Lumbar Enlargement Of Spinal Cord Research Articles

Electroacupuncture at "Jiaji" (EX-B2) induced down-regulation of OX-42 and P2X4 in lumbar spinal cord contributes to its analgesic after-effect in rats with chronic constriction injury

To investigate the effect of electroacupuncture (EA) at "Jiaji" (EX-B2) at different time points on the expression of OX-42 (a monoclonal antibody with specific expression of complement receptor-3 in spinal microglial cells) and purinergic receptor P2X4 (P2X4) in rats with chronic constriction injury (CCI) of the sciatic nerve, as well as the possible after-effect mechanism of EA analgesia in neuropathic pain. Sprague-Dawley rats were randomly divided into blank group, model group, immediately after EA group, 0.5-hour after EA group, 1-hour after EA group, 2-hour after EA group, 4-hour after EA group, 12-hour after EA group, and 24-hour after EA group, with 6 rats in each group. The rats in the model group and the EA groups were used to establish a model of CCI-induced neuropathic pain, and those in the immediately after EA group and the 0.5, 1, 2, 4, 12 and 24 hours after EA groups were treated with EA at bilateral L3 and L5 "Jiaji" points for 20 min after 7 d of modeling. Samples were collected immediately and at 0.5, 1, 2, 4, 12, and 24 hours after EA, and for the rats in the blank group and the model group, samples were collected after fixation the rats for 20 min. Heat pain threshold was observed before and after intervention, and immunohistochemistry was used to measure the protein expression of OX-42 and P2X4 in the spinal cord lumbar enlargement. After 7 days of modeling (before intervention), compared with the blank group, the heat pain threshold had a significant reduction in the model group and the EA groups (P<0.01). Compared with the model group after intervention, the immediately after EA group and the 0.5, 1 and 2 hours after EA groups had a significant increase in heat pain threshold (P<0.05). Compared with the model group, immediately after EA, the 0.5, 1 and 2 hours after EA groups had a significant reduction in the protein expression of OX-42 (P<0.01), and immediately after EA, the 0.5 and 1 hour after EA groups had a significant reduction in the protein expression of P2X4 (P<0.01). EA at "Jiaji" points can significantly increase heat pain threshold and down-regulate the protein expression of OX-42 and P2X4 in the spinal cord of CCI rats. The analgesic effect can last for 2 h.

Effects of intrathecal injection Roscovitine on the pain behavior and the expression of ERK1/2 and PPARγ protein in spinal cord in rats with neuropathic pain

Objective To investigate the effects of intrathecal injection of Roscovitine on the pain behavior and the expression of pERK1/2 and pPPARγ receptor in spinal cord in rats with neuropathic pain. Methods Fifty-two male adult Sprague-Dawley rats of 220~250 g were randomly divided into 4 groups: Sham+ DMSO group (dimethyl sulfoxide, S+ D group), Sham+ Roscovitine (S+ R) group, CCI+ DMSO group (I+ D), CCI+ Roscovitine (I+ R) group. The corresponding drugs were administered by intrathecal injection from the seventh day after CCI once a day for 14 days. The right hind paw mechanical threshold value(PMWT) was measured by Von Frey filament and paw thermal withdrawal latency(PWTL) was measured by Hargreaves methods before 1 day and 3 d, 7 d, 10 d , 14 d after surgery. The spinal cord lumbar enlargement was taken at 14 d after surgery, and Cdk5, p35, phosphorylated ERK protein(pERK), total ERK protein(ERK), phosphorylated PPARγ protein (pPPARγ) and total PPARγ protein (PPARγ) were detecteded by Western blot. Results Roscovitine was administered by intrathecal injection alleviated mechanical allodynia and thermal hyperalgesia of CCI rats.Compared with I+ D group, PWMT in I+ R group at 7 d, 10 d, 14 d after administration((4.65±0.08)g, ( 6.47±0.12)g, ( 7.90±0.19)g, ) vs ((3.71±0.06)g, (2.45±0.17)g, ( 2.31±0.15)g)(Fgroup=505.71, P<0.05, Ftime×group=15.33, P<0.05) , PWTL((9.22±0.33)s, (13.52±0.43)s, (12.63±0.88)s) vs ((8.02±0.20)s, (5.90±0.28)s, (5.40±0.38)s) (Fgroup=355, P<0.05, Ftime×group=8.589, P<0.05) were significantly increased. Compared with I+ D group(p35(0.58±0.02), pERK (1.12±0.13), pPPARγ (0.77±0.16)), p35 (0.46±0.04, F=11.06, P<0.05) and pERK(0.79±0.11, F=22.91, P<0.05) in I+ R group, were significantly dereased, and the expression of pPPARγ(0.99±0.13, F=17.62, P<0.05))was significantly increased. Conclusion Intrathecal injection Roscovitine can alleviate both mechanical allodynia and thermal hyperalgesia in rats with neuropathic pain, and its mechanisms may be related to the inhibition of Cdk5/p35 and ERK activity, enhancement of PPARγ activity in the spinal dorsal horn. Key words: Neuropathic pain; Cyclin-dependent protein kinases 5; Extracellular signal-regulated kinase1/2; Peroxisome proliferator-activated receptor gamma; Spinal dorsal horn

Intrathecal injection of AG-490 reduces bone-cancer-induced spinal cord astrocyte reaction and thermal hyperalgesia in a mouse model

To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP). Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490. Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05). Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.

Involvement of endoplasmic reticulum stress in formalin-induced pain is attenuated by 4-phenylbutyric acid.

BackgroundEndoplasmic reticulum (ER) stress is involved in many neurological and inflammatory responses. Peripheral inflammatory responses can induce central sensitization and trigger inflammatory pain. However, there is little research on the relationship between ER stress and inflammatory pain. In this study, we examined whether the ER stress response is involved in peripheral inflammatory pain using a formalin-induced rat pain model.MethodsRats were divided into the following five groups: control, formalin, formalin + vehicle, formalin + 4-phenylbutyric acid (4-PBA) (40 mg/kg) and formalin + 4-PBA (100 mg/kg). Formalin-induced pain was assessed behaviorally by recording licking activity. The expression levels of immunoglobulin-binding protein (BIP), activating transcription factor-6 (ATF6), phosphorylated inositol-requiring enzyme-1 (p-IRE1), phosphorylated protein kinase RNA-like ER kinase (p-PERK) and c-fos were quantitatively assessed by Western blot, and the distribution of BIP, ATF6 and c-fos in the lumbar enlargement of spinal cord were identified by immunohistochemistry in spinal dorsal horn slices. In addition, the concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the spinal cord were tested by biochemical measurement and enzyme-linked immunosorbent assay (ELISA), respectively.ResultsIntraperitoneal injection of 4-PBA at the dose of 100 mg/kg before formalin injection significantly decreased nociceptive behavior in the second phase compared with control, formalin, formalin + vehicle and formalin + 4-PBA (40 mg/kg) (P<0.05). Western blot showed that formalin injection significantly upregulated the expression of BIP, ATF6, p-PERK and c-fos in the spinal cord. This upregulation was reduced by peritoneal injection of 4-PBA (P<0.05), while expression of p-IRE1 was not altered by formalin treatment. Immunohistochemistry revealed markedly increased staining density for BIP, ATF6 and c-fos in the superficial spinal dorsal horn after formalin injection. This was significantly decreased by administration of 4-PBA (P<0.05). Compared with the formalin + vehicle group, 4-PBA inhibited the release of NO and PGE2 in the spinal cord (P<0.05).ConclusionThese results suggest that ER stress is involved in formalin-induced inflammatory pain and that inhibition of ER stress may attenuate central sensitization induced by peripheral inflammatory stimulation.

Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats

Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-stimulating factor (G-CSF) could attenuate NeP in several SCI patients. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated.The purpose of the present study was to elucidate the therapeutic effect and mechanism of action of granulocyte colony-stimulating factor for SCI-induced NeP.T9 level contusive SCI was introduced to adult male Sprague Dawley rats. Three weeks after injury, rats received intraperitoneal recombinant human G-CSF (15.0μg/kg) for 5days. Mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry and western blot analysis were performed in spinal cord lumbar enlargement samples.Testing with von Frey filaments showed significant increase in the paw withdrawal threshold in the G-CSF group compared with the vehicle group 4weeks, 5weeks, 6weeks and 7weeks after injury. Immunohistochemistry for CD11b (clone OX-42) revealed that the number of OX-42-positive activated microglia was significantly smaller in the G-CSF group than that in the vehicle rats. Western blot analysis indicated that phosphorylated-p38 mitogen-activated protein kinase (p38MAPK) and interleukin-1β expression in spinal cord lumbar enlargement were attenuated in the G-CSF-treated rats compared with that in the vehicle-treated rats.The present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced NeP, possibly through the inhibition of microglial activation and the suppression of p38MAPK phosphorylation and the upregulation of interleukin-1β.

Basic Research on Neuronal Activation Induced by Intradiscal Pain Stimulation into Degenerative ?Intervertebral Disc Using Capsaicin

Introduction Previously, it was reported that pain perception is related to not only DRG but also to the reaction of the central nervous system; however, the reaction of the dominant nervous system to pain stimulus in the intervertebral discs is unknown. The purpose of this research was to verify the mechanism of reaction of the nervous system, which controls the intervertebral discs, using chemical pain stimulus. Materials and Methods We used 6-week-old Sprague Dawley rats, and injected 20 µL capsaicin solution, which contains 2% Fluoro-Gold (FG), into the L5/6 intervertebral disc. FG is an antidromic neuronal tracer. Seven days after injection, we prepared perfusion fixation of the rats and extracted DRGs from L1 to L6 along with the lumbar enlargement of spinal cord. At the DRG, we detected CGRP, which is a nociceptive pain-related protein, by immunostaining, and measured the distribution of the expression. At the lumbar enlargement of the spinal cord, we detected Iba1, which is a marker for pain stimulus-activated micro glia, and compared Iba 1 expression in the capsaicin group and vehicle group, in which we injected only 20 µL saline solution (each is n = 5). Results The rate of distribution of CGRP positive cells in the FG positive rats was highest at the L2 DRG, and the rate was higher in the capsaicin group (54.9%) than in vehicle group (27.1%), with a statistically significant difference ( p &lt; 0.05). The number of Iba1 positive micro glia cells per 1.4 × 10 mm2 was also larger in the capsaicin group (31.7 cells) than in the vehicle group (14.8 cells), with a statically significant difference ( p &lt; 0.05) Conclusion The results of this experiment suggested that the chemical stimulus to the L5/6 intervertebral disc resulted in an elevated CGRP expression that caused pain around the L2 DRG area, and the mechanism of pain perception is strongly related to the activation of microglia at the posterior horn of the spinal cord. Disclosure of Interest None declared

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 microg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 +/- 0.02 for CNTF-treated rats vs 0.53 +/- 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18% (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87%, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91% over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

RSEP1 is a novel gene with functional involvement in neuropathic pain behaviour

Neuropathic pain from nerve injury by trauma, disease or surgery often causes prolonged suffering. To explore the molecular mechanisms that underlie neuropathic pain, we used mRNA from the L4--5 segments of the lumbar spinal cord of rats with chronic constriction injury (CCI)-induced neuropathic pain, and differentially screened a cDNA library from the rat brain. A novel gene, termed RSEP1 (Rat Spinal cord Expression Protein 1), was identified. Northern blots revealed that RSEP1 was expressed mainly in the central nervous system including the cerebral cortex, hippocampus, brainstem and spinal cord, as well as in the kidney and ovary. In situ hybridization showed a high level of RSEP1 expression in the CA1, CA3 and dentate gyrus regions of the hippocampus and the small sensory neurons in the dorsal horn, as well as the large neurons in the ventral horn of the spinal cord. Intrathecal injection of RSEP1 antisense oligonucleotide into the spinal cord lumbar enlargement attenuated neuropathic pain behaviours in CCI rats, suggesting a functional involvement of RSEP1 in neuropathic pain.

Insulin-like growth factor 1-induced alterations in lumbospinal monoamine dynamics.

A wealth of evidence indicates that insulin-like growth factor (IGF-1) is involved in neurotransmitter release, synaptic plasticity, morphogenesis and regulation of gene expression. RT-PCR and immunocytochemical-based techniques revealed that IGF-1 and its receptor are highly expressed by different neuronal elements of the spinal cord lumbar enlargement. Accordingly, the present study intended to examine lumbospinal monoamine dynamics in the context of the neurotrophic factor IGF-1. Spinal release of norepinephrine (NE) represented by 3-methoxy-4-hydroxyphenylglycol (MHPG)/NE ratio was enhanced by IGF-1. This action of IGF-1 was associated with a similar increase in both tyrosine hydroxylase (TH) immunoreactivity and the level of its mRNA. In contrast, neuronal contents of serotonin and its metabolite 5-hydroxyindoleacetic acid in IGF-1-treated animals remained at control level. Genistein, a tyrosine kinase inhibitor, which by itself had no effect on NE metabolism, abolished the induction effect of IGF-1 on TH and MHPG/NE ratio. Our results suggest that IGF-1 augments the lumbospinal noradrenergic system by an intracellular mechanism involving a receptor-linked tyrosine kinase. The physiological consequences of the IGF-1 actions are discussed in terms of neuroprotection and nociception.