psychophysical measures in healthy humans.

Animal studies have previously shown that deep dorsal horn neurons play a role in the processing of spatial characteristics of nociceptive information in mammals. Human studies have supported the role of the spinal neurons; however, the mechanisms involved, and its significance, remain to be clarified. The aim of this study was to investigate spatial aspects of the spinal integration of concurrent nociceptive electrical stimuli in healthy humans using the Nociceptive Withdrawal Reflex (NWR) as an objective indication of spinal nociceptive processing. Fifteen healthy volunteers participated in the study. Electrical stimuli were delivered, using five electrodes located across the sole of the foot in a mediolateral disposition, as a single or double simultaneous stimuli with varying Inter‐Electrode Distances (IEDs). The stimulation intensity was set at 1.5× NWR threshold (TA muscle). The size of the NWR was quantified in the 60–180 ms poststimulus window as a primary outcome measure. Psychophysical measures were secondary outcomes. Single stimulation elicited significantly smaller NWRs and perceived intensity than double stimulation (p < .01), suggesting the presence of spatial summation occurring within the spinal processing. During double stimulation, increasing the inter‐electrode distance produced significantly smaller NWR sizes (p < .05) but larger pain intensity ratings (p < .05). By the NWR, spatial summation was shown to affect the nociceptive processing within the spinal cord. The inhibited motor response obtained when simultaneously stimulating the medial and lateral side of the sole of the foot suggests the presence of an inhibitory mechanism with a functional, behaviorally oriented function.

Spinal neuronal excitability and neuroinflammation in a model of chemotherapeutic neuropathic pain: targeting the resolution pathways

BackgroundNeuroinflammation is a critical feature of sensitisation of spinal nociceptive processing in chronic pain states. We hypothesised that the resolvin pathways, a unique endogenous control system, may ameliorate aberrant spinal processing of somatosensory inputs associated with chemotherapy-induced neuropathic pain (CINP).MethodThe paclitaxel (PCX) model of CINP was established in male Sprague-Dawley rats and compared to control rats (n = 23 and 22, respectively). Behavioural pain responses were measured, and either single unit electrophysiological recordings of dorsal horn wide dynamic range (WDR) neurones were performed, or mRNA microarray analysis of the dorsal horn of the spinal cord was undertaken.ResultsPCX rats exhibited significant changes in behavioural responses to mechanical and cold stimuli. A higher proportion of WDR neurones in PCX rats were polymodal (generating post-discharge following a non-noxious mechanical stimulus, responding to non-noxious cold and exhibiting spontaneous activity) compared to control (p < 0.05). Microarray analysis revealed changes in proinflammatory pathways (Tlr, Tnfrsf1a, Nlrp1a, Cxcr1, Cxcr5, Ccr1, Cx3cr1) and anti-inflammatory lipid resolvin pathways (Alox5ap, Cyp2j4 and Ptgr1) compared to control (p < 0.05). Ingenuity pathway analysis predicted changes in glutamatergic and astrocyte signaling in the PCX group. Activation of the resolvin system via the spinal administration of aspirin-triggered resolvin D1 (AT-RvD1) markedly inhibited (73 ± 7% inhibition) normally non-noxious mechanically (8 g) evoked responses of WDR neurones only in PCX rats, whilst leaving responses to noxious mechanically induced stimuli intact. Inhibitory effects of AT-RvD1were comparable in magnitude to spinal morphine (84 ± 4% inhibition).ConclusionThe PCX model of CINP was associated with mechanical allodynia, altered neuronal responses and dysregulation of pro- and anti-inflammatory signalling in the spinal dorsal horn. The resolvin AT-RvD1 selectively inhibited low weight mechanical-evoked responses of WDR neurones in PCX rats, but not in controls. Our data support the targeting of spinal neuroinflammation via the activation of the resolvin system as a new therapeutic approach for CINP.

Differential expression of Na+/K+/Cl\u2212 cotransporter 1 in neurons and glial cells within the superficial spinal dorsal horn of rodents

Although convincing experimental evidence indicates that Na+/K+/Cl− cotransporter 1 (NKCC1) is involved in spinal nociceptive information processing and in the generation of hyperalgesia and allodynia in chronic pain states, the cellular distribution of NKCC1 in the superficial spinal dorsal horn is still poorly understood. Because this important piece of knowledge is missing, the effect of NKCC1 on pain processing is still open to conflicting interpretations. In this study, to provide the missing experimental data, we investigated the cellular distribution of NKCC1 in the superficial spinal dorsal horn by immunohistochemical methods. We demonstrated for the first time that almost all spinal axon terminals of peptidergic nociceptive primary afferents express NKCC1. In contrast, virtually all spinal axon terminals of nonpeptidergic nociceptive primary afferents were negative for NKCC1. Data on the colocalization of NKCC1 with axonal and glial markers indicated that it is almost exclusively expressed by axon terminals and glial cells in laminae I–IIo. In lamina IIi, however, we observed a strong immunostaining for NKCC1 also in the dendrites and cell bodies of PV-containing inhibitory neurons and a weak staining in PKCγ-containing excitatory neurons. Our results facilitate further thinking about the role of NKCC1 in spinal pain processing.

Comparison between Intrathecal Nalbuphine and Bupivacaine versus Fentanyl and Bupivacaine For Postoperative Analgesia After Lower Abdominal Surgeries

Abstract The use of opioids in intrathecal or epidural anesthesia has become popular to optimize postoperative analgesia. However, opioid-induced side effects, such as respiratory depression, nausea, vomiting, urinary retention and pruritus, limit their use. Several investigations have shown that intrathecal or epidural administration of opioids produces a dose dependent modulation of spinal nociceptive processing in animals and humans and is not associated with sedation. Aim of Study The purpose of this study was to assess the spinally mediated analgesic effects postoperativally of intrathecal Nalbuphine plus bupivacaine after lower abdominal surgeries in comparison to intrathecal bupivacaine plus fentanyl Patients &amp; Methods : 60 patients came to Ain-shams Hospital for lower abdominal surgeries, They were randomly allocated into two equal groups (30 patients each) group F and group N. Group F : Bupivacaine-Fentanyl Patients received an intrathecal injection of 3 ml of 0.5% heavy (hyperbaric) bupivacaine plus 0.5 ml (25 µg) fentanyl. Group N: Bupivacaine-Nalbuphin Patients received an intrathecal injection of 3 ml of 0.5% heavy (hyperbaric) bupivacaine plus 0.5 ml (0.5 mg) Nalbuphine. All patients were assessed and monitored for: Hemodynamics: ECG for heart rate, and non-invasive arterial pressure, respiratory rate and arterial oxygen saturation, onset of sensory block, onset of motor block, duration of analgesia &amp; 1st rescue analgesia, duration of motor block, adverse effects as: hypotension, bradycardia, respiratory depression, pruritus, shivering, nausea and vomiting were recorded. Results &amp; Conclusion Addition of a small dose of nalbuphine or fentanyl to bupivacaine in spinal anesthesia moderately prolonged the time of postoperative analgesia the duration of postoperative analgesia is nearly the same in nalbuphine group &amp; the fentanyl group. There is more rapid onset of motor block in fentanyl group than in nalbuphine group but with no difference in sensory or motor block regression. No respiratory depression or degree of sedation occurs in both groups. Hemodynamic stability &amp; side effects incidence are the same in both groups

SNAP25/syntaxin4/VAMP2/Munc18-1 Complexes in Spinal Dorsal Horn Contributed to Inflammatory Pain

SNAP25/syntaxin4/VAMP2/Munc18-1 Complexes in Spinal Dorsal Horn Contributed to Inflammatory Pain

Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse

Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch. Although it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory "microcircuits" within the region. As a result, the goal of this study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique, followed by RNA-seq analysis. Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared with non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1). In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH. Differential gene expression analysis comparing pDyn nuclei across the 3 ages revealed sets of genes that were significantly upregulated (such as Cartpt, encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1, encoding the receptor for neuropeptides B/W) during postnatal development. Collectively, these results provide new insight into the potential molecular mechanisms underlying the known age-dependent changes in spinal nociceptive processing and pain sensitivity.

Differential suppression of the ipsi- and contralateral nociceptive reflexes in the neonatal rat spinal cord by agonists of µ-, δ- and κ-opioid receptors

Differential suppression of the ipsi- and contralateral nociceptive reflexes in the neonatal rat spinal cord by agonists of µ-, δ- and κ-opioid receptors

High-threshold primary afferent supply of spinal lamina X neurons.

The spinal gray matter region around the central canal, lamina X, is critically involved in somatosensory processing and visceral nociception. Although several classes of primary afferent fibers terminate or decussate in this area, little is known about organization and functional significance of the afferent supply of lamina X neurons. Using the hemisected ex vivo spinal cord preparation, we show that virtually all lamina X neurons receive primary afferent inputs, which are predominantly mediated by the high-threshold Aδ- fibers and C-fibers. In two-thirds of the neurons tested, the inputs were monosynaptic, implying a direct targeting of the population of lamina X neurons by the primary nociceptors. Beside the excitatory inputs, 48% of the neurons also received polysynaptic inhibitory inputs. A complex pattern of interactions between the excitatory and inhibitory components determined the output properties of the neurons, one-third of which fired spikes in response to the nociceptive dorsal root stimulation. In this respect, the spinal gray matter region around the central canal is similar to the superficial dorsal horn, the major spinal nociceptive processing area. We conclude that lamina X neurons integrate direct and indirect inputs from several types of thin primary afferent fibers and play an important role in nociception.

Polymelia in a chimeric Simmental calf: nociceptive withdrawal reflex, anaesthetic and analgesic management, anatomic and genetic analysis

BackgroundPolymelia is a congenital defect characterized by one or more supernumerary legs. The genetics and aetiology of this condition in cattle have not yet been thoroughly investigated even though several case reports do exist. The model of the nociceptive withdrawal reflex (NWR) has been characterized in several species to study spinal nociceptive processing. It is a polysynaptic spinal reflex that can be elicited by noxious electrical stimulation and recorded by electromyography. Thorough nociceptive examination and preventive analgesic management has not yet been an aspect in the perioperative management of polymelia cases.Case presentationA 4-month-old female Simmental calf was presented with notomelia. The animal was in good health and showed no neurologic deficiencies. Preoperatively, computed tomography was performed to gain more detailed anatomical information. To evaluate the sensitivity of the accessory limb, NWR testing was performed and revealed a connection of the afferent reflex pathway of the accessory limb to the efferent of the normal limb. The accessory limb was surgically removed under general anaesthesia. Intensive care included multimodal pain therapy adapted to the pain intensity scored during regular pain assessment. A gross anatomical dissection as well as a genetic analysis of the accessory limb were performed postoperatively. The calf was identified as a chimera.ConclusionThis calf was successfully relieved of its accessory limb. Chimerism has not been described in the congenital defect polymelia. As the accessory limb was pain sensitive and a common nociceptive reflex pathway was identified, thorough perioperative pain management was performed with the intention to prevent chronic neuropathic pain development.

PC-FACS

PC-FACS

Modality selective roles of pro-nociceptive spinal 5-HT2A and 5-HT3 receptors in normal and neuropathic states

Descending brainstem control of spinal nociceptive processing permits a dynamic and adaptive modulation of ascending sensory information. Chronic pain states are frequently associated with enhanced descending excitatory drive mediated predominantly through serotonergic neurones in the rostral ventromedial medulla. In this study, we examine the roles of spinal 5-HT2A and 5-HT3 receptors in modulating ascending sensory output in normal and neuropathic states. In vivo electrophysiology was performed in anaesthetised spinal nerve ligated (SNL) and sham-operated rats to record from wide dynamic range neurones in the ventral posterolateral thalamus. In sham rats, block of spinal 5-HT3Rs with ondansetron revealed tonic facilitation of noxious punctate mechanical stimulation, whereas blocking 5-HT2ARs with ketanserin had minimal effect on neuronal responses to evoked stimuli. The inhibitory profiles of both drugs were altered in SNL rats; ondansetron additionally inhibited neuronal responses to lower intensity punctate mechanical stimuli and noxious heat evoked responses, whereas ketanserin inhibited innocuous and noxious evaporative cooling evoked responses. Neither drug had any effect on dynamic brush evoked responses nor on spontaneous firing rates in both sham and SNL rats. These data identify novel modality and intensity selective facilitatory roles of spinal 5-HT2A and 5-HT3 receptors on sensory neuronal processing within the spinothalamic-somatosensory cortical pathway.