L5 Spinal Nerve Research Articles

Effect of dexmedetomidine on high mobility group box 1 protein expression in the spinal dorsal born of rats underwent spinal nerve ligation

Objective To investigate the effect of dexmedetomidine (DEX) on high mobility group box 1 protein (HMGB1)expression in the L4-5 dorsal horns of rats underwent spinal nerve ligation.Methods Thirty male SD rats were randomly divided into three groups(n=10):sham operation group(Sham group),L5 spinal nerve ligation group(SNL group) and DEX-treatment group(DEX group).After intrathecal catheter implantation between L4-5 vertebrae,the rats in SNL group and DEX group were underwent L5 spinal nerve ligation.The surgical procedure for the the rats in Sham group was identical except nerve ligation.Then DEX (2 μg/10 μl) was injected intrathecally via intrathecal catheter in the rats of DEX group after L5 spinal nerve ligation,the same volume of normal saline was injected intraperitoneally in rats of Sham group and SNL group.Expression of HMGB1 in the spinal dorsal horn was measured by Western blot.Results Compared to Sham group,the paw withdrawal threshold(PWT) and paw withdrawal latency (PWL) of rats in SNL group were significantly decreased from 1 d and 3 d after SNL,respectively,and lasted for 7 d (P＜0.05).On day 7 after SNL,the PWT(12.9±1.6) g and PWL(8.3±0.9) s were decreased 67％ and 41％,respectively(P＜0.05),with the levels of HMGB1 expression increased 84％(P＜0.05).Compared to SNL group,the PWT and PWL of rats in DEX group were significantly increased (P＜0.05).On day 7 after SNL,the PWT(22.3±3.2) g and PWL(10.4±1.1) s were increased 73％ and 25％,respectively(P＜0.05),with the levels of HMGB1 expression decreased 29％(P＜0.05).Conclusions Increase of HMGB1 expression in spinal dorsal horn was involved in SNL-induced neuropathic pain.DEX injected intrathecally ameliorated SNL-induced mechanical allodynia and thermal hyperalgesia,the effect may be related to inhibition of HMGB1 expression by DEX. Key words: Dexmedetomidine; Spinal nerve ligation; Neuropathic pain; High mobility group box 1 protein

The effect of intrathecal curcumin on mechanical allodynia in rats after L5 spinal nerve ligation

The effect of intrathecal curcumin on mechanical allodynia in rats after L5 spinal nerve ligation

G protein-coupled receptor, family C, group 5 (GPRC5B) downregulation in spinal cord neurons is involved in neuropathic pain.

BackgroundG protein-coupled receptor, family C, group 5 (GPRC5B), a retinoic acid-inducible orphan G-protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins presumably related in non-canonical Wnt signaling. In this study, we investigated altered GPRC5B expression in the dorsal horn of the spinal cord after spinal nerve injury and its involvement in the development of neuropathic pain.MethodsAfter induction of anesthesia by intraperitoneal injection of pentobarbital (35 mg /kg), the left L5 spinal nerve at the level of 2 mm distal to the L5 DRG was tightly ligated with silk and cut just distal to the ligature. Seven days after nerve injury, animals were perfused with 4% paraformaldehyde, and the spinal cords were extracted and post-fixed at 4℃ overnight. To identify the expression of GPRC5B and analyze the involvement of GPRC5B in neuropathic pain, immunofluorescence was performed using several markers for neurons and glial cells in spinal cord tissue.ResultsAfter L5 spinal nerve ligation (SNL), the expression of GPRC5B was decreased in the ipsilateral part, as compared to the contralateral part, of the spinal dorsal horn. SNL induced the downregulation of GPRC5B in NeuN-positive neurons in the spinal dorsal horn. However, CNPase-positive oligodendrocytes, OX42-positive microglia, and GFAP-positive astrocytes were not immunolabeled with GPRC5B antibody in the spinal dorsal horn.ConclusionsThese results imply that L5 SNL-induced GPRC5B downregulation may affect microglial activation in the spinal dorsal horn and be involved in neuropathic pain.

Management of Patients with L2 Spinal Nerve Root Block Who Are Suffering from Low Back and Referred Pain

Study Design: Prospective clinical study. Objectives:The aim of the study was to verify the effectiveness of L2 spinal nerve root block for patients who are suffering from low back pain and referred pain with compound causes. Summary of Literature Review: Most of low back pain and referred pain arises from discs, facet joints and sacroiliac joints. All structures above have the pain perception pathway through sympathetic nerves with a connection to L2 spinal nerves. Materials and Methods: We selected patients with low back pain and referred pain for more than 2 weeks. Each 50 patients were randomly allocated to an experimental and a control group.The experimental group underwent a L2 spinal nerve root block with 2cc of 0.25% bupibacaine at the symptom dominant side. The control group underwent a skin infiltration with 2cc of 2% lidocaine only. The pain was assessed with a visual analogue scale (VAS) consisting of 100 points at 5minutes, day 1, day 3, day 5, day 7 and day 30 postprocedure. Results: Both groups showed a significant improvement after the procedures(p=0.000). However, a greater improvement was shown in the experimental group(p=0.000). In the individual analysis, the experimental group had improved as time elapsed and the significancewas maintained until 30 days. However, in control group, the significance was lost at day 30. Conclusion: L2 spinal nerve root block is recognized to reduce the low back and referred pains which arise from compound causes in a degenerative spinal disease.

Fifth lumbar spinal nerve injury causes neurochemical changes in corresponding as well as adjacent spinal segments: A possible mechanism underlying neuropathic pain

Previous investigations of the anatomical basis of the neuropathic-like manifestations in the spinal nerve ligation animal model have shown that the central terminations of the unmyelinated primary afferents of L5 spinal nerve are not restricted to the corresponding L5 spinal segment, and rather extend to two spinal segments rostrally and one segment caudally where they intermingle with primary afferents of the adjacent L4 spinal nerve. The aim of the present study was to investigate the neurochemical changes in the dorsal horn of the spinal cord and DRGs after L5 nerve injury in rats. In the first experiment, the right L5 nerve was ligated and sectioned for 14 days, and isolectin B4 (IB4, a tracer for unmyelinated primary afferents) was injected into the left L5 nerve. The results showed that the vasoactive intestinal peptide (VIP) was up-regulated in laminae I–II of L3–L6 spinal segments on the right side in exactly the same areas where IB4 labelled terminals were revealed on the left side. In the second experiment, L5 was ligated and sectioned and the spinal cord and DRGs were stained immunocytochemically with antibodies raised against various peptides known to be involved in pain transmission and hyperalgesia. The results showed that L5 nerve lesion caused down-regulation of substance P, calcitonin-gene related peptide and IB4 binding and up-regulation of neuropeptide Y and neurokinin-1 receptor in the dorsal horn of L4 and L5 spinal segments. Similar neurochemical changes were observed only in the corresponding L5 DRG with minimal effects observed in L3, L4 and L6 DRGs. Although, L5 nerve injury caused an up-regulation in NPY, no change in SP and CGRP immunoreactivity was observed in ipsilateral garcile nucleus. These neuroplastic changes in the dorsal horn of the spinal cord, in the adjacent uninjured territories of the central terminations of the adjacent uninjured nerves, might explain the mechanism of hyperalgesia after peripheral nerve injury.

Anti-allodynic effects of obtusifolin and gluco-obtusifolin against inflammatory and neuropathic pain.

Inflammatory pain and neuropathic pain are major health issues that represent considerable social and economic burden worldwide. In this study we investigated the potential of obtusifolin and gluco-obtusifolin, two anthraquinones found in the seeds of the widely used traditional Chinese medical botanical Cassia obtusifolia, to reduce neuropathic and inflammatory pain. The potential analgesic effects of obtusifolin and gluco-obtusifolin were evaluated by mice formalin test and complete Freund's adjuvant (CFA)-induced nociceptive behaviors in rats. Chronic constriction injury (CCI), L5 spinal nerve ligation (L5 SNL), diabetes, and chemotherapeutics inducing allodynia were used to test whether repeated treatment with obtusifolin and gluco-obtusifolin ameliorated neuropathic pain. Finally, we explored whether obtusifolin and gluco-obtusifolin altered the degree of neuroinflammation in rat spinal cord after CFA administration and CCI induction. Obtusifolin and gluco-obtusifolin (0.25, 0.5, 1, and 2 mg/kg) reduced licking/biting time in dose-dependent manner in phase 2 of formalin-induced behavior in mice. Furthermore, repeated administration of obtusifolin and gluco-obtusifolin (0.25, 0.5, 1, and 2 mg/kg) reversed mechanical allodynia induced by CFA, CCI, L5 SNL, diabetes, and oxaliplatin in a dose-dependent manner in rats. Levels of activated nuclear factor-kappa B (NF-κB) and proinflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α)) in lumbar spinal cord were elevated in rats following CFA treatment and CCI induction, and obtusifolin and gluco-obtusifolin significantly inhibited these effects. Our results demonstrate that obtusifolin and gluco-obtusifolin produce significant antinociceptive action in rodent behavioral models of inflammatory/neuropathic pain, and that this activity is associated with modulation of neuroinflammation in spinal cord.

Variability in C-type lectin receptors regulates neuropathic pain-like behavior after peripheral nerve injury.

IntroductionNeuropathic pain is believed to be influenced in part by inflammatory processes. In this study we examined the effect of variability in the C-type lectin gene cluster (Aplec) on the development of neuropathic pain-like behavior after ligation of the L5 spinal nerve in the inbred DA and the congenic Aplec strains, which carries seven C-type lectin genes originating from the PVG strain.ResultsWhile both strains displayed neuropathic pain behavior early after injury, the Aplec strain remained sensitive throughout the whole study period. Analyses of several mRNA transcripts revealed that the expression of Interleukin-1β, Substance P and Cathepsin S were more up-regulated in the dorsal part of the spinal cord of Aplec rats compared to DA, indicating a stronger inflammatory response. This notion was supported by flow cytometric analysis revealing increased infiltration of activated macrophages into the spinal cord. In addition, macrophages from the Aplec strain stimulated in vitro displayed higher expression of inflammatory cytokines compared to DA cells. Finally, we bred a recombinant congenic strain (R11R6) comprising only four of the seven Aplec genes, which displayed similar clinical and immune phenotypes as the Aplec strain.ConclusionWe here for the first time demonstrate that C-type lectins, a family of innate immune receptors with largely unknown functions in the nervous system, are involved in regulation of inflammation and development of neuropathic pain behavior after nerve injury. Further experimental and clinical studies are needed to dissect the underlying mechanisms more in detail as well as any possible relevance for human conditions.

Phenotypic Identification of Spinal Cord-Infiltrating CD4+ T Lymphocytes in a Murine Model of Neuropathic Pain.

BackgroundNeuropathic pain is one of the most devastating kinds of chronic pain. Neuroinflammation has been shown to contribute to the development of neuropathic pain. We have previously demonstrated that lumbar spinal cord-infiltrating CD4+ T lymphocytes contribute to the maintenance of mechanical hypersensitivity in spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we further examined the phenotype of the CD4+ T lymphocytes involved in the maintenance of neuropathic pain-like behavior via intracellular flow cytometric analysis and explored potential interactions between infiltrating CD4+ T lymphocytes and spinal cord glial cells.ResultsWe consistently observed significantly higher numbers of T-Bet+, IFN-γ+, TNF-α+, and GM-CSF+, but not GATA3+ or IL-4+, lumbar spinal cord-infiltrating CD4+ T lymphocytes in the L5Tx group compared to the sham group at day 7 post-L5Tx. This suggests that the infiltrating CD4+ T lymphocytes expressed a pro-inflammatory type 1 phenotype (Th1). Despite the observation of CD4+ CD40 ligand (CD154)+ T lymphocytes in the lumbar spinal cord post-L5Tx, CD154 knockout (KO) mice did not display significant changes in L5Tx-induced mechanical hypersensitivity, indicating that T lymphocyte-microglial interaction through the CD154-CD40 pathway is not necessary for L5Tx-induced hypersensitivity. In addition, spinal cord astrocytic activation, represented by glial fibillary acidic protein (GFAP) expression, was significantly lower in CD4 KO mice compared to wild type (WT) mice at day 14 post-L5Tx, suggesting the involvement of astrocytes in the pronociceptive effects mediated by infiltrating CD4+ T lymphocytes.ConclusionsIn all, these data indicate that the maintenance of L5Tx-induced neuropathic pain is mostly mediated by Th1 cells in a CD154-independent manner via a mechanism that could involve multiple Th1 cytokines and astrocytic activation.

Temporal changes in MrgC expression after spinal nerve injury

Mas-related G-protein-coupled receptor subtype C (MrgC) may play an important role in pain sensation. However, the distribution of MrgC receptors in different subpopulations of rodent dorsal root ganglion (DRG) neurons has not been clearly demonstrated owing to a lack of MrgC-selective antibody. It is also unclear whether peripheral nerve injury induces different time-dependent changes in MrgC expression in injured and uninjured DRG neurons. Here we showed that MrgC immunoreactivity is distributed in both IB4-positive (non-peptidergic) and calcitonin gene-related peptide-positive (peptidergic) DRG neurons in mice and rats. Importantly, the MrgC mRNA level and MrgC immunoreactivity were both decreased in the injured L5 DRG compared to corresponding levels in the contralateral (uninjured) DRG in rats on days 14 and 30 after an L5 spinal nerve ligation. In contrast, mRNA and protein levels of MrgC were increased in the adjacent uninjured L4 DRG. Thus, nerve injury may induce temporal changes in MrgC expression that differ between injured and uninjured DRG neurons. In animal behavior tests, chronic constriction injury of the sciatic nerve induced mechanical pain hypersensitivity in wild-type mice and Mrg-clusterΔ−/− mice (Mrg KO). However, the duration of mechanical hypersensitivity was longer in the Mrg KO mice than in their wild-type littermates, indicating that activation of Mrgs may constitute an endogenous mechanism that inhibits the maintenance of neuropathic pain in mice. These findings extend our knowledge about the distribution of MrgC in rodent DRG neurons and the regulation of its expression by nerve injury.

MrgC agonism at central terminals of primary sensory neurons inhibits neuropathic pain

Chronic neuropathic pain is often refractory to current pharmacotherapies. The rodent Mas-related G-protein-coupled receptor subtype C (MrgC) shares substantial homogeneity with its human homologue, MrgX1, and is located specifically in small-diameter dorsal root ganglion neurons. However, evidence regarding the role of MrgC in chronic pain conditions has been disparate and inconsistent. Accordingly, the therapeutic value of MrgX1 as a target for pain treatment in humans remains uncertain. Here, we found that intrathecal injection of BAM8-22 (a 15-amino acid peptide MrgC agonist) and JHU58 (a novel dipeptide MrgC agonist) inhibited both mechanical and heat hypersensitivity in rats after an L5 spinal nerve ligation (SNL). Intrathecal JHU58-induced pain inhibition was dose dependent in SNL rats. Importantly, drug efficacy was lost in Mrg-cluster gene knockout (Mrg KO) mice and was blocked by gene silencing with intrathecal MrgC siRNA and by a selective MrgC receptor antagonist in SNL rats, suggesting that the drug action is MrgC dependent. Further, in a mouse model of trigeminal neuropathic pain, microinjection of JHU58 into ipsilateral subnucleus caudalis inhibited mechanical hypersensitivity in wild-type but not Mrg KO mice. Finally, JHU58 attenuated the miniature excitatory postsynaptic currents frequency both in medullary dorsal horn neurons of mice after trigeminal nerve injury and in lumbar spinal dorsal horn neurons of mice after SNL. We provide multiple lines of evidence that MrgC agonism at spinal but not peripheral sites may constitute a novel pain inhibitory mechanism that involves inhibition of peripheral excitatory inputs onto postsynaptic dorsal horn neurons in different rodent models of neuropathic pain.

Neuromodulation attenuates bladder hyperactivity in a rat cystitis model

BackgroundWe investigated the regulation of urinary bladder function by electrical stimulation of the L6 spinal nerve (SN) using cystometry in normal rats and in rats with cystitis induced by intravesical infusion of dilute acetic acid.MethodsIn anesthetized rats, a cannula was placed into the bladder dome for saline/acetic acid infusion and intravesical pressure monitoring. Threshold pressure (TP), basal pressure (BP) and inter-contraction interval (ICI) were measured from the bladder pressure recording and void volume (VV) was measured by weighing the voided fluid.ResultsComparison of cystometrograms obtained with infusion of saline or acetic acid showed that acetic acid decreases TP, ICI and VV. These excitatory effects, characteristic of acetic acid induced bladder hyperactivity, were significantly reversed by bilateral SN stimulation (P <0.05, vs pre-stimulation, Student t-test). In saline perfused rats, one hour of bilateral SN stimulation at 10 Hz and at motor threshold (0.19 ± 0.01 milliamps) increased ICI (265%) and VV (217%). In rats perfused with acetic acid, the corresponding increases produced by SN stimulation were 350% for ICI and 383% for VV. The percentage increases in the acetic acid-treated group were not significantly higher than those in saline-treated group.ConclusionsUsing continuous flow cystyometry, we find that SN stimulation can produce effects on micturition consistent with its effects on isovolumetric model, and consistent with the therapeutic effect observed with InterStim® therapy in overactive bladder patients. Although the effect of SN stimulation was slightly greater in bladder irritated over normal rats, the difference was not statistically significant.

Artemin induced functional recovery and reinnervation after partial nerve injury

Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.

Role of autophagy in development of neuropathic pain in rats

Objective To evaluate the role of autophagy in the development of neuropathic pain (NP) in rats.Methods Thirty male Sprague-Dawley rats,weighing 200-220 g,were randomly divided into 3 groups (n =10 each) using a random number table:sham operation group (sham group),NP group and rapamycin (autophagy inducer) group (Rap group).Intrathecal catheter was inserted into the subarachnoid space and advanced caudally until the tip reached L4,5 segment.NP was induced by ligation of the left L5 spinal nerve (SNL) in NP and Rap groups.The L5 spinal nerve was only exposed,but not ligated in group sham.At 30 min before ligation and 2 days after operation,rapamycin 60 μg was injected intrathecally via the intrathecal catheter in Rap group,while the equal volume of vehicle (5％ dimethyl sulfoxide) was injected in sham and NP groups.The mechanical and thermal pain thresholds were measured on 1,3,5 and 7 days after ligation (T-4).The rats were sacrificed after the last measurement of pain threshold at T4.The ipsilateral L5 segment of spinal dorsal horn was removed for examination of autophagosomes (using transmission electron microscope) and for determination of the expression of LC3 Ⅱ and p62 (by Western blot) and content of IL-1β (by ELISA).Results Compared with sham group,the mechanical pain threshold at each time point and thermal pain threshold at T2-4 were significantly decreased,and the LC3 Ⅱ and p62 expression and IL-1β content were increased at T4 in group NP (P ＜ 0.05).Compared with NP group,the mechanical pain threshold at each time point,thermal pain threshold at T2-4 and LC3 Ⅱ expression at T4 were significantly increased,and the p62 expression and IL-1β content were decreased at T4 in group Rap (P ＜ 0.05).Microscopic examination showed that autophagosomes were observed in the spinal dorsal horn in NP and Rap groups,and the damage to organelles was lighter in Rap group than in NP group.Conclusion The development of NP is related to autophagic dysfunction in rats. Key words: Autophagy; Neuralgia

Behavioral and neuropathological changes in animal models of chronic painful scar

BackgroundLong-lasting limb pain or back pain after surgery occasionally develops into chronic pain that leads to lower activity and a poorer quality of life for many patients. To determine the histopathological and neuropathological mechanisms that cause persistent post-operative pain, we developed an original animal model with sustained painful scars and then examined pain-related behavior and the pathological alteration of peripheral tissues and spinal nerves associated with the model. MethodsThe animal model (Scar group) was prepared in rats by extensively stripping subcutaneous tissue from the plantar in the hind paw followed by subsequent examination of pain-related behavior over the next 12weeks. Thereafter, we conducted histological staining of the scar tissues, immunohistochemical staining of c-Fos (L5 dorsal horn), and electron microscopic analysis of the L5 spinal nerve fibers/dorsal roots. ResultsThe mechanical pain threshold decreased specifically in the ipsilateral plantar in animals with scar. This state was maintained for 12weeks. A collagen layer developed from fibers derma to the muscular layer in the scar tissue in which many fibroblasts were observed. No statistical differences were found for the areas of the c-Fos-immunoreactive (c-Fos-IR) neurons in the ipsilateral and contralateral sides of the L5 level of the dorsal horn in both the Scar group and Pinhole (sham operation) group. However, myelin sheath fragmentation of the nerve fibers was observed in the ipsilateral dorsal root at the L5 position. ConclusionsWe created a persistent painful scar model through extensive injury of the peripheral tissues. Fibrotic thickening of the cutaneous tissues, possible sensitization, and partial degradation of the spinal nerve related to the painful scar were observed. This model should enable us to better understand the mechanism of sensitization caused by painful scar and investigate new methods for treating painful scars in humans.

The role of alpha-2 adrenoceptor subtype in the antiallodynic effect of intraplantar dexmedetomidine in a rat spinal nerve ligation model

The purpose of this study was to examine the effects of intraplantar dexmedetomidine to relieve neuropathic pain and determine the role of peripheral α2-adrenoceptors. Neuropathic pain was induced by ligating the L5 and L6 spinal nerves in male Sprague–Dawley rats, and mechanical allodynia was assessed using von Frey filaments. Several antagonists were injected into the hindpaws to evaluate the mechanisms of action of dexmedetomidine, a nonselective α2-adrenoceptor antagonist yohimbine, an α2A-adrenoceptor antagonist BRL 44408, an α2B-adrenoceptor antagonist ARC 239, and a α2C-adrenoceptor antagonist JP 1302. The expression of α2A-adrenoceptor, α2B-adrenoceptor, and α2C-adrenoceptor genes in the lumbar segment of the spinal cord and the plantar skin of the nerve-injured leg was detected by reverse transcription-polymerase chain reaction. Ipsilateral intraplantar injection of dexmedetomidine produced dose-dependent antiallodynia. Ipsilateral, but not contraleral, intraplantar injection of yohimbine reversed the antinociception of dexmedetomidine. Intraplantar BRL 44408, ARC 239, and JP 1302 reversed the antinociception of dexmedetomidine. The expression levels of α2-adrenoceptor genes in the lumbar spinal cord did not differ between rats with neuropathic pain and naïve rats. The expression levels of α2B-adrenoceptor and α2C-adrenoceptor genes of plantar skin were upregulated significantly in the model group, whereas α2A-adrenoceptor expression was unchanged. These results suggest that intraplantar injection of dexmedetomidine produced an antiallodynic effect in spinal nerve ligation-induced neuropathic pain. All three types of peripheral α2A, α2B, and α2C-adrenoceptors were involved in the antiallodynic mechanism of dexmedetomidine.

Differentiation and interaction of tibial versus spinal nerve stimulation for micturition control in the rat

AimsTo determine time course of the bladder inhibitory response to unilateral or bilateral stimulation of the tibial nerve (TN) and spinal nerve (SN) as well as the interaction of stimulation at these two sites.MethodsIn anesthetized female rats, a wire electrode was placed under either one or both of the TN or L6 SN. A cannula was placed into the bladder via the urethra. Saline infusion induced bladder rhythmic contraction (BRC).ResultsCompared to SN neuromodulation, TN neuromodulation is less efficacious. The first 5-min stimulation at three times motor threshold on the SN and TN decreased the BRC frequency to 9% and 69% of controls, respectively. In contrast to SN stimulation, bilateral TN neuromodulation is not more effective than unilateral and sustained TN stimulation results in an apparent desensitization of the bladder response. If a 15-min TN stimulation was applied, BRCs were shutdown only during the first 5 min of stimulation. If a 5-min stimulation, using sufficient current to abolish BRC, is repeated, at least 20 min between stimulations was required in order for the responses to the first and second stimulations to be equivalent. Finally, stimulation of the TN combined with SN never produced a significantly greater effect than TN or SN stimulation alone.ConclusionsBased on the current experiments, it would appear that SN neuromodulation of bladder activity is preferable to TN stimulation and there is no evidence to suggest that stimulation at both sites would offer a therapeutic advantage over spinal stimulation alone. Neurourol. Urodynam. 34:92–97, 2015. © 2013 The Authors. Neurourology & Urodynamics published by Wiley Periodicals, Inc.

A rat model for chronic spinal nerve root compression

The pathophysiology of radiculopathy associated with lumbar spinal stenosis and lumbar disc herniation is incompletely understood. The goal of the present study was to establish a chronic spinal nerve root compression model that can mimic lumbar disc herniation or spinal stenosis using silicone tube compression. We also try to link the pathology changes of damaged nerve root with the reaction of microglia in spinal cord in same rat at different time points. Thirty rats were used in this study. The L5 nerve roots (dorsal and ventral) were exposed by hemilaminectomy; the diameter of the L5 nerve root was measured at the 2mm proximal from the dorsal root ganglia. The dorsal and ventral nerve roots of L5 were compressed using a silicone tube, and the sham group was only exposed dorsal and ventral roots of L5. Five rats from the sham group were perfused at 8days after surgery, and 25 rats from the model groups were perfused at 3, 8, 12, 45days, and 5months after surgery, each model group was composed of 5 rats according to the time point. The L5 spinal cord segments and nerve root that compressed by silicone tube were harvested from the same rat. Microglia and neuron in the spinal cord were stained by immunohistochemistry, and the nerve root was shown by electron microscope. In sham-operated rat, the arrangement of axon and myelin sheath is normal, the ventral root is mainly composed of large axon (>6μm) and it is composed of 46.3% of all the axons of the ventral root; the average myelin thickness of large axon is 1.86μm; the dorsal root is mainly composed of medium (2-3.9 or 4-5.9μm) axons and they are composed of 79.1% of all the axons of the dorsal root; the average myelin thickness of this category is 0.94 or 1.55μm. The average myelin thickness of large axon in ventral root reduced to 0.97 and 1.19μm from more than 1.86μm after compression for 3 and 8days separately. Most of myelin sheath disappeared after 12days of compression; the myelin sheath was partly restored at 45days after compression which the myelin sheath thickness of large axons in ventral root was 0.47μm. The medium category in dorsal root reduced to 0.59 or 0.72μm from 0.94μm, and 1.55μm after compression for 3days (p<0.05 to p<0.0001). The medium category axon in dorsal root is also 0.47μm after compression for 45days (p≤0.0001). The myelin sheath was almost totally restored at the 5months of compression; the myelin sheath thickness returned to normal and the axons were intact in structure under EM. The number of Iba1-positive microglia increased by 18.69, 40.44, and 18.49% after compression for 3, 8, and 12days separately in the ipsilateral dorsal horn and 21.26, 32.15, 22.87% in ventral horns, and the activation of microglia was also prominent in contralateral sides of the dorsal and ventral horn at 8days time point. The microglia cell reconverted to resting status after compression for 45days or 5months. The chronic spinal nerve root compression with silicone tube produces a recoverable damage to nerve root, which produces recoverable microglial activation in the spinal cord. These results demonstrated that the chronic spinal nerve root compression with silicone tube could mimic the pathological changes of lumbar spinal stenosis or lumbar disc herniation.

The Effect of Urinary Trypsin Inhibitor Against Neuropathic Pain in Rat Models

BackgroundNerve injury sometimes leads to chronic neuropathic pain associated with neuroinflammation in the nervous system. In the case of chronic neuropathic pain, the inflammatory and algesic mediators become predominant and result in pain hypersensitivity following nervous system damage. It is well known that urinary trypsin inhibitor (ulinastatin, UTI) has an anti-inflammatory activity. Recently, the neuroprotective action of UTI on the nervous system after ischemic injury has been reported. Thus, we evaluated the neuroprotective effect of ulinastatin in a rat model of neuropathic pain.MethodsNeuropathic pain was induced with L5 spinal nerve ligation (SNL) in male Sprague-Dawley rats weighing 100-120 g. The rats were divided into 3 groups, with n = 8 in each group. The rats in the control group (group 1) were administered normal saline and those in group 2 were administered UTI (50,000 U/kg) intravenously through the tail vein for 3 days from the day of SNL. Rats in group 3 were administered UTI (50,000 U/kg) intravenously from the 5th day after SNL. The paw withdrawal threshold was measured using the von Frey test for 3 days starting from the 5th day after SNL.ResultsThe paw withdrawal thresholds were significantly increased in the rats of group 2 compared to the other groups (P < 0.05).ConclusionsUlinastatin, which was administered for 3 days after SNL, increased the paw withdrawal threshold and it could have a neuroprotective effect in the rat model of neuropathic pain.

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors*♦

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

Up-regulation of spinal microglial Iba-1 expression persists after resolution of neuropathic pain hypersensitivity

Spinal microglial activation plays a major role in the development of neuropathic pain following peripheral nerve injury. We here provide evidence for an elevated expression of the microglial marker Iba-1 in the lumbar dorsal horn ipsilateral to L5 spinal nerve transection that persists for at least 14 weeks, a time at which mechanical hypersensitivity had fully resolved. Iba-1 expression was, however; significantly lower than at 4 weeks. We therefore conclude that microglia remain partly activated beyond the phase of pain hypersensitivity. Thus, the relation between microglial cells and neuropathic pain outcome is subject to change over time after nerve injury.