Spinal Cord Astrocytes Research Articles

Gene therapy for neuropathic pain induced by spared nerve injury with naked plasmid encoding hepatocyte growth factor.

Neuropathic pain (NP) is a refractory disease in the clinic with a tremendous impact on the quality of life of patients. Gene therapy is a potential strategy for the management of NP. In the present study, we examined the analgesic effect and mechanism of hepatocyte growth factor (HGF) in vitro and in vivo. We examined the proinflammatroy gene changes in lipopolysaccharide (LPS)-induced microglia BV2 cells with a quantitative real-time polymerase chain reaction of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Mechanical stimulation tests were performed five times at 5-min intervals to assess pain thresholds using Von Frey Hair in mice following spared nerve injury (SNI). The glial cell activation of spinal cord was examined by western blotting. Statistical significance was determined by a Tukey's test and a paired t-test. We found that recombinant human HGF protein suppressed LPS-induced BV2 cell activation in vitro, marked by the down-regulation of IL-1β, IL-6, TNF-α and iNOS expression, as well as decrease of nitric oxide production. Moreover, intrathecal injection of naked plasmid encoding HGF gene (pUDK-HGF) significantly attenuated SNI-induced pain behaviors in mice by direct inhibition of spinal cord microglia and astrocyte activation. The results of the present study indicate that pUDK-HGF can reduce cytotoxicity products released from activated glial cells, which may provide a promising therapeutic strategy for treating NP.

Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-\u03baB signaling in an IL-6-dependent manner

BackgroundSpinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R).MethodsThe post-natal day 1–2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett’s test.ResultsThe OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated—at least in part—via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes.ConclusionsHMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses.

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

Attenuation of mechanical pain hypersensitivity by treatment with Peptide5, a connexin-43 mimetic peptide, involves inhibition of NLRP3 inflammasome in nerve-injured mice

Connexin43 (Cx43) hemichannels in spinal cord astrocytes are implicated in the maintenance of neuropathic pain following peripheral nerve injury. Peptide5 is a Cx43 mimetic peptide that blocks hemichannels. In this study, we investigated the effects of spinal delivery of Peptide5 on mechanical pain hypersensitivity in two mouse models of neuropathic pain, peripheral nerve injury and chemotherapy-induced peripheral neuropathy (CIPN). We demonstrated that 10days following a chronic constriction injury (CCI) of the sciatic nerve, Cx43 expression, co-localised predominantly with astrocytes, was increased in the ipsilateral L3–L5 lumbar spinal cord. An intrathecal injection of Peptide5 into nerve-injured mice, on day 10 when pain was well-established, caused significant improvement in mechanical pain hypersensitivity 8h after injection. Peptide5 treatment resulted in significantly reduced Cx43, and microglial and astrocyte activity in the dorsal horn of the spinal cord, as compared to control saline-treated CCI mice. Further in vitro investigations on primary astrocyte cultures showed that 1h pre-treatment with Peptide5 significantly reduced adenosine triphosphate (ATP) release in response to extracellular calcium depletion. Since ATP is a known activator of the NOD-like receptor protein 3 (NLRP3) inflammasome complex, a key mediator of neuroinflammation, we examined the effects of Peptide5 treatment on NLRP3 inflammasome expression. We found that NLRP3, its adaptor apoptosis-associated spec-like protein (ASC) and caspase-1 protein were increased in the ipsilateral spinal cord of CCI mice and reduced to naïve levels following Peptide5 treatment. In the models of oxaliplatin- and paclitaxel-induced peripheral neuropathy, treatment with Peptide5 had no effect on mechanical pain hypersensitivity. Interestingly, in these CIPN models, although spinal Cx43 expression was significantly increased at day 13 following chemotherapy, NLRP3 expression was not altered. These results suggest that the analgesic effect of Peptide5 is specifically achieved by reducing NLRP3 expression. Together, our findings demonstrate that blocking Cx43 hemichannels with Peptide5 after nerve injury attenuates mechanical pain hypersensitivity by specifically targeting the NLRP3 inflammasome in the spinal cord.

Effects of gaboxadol on the pain and spinal astrocyte activation of rats with neuropathological pain

Objective To explore the effects of subcutaneous injection of γ aminobutyric acid(GABA) A receptor agonist gaboxadol for the pain threshold of rats with neuropathological pain(NP) and the activation of spinal astrocytes. Methods A NP model was established in rats through chronic constriction injury (CCI) of the sciatic nerve. Thirty-six male SD rats were randomized into three groups(n=12): a sham group (group S), a CCI group (group C) and a CCI+gaboxadol group (group G). Then, normal saline or 10 mg/kg gaboxadol was subcutaneously injected in group S, group C or group G respectively, once a day, from Days 10 to 14 after operation. 8 rats were randomly selected in each group, and paw withdrawal mechanical threshold(PWMT) was measured one day before operation and on day 3 and 10 after operation, and 0.5, 1, 2, 4, 8, 12 h after final injection. 4 rats were selected in each group, and they were killed sacrificed 2 h after final injection and the expression of astrocytes in the spinal cord was measured by immunofluorescence. Results Compared with group S, rats in group C started to show decreases in PWMT on day 3 after operation [(13.46±1.67) g vs(4.65±0.46) g], which was maintained until 12 h after injection (P<0.05). Compared with group S, the number of astrocyte cells in group C[(103±7) vs (402±20)] were significantly increased 2 h after injection (P<0.05). Compared with group C, group G showed remarkable increases in PWMT [(2.73±0.57) g vs (7.31±0.55) g] 0.5 h after injection, which was maintained until 8 h after injection (P<0.05). Compared with group C, the number of astrocyte cells in group G(251±20) were significantly decreased 2 h after injection(P<0.05). Conclusions Subcutaneous injection of gaboxadol relieves the mechanical pain in rats, which may be associated with inhibited activation of astrocytes in the spinal cord. Key words: Neuropathic pain; γaminobutyric acid A receptor; Astrocyte; Spinal cord

PPP1CC is associated with astrocyte and microglia proliferation after traumatic spinal cord injury in rats

Reactive astrogliosis and microgliosis after spinal cord injury (SCI) contribute to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte and microglia proliferation upon injury remain partially understood. Protein phosphatase 1, catalytic subunit, gamma isozyme (PPP1CC) participates in cell proliferation, differentiation and apoptosis. However, the expression and functions of PPP1CC following SCI are still unknown. In this study, an acute spinal cord contusion injury model in adult rats was established to investigate the potential role of PPP1CC during the pathological process of SCI. The palpable expression increase of PPP1CC after SCI was found by western blot and immunohistochemistry staining. Double immunofluorescence staining showed that PPP1CC expression mainly increased in astrocytes and microglia, as well as PPP1CC and proliferating cell nuclear antigen (PCNA) co-localized in astrocytes and microglia. Furthermore, PCNA expression also elevated after SCI in a similar manner as PPP1CC. In vitro, PPP1CC and PCNA expression in primary rat spinal cord astrocytes and microglia changed in a similar concentration- and time-dependent manner according to LPS treatment. In addition, PPP1CC knockdown in astrocytes and microglia resulted in the decrease of PCNA expression and the number of Brdu positive cells after LPS stimulation, showing that PPP1CC promoted astrocyte and microglia proliferation after inflammation. In a word, PPP1CC might be associated with astrocyte and microglia proliferation after SCI, implying that PPP1CC is a potential molecular target for the therapy of SCI.

Evaluation of the neonatal streptozotocin model of diabetes in rats: Evidence for a model of neuropathic pain.

The purpose of this study was to evaluate the participation of satellite glial cells (SGC), microglia and astrocytes in a model of streptozotocin-induced diabetes initiated in neonatal rats (nSTZ) and to determine the pharmacological profile for pain relief. nSTZ was used to induce experimental diabetes. Von Frey filaments were used to assess tactile allodynia. Drugs were given by systemic administration. Western blotting and immunohistochemistry were used to determine protein expression and cellular localization. nSTZ produced mild hyperglycemia, weight loss, glucose intolerance, and reduction of nerve conduction velocity of C fibers. Moreover, nSTZ enhanced activating transcription factor 3 (ATF3) immunoreactivity in dorsal root ganglia (DRG) and sciatic nerve of adult rats. ATF3 was found in SGC (GFAP+ cells) surrounding DRG at week 16. Late changes in ATF3 immunoreactivity in DRG correlated with up-regulation of ATF3 and GFAP protein expression. nSTZ increased GFAP and OX-42 immunoreactivity and percentage of hypertrophied and ameboid microglia in the spinal dorsal horn. These changes correlated with the presence of mechanical hypersensitivity (tactile allodynia). Administration of gabapentin (30-100mg/kg, po) and metformin (200mg/kg/day, po for 2 weeks) alleviated tactile allodynia, whereas morphine (1-3mg/kg, ip) had a modest effect. Results suggest that nSTZ leads to activation of SGC, microglia and astrocytes in DRG and spinal cord. Pharmacological profile in the nSTZ model resembles diabetic neuropathic pain in humans. Our findings support the conclusion that the nSTZ rat model has utility for the study of a long-lasting diabetic neuropathic pain.

Carbon Monoxide-Releasing Molecule-2 Inhibits Connexin 43-Hemichannel Activity in Spinal Cord Astrocytes to Attenuate Neuropathic Pain.

Carbon monoxide-releasing molecule (CORM-2) acts as a carbon monoxide (CO) deliverer in a more controlled manner without altering carboxyhemoglobin level and exerts potential function in inhibiting inflammation and/or acute nociception. However, the regulatory mechanism of CORM-2 on spinal nerve ligation (SNL)-induced neuropathic pain is not currently clear. Our study aims to investigate the role of CORM-2 in neuropathic pain and the underlying mechanism. We found that spinal cord astrocytes were dramatically activated on day 7 after SNL. L-α-aminoadipate (L-α-AA), an astroglial toxin, reversed SNL-induced astrocyte activation at sub-toxic dose. Intrathecal administration of CO donor CORM-2 induced antiallodynic and antihyperalgesic effects in neuropathic animals induced by SNL and suppressed SNL-induced spontaneous excitatory postsynaptic current (EPSC) frequency in lamina II neurons of spinal cord slices. CORM-2 administration markedly inhibited SNL-induced connexin 43 (Cx43) expression, hemichannel function, and gap junction function on spinal astrocyte membranes. Moreover, exogenous CORM-2 could attenuate HO-1 expression, while overexpressed heme oxygenase-1 (HO-1) increased intracellular CO production, attenuated Cx43 expression, hemichannel function, and gap junction function on spinal astrocyte membranes. Additionally, Cx43 over-expression markedly reduced CORM-2-induced mechanical threshold and thermal hyperalgesia and elevated CORM-2-induced spontaneous EPSC frequency. In conclusion, CORM-2 attenuated SNL-induced neuropathic pain via suppressing Cx43-hemichannel function, which may contribute to understanding of the pathology of neuropathic pain.

Influences of intraperitoneal injection of dexmedetomidine on activation of spinal glial cells in diabetic neuropathic pain rats

Objective Exploring the influences of intraperitoneal injection of dexmedetomidine(Dex) on activation of spinal glial cells and expression of inflammatory factors in rats with diabetic neuropathic pain(DNP). Methods Thirty two health male SD rats, weight 180-200 g, were divided into four groups (n=8) according the random number table method. The four groups included the control group(group C), diabetic neuropathic pain group(group DNP), group Dex and α2 receptor antagonist group(group YOH). Streptozotocin(STZ) was used to establish the DNP model in rats. Starting from 28 d after STZ injection, Dex group was given intraperitoneal injection of Dex(50 μg/kg) for consecutive 7 d, while group YOH was given intraperitoneal injection of Yohimbine(0.1 mg/kg) 30 min before intraperitoneal injection of Dex(50 μg/kg) for consecutive 7 d. Mechanical withdrawal threshold (MWT) was determined in each group of rats 29-35 d after STZ injection. Rats were sacrificed after MWT detection on 35 d. Immunofluorescence double-staining was used to detect activation of microglia and astrocytes in rat spinal cord of each group. ELISA was used to measure TNF-α and IL-1 expression in rat spinal cord of each group. Results Compared with group C, rat body weights were decreased while blood glucose values were increased in group DNP, group Dex and group YOH(P 0.05). Compared with group C, MWT at all time points decreased in group DNP and group YOH(P 0.05). Conclusions Dex can relieve hyperalgesia in rats with diabetic neuropathy pain. The underlying mechanism may be related to inhibition of microglia activation and reduction of the inflammatory reaction in spinal cord. Key words: Dexmedetomidine; Diabetic neuropathic pain; Microglia; Astrocyte; Inflammatory reaction

Potentiation of spinal glutamatergic response in the neuron-glia interactions underlies the intrathecal IL-1β-induced thermal hyperalgesia in rats.

We previously demonstrated that intrathecal IL-1β upregulated phosphorylation of p38 mitogen-activated protein kinase (P-p38 MAPK) and inducible nitric oxide synthase (iNOS) in microglia and astrocytes in spinal cord, increased nitric oxide (NO) release into cerebrospinal fluid, and induced thermal hyperalgesia in rats. This study investigated the role of spinal glutamatergic response in intrathecal IL-1β-induced nociception in rats. The pretreatment effects of MK-801 (5μg), minocycline (20μg), and SB203580 (5μg) on intrathecal IL-1β (100ng) in rats were measured by behavior, Western blotting, CSF analysis, and immunofluorescence studies. IL-1β increased phosphorylation of NR-1 (p-NR1) subunit of N-methyl-D-aspartate receptors in neurons and microglia, reduced glutamate transporters (GTs; glutamate/aspartate transporter by 60.9%, glutamate transporter-1 by 55.0%, excitatory amino acid carrier-1 by 39.8%; P<.05 for all), and increased glutamate (29%-133% increase from 1.5 to 12hours; P<.05) and NO (44%-101% increase from 4 to 12hours; P<.05) levels in cerebrospinal fluid. MK-801 significantly inhibited all the IL-1β-induced responses; however, minocycline and SB203580 blocked the IL-1β-downregulated GTs and elevated glutamate but not the upregulated p-NR1. The enhanced glutamatergic response and neuron-glia interaction potentiate the intrathecal IL-1β-activated P-p38/iNOS/NO signaling and thermal hyperalgesia.

Spinal CCL2 Promotes Central Sensitization, Long-Term Potentiation, and Inflammatory Pain via CCR2: Further Insights into Molecular, Synaptic, and Cellular Mechanisms.

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2+) neurons. CCL2 increased NMDA-induced currents in CCR2+/VGLUT2+ neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.

Sophorolipid Butyl Ester Diacetate Does Not Affect Macrophage Polarization but Enhances Astrocytic Glial Fibrillary Acidic Protein Expression at Micromolar Concentrations in Vitro.

Peritoneal macrophages (PMACs) and spinal cord astrocytes were exposed to varying concentrations of soluble sophorolipid butyl ester diacetate (SLBEDA) in vitro. Macrophages and astrocytes demonstrated no decrease in viability in response to SLBEDA. Studying pro- and anti-inflammatory genes, PMACs did not show a shift toward a pro-inflammatory phenotype. However, at higher concentrations (3 and 30 μM), astrocytes showed an increase in their expression of glial acidic fibrillary protein. This novel category of compounds poses low risk to PMAC and astrocyte viability; however, the effect on PMAC polarization and astrocyte reactivity requires more elucidation.

Edaravone protects against oxygen-glucose-serum deprivation/restoration-induced apoptosis in spinal cord astrocytes by inhibiting integrated stress response.

We previously found that oxygen-glucose-serum deprivation/restoration (OGSD/R) induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2-alpha (eIF2α) and activating transcription factor 4 (ATF4). We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone (0.1, 1, 10, 100 μM) treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated (p)-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

Continuous Intrathecal Infusion of Cannabinoid Receptor Agonists Attenuates Nerve Ligation-Induced Pain in Rats.

Cannabinoid receptors (CB1R/CB2R) are known to play important roles in pain transmission. In this study, we investigated the effects of continuous intrathecal infusion of CB1/2R agonists in the L5/6 spinal nerve ligation pain model. Under isoflurane anesthesia, rats received nerve ligation and intrathecal catheter connected to an infusion pump. After surgery, saline (1 μL/h), CB1/2R agonist WIN55,212-2, CB1R agonist ACEA, or CB2R agonist AM1241 (1 μmol/h) was given intrathecally for 7 days. The mechanical and thermal sensitivities of rat hindpaw were determined by von Frey hair and radiant heat tests. The expression of CB1/2R and protein levels of CB1/2R, Iba1, glial fibrillary acidic protein, and tumor necrosis factor α were examined by immunofluorescence study and Western blotting. On postligation day 7, rats that received WIN55,212-2, ACEA or AM1241 had significantly higher mean withdrawal thresholds (6.8, 8.4, and 10.2 g) and latencies (6.3, 7.3, and 9.1 seconds) than did saline-treated rats (1.7 g, 2.2 seconds). Cannabinoid receptors were expressed not only in IB4 (isolectin B4) and CGRP (calcitonin gene-related peptide) dorsal root ganglion neurons, their central terminals, and peripheral axons, but also in neurons, microglia, and astrocytes in spinal cord. Cannabinoid receptor agonists enhanced nerve ligation-induced up-regulation of cannabinoid receptor in spinal cord and dorsal root ganglion. Treatment with WIN55,212-2 or AM1241, but not ACEA, markedly reduced nerve ligation-induced up-regulation of Iba1, glial fibrillary acidic protein, and tumor necrosis factor α in spinal cord. Continuous intrathecal infusion of CB1/2R agonists elicits antinociception in the pain model. The mechanisms might involve their actions on neurons and glial cells. CB2R, but not CB1R, seems to play an important role in the regulation of nerve injury-induced neuroinflammation.

Spatiotemporal Patterns of RING1 Expression after Rat Spinal Cord Injury

Ring finger protein 1 (RING1) is a RING domain characterized protein belonging to the RING finger family. It is an E3 ubiquitin-protein ligase that mediated monoubiquitination of histone H2A and the core component of PRC1 complex, which is the repressive multiprotein complex of Polycomb group (PcG). Previous studies showed the important tumorigenic role of RING1 via promoting cell proliferation and the crucial function in maintaining transcriptional program stability during development. However, its mechanism for spinal cord injury (SCI) is still unknown. In our research, we established an acute SCI model in adult rats and studied the expression and function profiles of RING1. RING1 protein level detected by western blot peaked at day 3 after trauma and then decreased gradually. Immunohistochemistry showed the increase of RING1 expression displayed in the white matter more obviously than in the gray matter. Furthermore, increased co-expression of RING1 and GFAP confirmed activated astrocytes in injured spinal cord via double immunofluorescence staining. Meanwhile, we also found the co-localization of PCNA, a famous marker of proliferative cells, with RING1 and GFAP, which indicated RING1 might play a role in astrocyte proliferation after SCI. In vitro studies, RING1 protein level in C6 cells increased after LPS challenge and RING1 was required for astrocyte proliferation and activation induced by LPS. In summary, we took a new insight into the function of RING1 in the cellular and molecular mechanism underlying the pathophysiology of SCI.

Changes in Neuroglial Activity in Multiple Spinal Segments after Caudal Epidural Pulsed Radiofrequency in a Rat Model of Lumbar Disc Herniation

Background: Herniated lumbar discs can induce sciatica by mechanical compression and/ or chemical irritation. It was recently reported that neuroglial cellular activity after pulsed radiofrequency (PRF) application to a single dorsal root ganglion (DRG) attenuated neuroglial activity at the corresponding spinal dorsal horn. Recently, caudal epidural PRF has been used to manage neuropathic pain, but evidence of molecular changes after the administration of caudal epidural PRF to attenuate neuropathic pain is lacking, and it has not been determined whether caudal epidural PRF affects neuroglial activity at different spinal levels. Objectives: Using immunohistochemical methods in a rat model of lumbar disc herniation, the authors investigated the effects of caudal epidural PRF administration on pain-related behavior, on the activations of microglia and astrocytes in spinal cord, and on the expressions of calcitonin gene-related peptide (CGRP) and Transient receptor potential vanilloid 1(TRPV1) in the DRG at the L3, L4, L5, L6, and S1 levels. Study Design: Controlled animal trial. Setting: University hospital laboratory. Methods: Forty-five Sprague-Dawley rats were randomly assigned to a sham-operated group (n = 10) or a nucleus pulposus (NP)-exposed group (n = 35). Rats in the NP-exposed group were further subdivided into a NP-exposed with sham stimulation group (the NP-nonPRF group; n = 13) or a NP exposed with caudal epidural PRF stimulation group (the NP-PRF group; n = 22). Pulsed radiofrequency was administered on postoperative day 10 (POD 10) by placing an electrode in the caudal epidural space through the sacral hiatus and administering 5 Hz of PRF current for 600 seconds (maximum tip temperature 42°C). Rats were tested for mechanical allodynia on POD 10 and on days 7 and 14 after caudal epidural PRF administration (post-PRF). At 14 days post-PRF, sections of the spinal cord from L3, L4, L5, L6, and S1 were immunostained for ionized calciumbinding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP), and DRGs from the same levels were immunostained for CGRP and TRPV1. Results: Mechanical withdrawal thresholds increased at 7 days post-PRF (P = 0.04), and the immunohistochemical expression of Iba1 in the L5 spinal dorsal horn and of CGRP in the L5 DRG were quantitatively reduced (P &lt; 0.001) at 14 days post-PRF. Furthermore, the upregulations of Iba1 at L3, L4, L6, and S1 dorsal horns and CGRP at L6 DRG were also attenuated by caudal epidural PRF (P &lt; 0.001). Limitation: We examined molecular changes only in ipsilateral lumbar regions and at 14 days post-PRF. Conclusion: Caudal epidural PRF reduced mechanical allodynia and downregulated microglia activity and CGRP expression at the lumbar disc herniated level and in adjacent lumbar spinal levels in a rat model of lumbar disc herniation. Key words: Caudal, pulsed radiofrequency, multisegmental, lumbar disc herniation, microglia, calcitonin gene-related peptide

NFAT5 protects astrocytes against oxygen-glucose-serum deprivation/restoration damage via the SIRT1/Nrf2 pathway.

Nuclear factor of activated T cells (NFAT) is a multifunctional cytokine family. NFAT5 was recently reported to be involved in many neuronal functions, but its specific function remains unclear. In this study, our aim is to investigate whether NFAT5 overexpression can protect astrocytes against oxygen-glucose-serum deprivation/restoration (OGSD/R) damage. In vivo, rats were subjected to ischemia-reperfusion injury, resulting in increased water content, infarct volume, and expression of NFAT5 protein in rat spinal cord. After primary culture for spinal cord astrocytes, the in vitro OGSD/R model was established. The results of the CCK8 assay and flow cytometry showed that, in the OGSD/R group, astrocyte cell viability was downregulated, but astrocyte apoptosis increased. Caspase 3 activity increased as well. Levels of NFAT5, as detected by real-time quantitative PCR and western blot, decreased under OGSD/R, as did SIRT1. Commercial kits for activity assays were used to show that OGSD/R inhibited SIRT1 activation but accelerated SOD activation after OGSD/R. Next, pcDNA-NFAT5 or NFAT5 siRNA was transfected into astrocytes. Overexpression of NFAT5 not only promoted the survival of the astrocytes and SIRT1 activation under OGSD/R but also inhibited cell apoptosis and SOD activation. Moreover, overexpression of NFAT5 apparently diminished histone acetylation and promoted the nuclear transport of Nrf2. Our results show that NFAT5 protects spinal astrocytes in a manner that depends on activation of the SIRT1/Nrf2 pathway. These findings present a novel potential molecular mechanism for NFAT5 therapy in the context of spinal cord injury.

Onset of Spinal Cord Astrocyte Precursor Emigration from the Ventricular Zone Involves the Zeb1 Transcription Factor

During spinal cord development, astrocyte precursors arise from neuroepithelial progenitors, delaminate from the ventricular zone, and migrate toward their final locations where they differentiate. Although the mechanisms underlying their early specification and late differentiation are being deciphered, less is known about the temporal control of their migration. Here, we show that the epithelial-mesenchymal transition regulator Zeb1 is expressed in glial precursors and report that loss of Zeb1 function specifically delays the onset of astrocyte precursor delamination from the ventricular zone, correlating with transient deregulation of the adhesion protein Cadherin-1. Consequently, astrocyte precursor invasion into the Zeb1-/- mutant white matter is delayed, and induction of their differentiation is postponed. These findings illustrate how fine regulation of adhesive properties influences the onset of neural precursor migration and further support the notion that duration of exposure of migrating astrocyte precursors to environmental cues and/or their correct positioning influence the timing of their differentiation.

Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress.

Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.