Rat Dorsal Horn Research Articles

Effect of Reboxetine Treatment on BDNF, Synaptophysin, and PSD-95 Levels in the Spinal Dorsal Horn of Rats with Diabetic Neuropathy

Objective: It is known that neuropathic pain is accompanied by alterations in the levels of neurotrophic factors and synaptic proteins in the microenvironment of the spinal dorsal horn. Such changes contribute to hyperalgesia and allodynia processes; thus, analgesic drugs can exert their pharmacological effects by affecting the expressions, levels, or functions of these endogenous substances. In this study, based on the knowledge that reboxetine (a selective noradrenaline reuptake inhibitor) has the potential for antihyperalgesic efficacy in diabetic neuropathy, we aimed to examine the probable effects of this drug on diabetes-induced changes in brain-derived neurotrophic factor (BDNF), synaptophysin (the pre-synaptic marker of synaptic integration), and postsynaptic density-95 (PSD-95) (the postsynaptic marker of synaptic integration) levels in the superficial laminae of the dorsal horn. 
 Methods: Experimental diabetes was induced by a single-dose injection of streptozotocin (STZ) (50 mg/kg) in rats. After four week-long induction period of painful diabetic neuropathy, rats were treated orally with 8 mg/kg reboxetine for two weeks. Hyperalgesia responses were evaluated by using the Randall–Selitto and Hargreave's tests. Following the pain tests, immunohistochemical studies were performed.
 Results: Two weeks of reboxetine administration increased the reduced paw withdrawal thresholds and shortened the paw withdrawal latencies of diabetic rats in neuropathic pain tests, indicating the antihyperalgesic efficacy of this drug. Moreover, augmented BDNF and synaptophysin levels in diabetic rats reversed by reboxetine treatment. However, there was no alteration in the densities of PSD-95, in both STZ-diabetic and reboxetine-treated STZ-diabetic rats. 
 Conclusion: The obtained results suggested that inhibition of central sensitization and modulation of spinal plasticity seem to be pharmacological mechanisms underlying reboxetine's antihyperalgesic effects on diabetic rats. However, further studies are still needed to clarify the exact mechanism of action.

Htr2b Promotes M1 Microglia Polarization and Neuroinflammation after Spinal Cord Injury via Inhibition of Neuregulin-1/ErbB Signaling.

The secondary injury of spinal cord injury (SCI) is dominated by neuroinflammation, which was caused by microglia M1 polarization. This study aimed to investigate the role and mechanism of Htr2b on neuroinflammation of SCI. The BV2 and HMC3 microglia were treated with lipopolysaccharide (LPS) or interferon (IFN)-γ to simulate in vitro models of SCI. Sprague-Dawley rats were subjected to the T10 laminectomy to induce animal model of SCI. Htr2b mRNA expression was measured by qRT-PCR. The expression of Htr2b and Iba-1 was detected by western blot and immunofluorescence. The expression of inflammatory cytokines in vitro and in vivo was also measured. Kyoto Encyclopedia of Genes and Genomes (KEGG) was employed to analyze Htr2b-regulated signaling pathways. Rat behavior was analyzed by the Basso, Beattie, and Bresnahan (BBB) and inclined plane test. Rat dorsal horn tissues were stained by hematoxylin-eosin (H&E) and Nissl to measure neuron loss. Htr2b was highly expressed in LPS- and IFN-γ-treated microglia and SCI rats. SCI modeling promoted M1 microglia polarization and increased levels of inflammatory cytokines. Inhibition of Htr2b by Htr2b shRNA or RS-127445 reduced the expression of Htr2b, Iba-1, and iNOS and suppressed cytokine levels. KEGG showed that Htr2b inhibited ErbB signaling pathway. Inhibition of Htr2b increased protein expression of neuregulin-1 (Nrg-1) and p-ErbB4. Inhibition of the ErbB signaling pathway markedly reversed the effect of Htr2b shRNA on M1 microglia polarization and inflammatory cytokines. Htr2b promotes M1 microglia polarization and neuroinflammation after SCI by inhibiting Nrg-1/ErbB signaling pathway.

NETO2-GluK2 interaction contributes to postoperative pain hypersensitivity through inducing PKCγ activation and synaptic incorporation of AMPA receptor GluR1 subunits in rat dorsal horn

Important roles in the initiation and maintenance of postoperative pain are played by the functional control of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the rat dorsal horn (DH). However, the mechanisms underpinning the cross-talk between spinal KA and AMPA receptors in postoperative pain are poorly understood. We hypothesized that after the rat’s plantar incision, the synaptic incorporation of AMPA receptor GluR1 subunits in the DH ipsilateral to the incision would increase due to the interaction between GluK2 and neuropilin tolloid-like 2 (NETO2). Our findings showed that incision stimuli caused severe pain responses, as measured by cumulative pain scores. GluK2-NETO2 but not GluK2-NETO1interaction was upregulated in ipsilateral dorsal horn neurons (DHNs) at 6 h post-incision. At 6 h post-incision, NETO2 small interfering ribonucleic acid (siRNA) intrathecal pretreatment increased mechanical withdrawal thresholds to von Freys and decreased ipsilateral paw cumulative pain scores. Further, PKCγactivation and synaptic abundance of GluK2 and GluR1 subunits in the ipsilateral DH were decreased by intrathecal pretreatment with NETO2 siRNA at 6 h post-incision. In conclusion, our findings imply that GluK2-NETO2 interaction could trigger PKCγactivation and the synaptic incorporation of AMPA receptor GluR1 subunits in rat DHs, which in turn led to the enhanced pain hypersensitivity after surgery. It sheds light on the interplay between KA and AMPA receptors in DHNs, which is thought to contribute to postoperative pain.

Effect of a Single Dose of Oxaliplatin on the Induction of Peripheral Neuropathy in a Rat Model: An in Vivo Electrophysiological Study.

The anticancer drug oxaliplatin is associated with peripheral neuropathy as a side effect accompanied by mechanical and cold allodynia. Although the superficial layer of the spinal cord dorsal horn is known to receive information primarily from peripheral pain nerves, to our knowledge, no in vivo electrophysiological analyses have been conducted to determine whether oxaliplatin administration increases the excitability of superficial layer neurons. Therefore, in vivo extracellular recordings were performed to measure action potentials in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose (6 mg/kg) of oxaliplatin. Action potentials were produced by mechanical stimulation with von Frey filaments to the hindlimb receptive fields. The results revealed that the firing frequency of action potentials increased relative to the intensity of mechanical stimulation, and that both deep and superficial layer neurons in the spinal cord dorsal horn increased significantly in oxaliplatin-treated compared with vehicle-treated rats, especially in the superficial layer. Several superficial layer neurons showed spontaneous firing that was not seen in vehicle-treated rats. In addition, a clear increase was seen in the firing frequency of neurons in the superficial layer of oxaliplatin-treated rats in response to a cold stimulus (here, the addition of acetone to the hindlimb receptive field). This study suggests that the superficial spinal cord dorsal horn strongly reflects the pain pathophysiology in peripheral neuropathy induced by oxaliplatin administration, and that the superficial layer neurons are useful for in vivo electrophysiological analysis using this pathological model.

Upregulation of lysine-specific demethylase 6B aggravatesinflammatory pain through H3K27me3 demethylation-dependent production of TNF-α in the dorsal root ganglia and spinal dorsal horn in rats.

Lysine-specific demethylase 6B (KDM6B) serves as a key mediator of gene transcription. It regulates expression of proinflammatory cytokines and chemokines in variety of diseases. Herein, the role and the underlying mechanisms of KDM6B in inflammatory pain were studied. The inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in rats. Immunofluorescence, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR were performed to investigate the underlying mechanisms. CFA injection led to upregulation of KDM6B and decrease in the level of H3K27me3 in the dorsal root ganglia (DRG) and spinal dorsal horn. The mechanical allodynia and thermal hyperalgesia following CFA were alleviated by the treatment of intrathecal injection of GSK-J4, and by microinjection of AAV-EGFP-KDM6B shRNA in the sciatic nerve or in lumbar 5 dorsal horn. The increased production of tumor necrosis factor-α (TNF-α) following CFA in the DRGs and dorsal horn was inhibited by these treatments. ChIP-PCR showed that CFA-induced increased binding of nuclear factor κB with TNF-α promoter was repressed by the treatment of microinjection of AAV-EGFP-KDM6B shRNA. These results suggest that upregulated KDM6B via facilitating TNF-α expression in the DRG and spinal dorsal horn aggravates inflammatory pain.

Exploring the effect of Tuina on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation based on NR2B/PSD-95 pathway

ObjectiveTo investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation.MethodsFifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention.ResultsCompared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn.ConclusionTuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.

Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain.

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-β1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-β1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.

Analgesic effect and mechanism of electroacupuncture on SNI rats based on microglia-BDNF-neuron signal

To observe the effect of electroacupuncture (EA) at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the activation of glial cells, the expression of brain-derived neurotrophic factor (BDNF), excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury (SNI) of sciatic nerve, and to explore the analgesic mechanism of EA on SNI. PartⅠ: Sixty SD rats were randomly divided into a sham operation group, a model group, an EA group and a sham EA group, 15 rats in each group. Except the sham operation group, the SNI rat model was established in the remaining groups. The rats in the sham operation group were only treated with incision without damaging the nerve. The rats in the EA group were treated with EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side, continuous wave, frequency of 2 Hz, current intensity of 1 mA, 30 minutes each time, once a day, for 14 days. The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side; the manipulation, EA parameters and treatment course were the same as the EA group. The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3, 7 and 14 days after modeling. Fourteen days after modeling, Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1 (Iba-1), glial fibrillary acidic protein (GFAP), BDNF and c-Fos in the spinal dorsal horn; the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining; immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn; Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons. PartⅡ: Thirty SD rats were randomly divided into a control group, a BDNF group and a BDNF+anti-TrkB group, 10 rats in each group. The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide (DMSO); the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO; the BDNF+anti-TrkB group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B (TrkB) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO. The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1, 3 and 7 days after injection. Seven days after injection, the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining. PartⅠ: Compared with the sham operation group, 3, 7 and 14 days after modeling, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased (P<0.05); 7 and 14 days after modeling, compared with the model group, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased (P<0.05). The expressions of Iba-1, GFAP, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group (P<0.05); the expressions of Iba-1, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group (P<0.05). PartⅡ: 3 and 7 days after intrathecal injection, the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group (P<0.05); the threshold of mechanical foot retraction reflex in the BDNF+anti-TrkB group was higher than that in the BDNF group (P<0.05). The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group (P<0.05); the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-TrkB group was lower than that in the BDNF group (P<0.05). The analgesic effect of EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord, thereby blocking the signal of microglia-BDNF-neuron, and finally reducing the excitability of neurons.

Upregulation of Hevin contributes to postoperative pain hypersensitivity by inducing neurexin1β/neuroligin1-mediated synaptic targeting of GluA1-containing AMPA receptors in rat dorsal horn

The astrocytes-secreted active molecule, Hevin considerably contributes in the transsynaptic bridge of neurexin1β/neuligin1 in excitatory synapse. Previous studies have demonstrated that activity-dependent synaptic recruitment of spinal neuroligin1 and GluA1-containing AMPA receptors (AMPARs) is involved in incisional, inflammatory and neuropathic pain. Here, we hypothesized that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1-mediated synaptic targeting of GluA1-containing AMPARs in spinal dorsal horns (DH). Our results showed that plantar incision induced significant postoperative pain behavior, which was described by the cumulative pain scores. At 1 d and 3 d post-incision, Hevin expression was considerably elevated in ipsilateral DHs, although it recovered to baseline value at 5 d following the incision. At 1 d post plantar incision, the neurexin1β/neuroligin1 interactions significantly increased in ipsilateral DHs in rats subjected to incision when compared with those in control rats. Intrathecal pretreatments of small interference RNA targeting Hevin substantially suppressed postoperative pain hypersensitivity and reduced the neurexin1β/neurolgin1 interaction as well as the synaptic targeting of GluA1 in ipsilateral spinal DHs. These data suggest that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1 interaction and subsequent synaptic targeting of GluA1-containing AMPARs in ipsilateral spinal DHs. It provides new insights into the role of Hevin-mediated trans-synaptic regulation in postoperative pain hypersensitivity, which would help develop a novel therapeutic strategy.

Phytohormone abscisic acid ameliorates neuropathic pain via regulating LANCL2 protein abundance and glial activation at the spinal cord.

Spinal neuroinflammation plays a critical role in the genesis of neuropathic pain. Accumulating data suggest that abscisic acid (ABA), a phytohormone, regulates inflammatory processes in mammals. In this study, we found that reduction of the LANCL2 receptor protein but not the agonist ABA in the spinal cord is associated with the genesis of neuropathic pain. Systemic or intrathecal administration of ABA ameliorates the development and pre-existence of mechanical allodynia and heat hyperalgesia in animals with partial sciatic nerve ligation (pSNL). LANCL2 is expressed only in microglia in the spinal dorsal horn. Pre-emptive treatment with ABA attenuates activation of microglia and astrocytes, ERK activity, and TNFα protein abundance in the dorsal horn in rats with pSNL. These are accompanied by restoration of spinal LANCL2 protein abundance. Spinal knockdown of LANCL2 gene with siRNA recapitulates the behavioral and spinal molecular changes induced by pSNL. Activation of spinal toll-like receptor 4 (TLR4) with lipopolysaccharide leads to activation of microglia, and over production of TNFα, which are concurrently accompanied by suppression of protein levels of LANCL2 and peroxisome proliferator activated-receptor γ. These changes are ameliorated when ABA is added with LPS. The anti-inflammatory effects induced by ABA do not requires Gi protein activity. Our study reveals that the ABA/LANCL2 system is a powerful endogenous system regulating spinal neuroinflammation and nociceptive processing, suggesting the potential utility of ABA as the management of neuropathic pain.

Effect of wheat-grain moxibustion on the expression of Beclin-1/GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy

To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy (CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR. A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time) on the basis of the model group; the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment; after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry; the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method; the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope. After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased (P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased (P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups. Wheat-grain moxibustion at "Dazhui" (GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.

Effect of electroacupuncture and pretreatment of electroacupuncture on pain sensitization and expression of P2X7R in spinal dorsal horn in rats with diabetic neuropathic pain

To observe the occurrence time of neuralgia and the expression of purinergic ligand-gated ion channel 7 receptor (P2X7R) in the dorsal horn of the spinal cord after intraperitoneal injection of streptozotocin (STZ) in diabetic rats, and to explore the effect of electroacupuncture (EA) and pretreatment of EA on the heat pain threshold and expression of P2X7R in the spinal dorsal horn in rats with diabetic neuropathic pain (DNP), and to explore the possible mechanism of EA for DNP. PartⅠ: Thirty male SD rats were randomly selected from 64 male SD rats as the control group; the remaining rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model, and 30 rats were successfully modeled as the model group. The control group and the model group were divided into three subgroups respectively at 7, 14 and 21 days, with 10 rats in each subgroup. Body mass, fasting blood glucose (FBG) and thermal pain threshold were recorded at 7, 14 and 21 days after injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot. PartⅡ: Eight SD rats were randomly selected from 35 male SD rats as the blank group, and the remaining 27 rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model. The 24 rats with successful diabetes model were randomly divided into a DNP group, an EA group and a pre-EA group, 8 rats in each group. Fifteen to 21 days after STZ injection, the EA group received EA at "Zusanli" (ST 36) and "Kunlun" (BL 60), continuous wave, frequency of 2 Hz, 30 min each time, once a day; the intervention method in the pre-EA group was the same as that in the EA group. The intervention time was 8 to 14 days after STZ injection. The body mass, FBG and thermal pain threshold were recorded before STZ injection and 7, 14 and 21 days after STZ injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot 21 days after injection. PartⅠ: Compared with the control group, in the model group, the body mass was decreased and FBG was increased 7, 14 and 21 days after STZ injection (P<0.01), and the thermal pain threshold was decreased 14 and 21 days after STZ injection (P<0.05), and the expression of P2X7R in spinal dorsal horn was increased 7, 14 and 21 days after STZ injection (P<0.05, P<0.01). PartⅡ: Compared with the blank group, in the DNP group, the body mass was decreased and fasting blood glucose were increased 7, 14 and 21 days after STZ injection (P<0.01). Compared with the DNP group, in the pre-EA group, the heat pain threshold was increased 14 and 21 days after STZ injection (P<0.05), while in the EA group, the heat pain threshold was increased 21 days after STZ injection (P<0.01), and the expression of P2X7R in the dorsal horn in the EA group and the pre-EA group was decreased (P<0.01). The diabetic neuropathic pain is observed 14 days after STZ injection. EA could not only treat but also prevent the occurrence of DNP, and its mechanism may be related to down-regulation of P2X7R expression in the dorsal horn of the spinal cord.

Dual PI3Kδ/γ Inhibitor Duvelisib Prevents Development of Neuropathic Pain in Model of Paclitaxel-Induced Peripheral Neuropathy.

The development of painful paclitaxel-induced peripheral neuropathy (PIPN) represents a major dose-limiting side effect of paclitaxel chemotherapy. Here we report a promising effect of duvelisib (Copiktra), a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, in preventing paclitaxel-induced pain-like behavior and pronociceptive signaling in DRGs and spinal cord dorsal horn (SCDH) in rat and mouse model of PIPN. Duvelisib blocked the development of mechanical hyperalgesia in both males and females. Moreover, duvelisib prevented paclitaxel-induced sensitization of TRPV1 receptors, and increased PI3K/Akt signaling in small-diameter DRG neurons and an increase of CD68+ cells within DRGs. Specific optogenetic stimulation of inhibitory neurons combined with patch-clamp recording revealed that duvelisib inhibited paclitaxel-induced weakening of inhibitory, mainly glycinergic control on SCDH excitatory neurons. Enhanced excitatory and reduced inhibitory neurotransmission in the SCDH following PIPN was also alleviated by duvelisib application. In summary, duvelisib showed a promising ability to prevent neuropathic pain in PIPN. The potential use of our findings in human medicine may be augmented by the fact that duvelisib is an FDA-approved drug with known side effects.SIGNIFICANCE STATEMENT We show that duvelisib, a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, prevents the development of paclitaxel-induced pain-like behavior in males and females and prevents pronociceptive signaling in DRGs and spinal cord dorsal horn in rat and mouse model of paclitaxel-induced peripheral neuropathy.

Upregulation of Hevin Contributes to Postoperative Pain Hypersensitivity by Inducing Neurexin1β/Neuroligin1-Mediated Synaptic Targeting of Glua1-Containing Ampa Receptors in Rat Dorsal Horn

Upregulation of Hevin Contributes to Postoperative Pain Hypersensitivity by Inducing Neurexin1β/Neuroligin1-Mediated Synaptic Targeting of Glua1-Containing Ampa Receptors in Rat Dorsal Horn

Effect of moxibustion on the expression of microglia and brain-derived neurotrophic factor protein in the spinal cord of rats with myofascial pain syndrome

To observe the effect of moxibustion at myofascial trigger points on microglia activation and the expression of brain-derived neurotrophic factor (BDNF) in the spinal cord of rats with myofascial pain syndrome (MPS), so as to explore the central mechanism of the analgesic effect of moxibustion. Twenty-four male SD rats were randomly divi-ded into control, model and moxibustion groups (n=8 in each group). The MPS model was established by strinking on fascia musculares and eccentric exercise. Rats in the moxibustion group were treated with mild moxibustion at myofascial trigger point for 15 min, once daily for 7 days. The thermal withdrawal latency (TWL) of rats was measured with a hot stabbing instrument. The pathological changes of the rat medial femoris muscle were observed after H.E. staining. The expressions of microglia marker (OX-42) and BDNF in the spinal dorsal horn were detected by immunohistochemistry and Western blot, separately. After modeling, the TWL of both the model and the moxibustion groups were significantly down-regulated (P<0.01)，and were significantly decreased in contrast to that of the control group (P<0.01). After treatment and compared with the model group, the TWL of the moxibustion group was significantly increased (P<0.01) in the moxibustion group. Rat's muscle fibers of the control group were uniform in thickness and arranged tightly and regularly. While in the model group, some fractures and connective structure tissue renewal, irregular arrangement, and inflammatory cell infiltration were seen. The morphology of muscle fibers in the moxibustion group was close to normal, and the arrangement was neat and orderly, with a small amount of inflammatory cell infiltration. Compared with the control group, the expression of OX-42 and BDNF in the spinal dorsal horn of rats in the model group was increased(P<0.01). Following the treatment, and in comparison with the model group, the expression of OX-42 and BDNF of moxibustion group was down-regulated(P<0.05). Moxibustion can significantly improve the injury of the medial femoral muscle and the TWL in MPS rats，which may be related to its effects in inhibiting the activation of spinal dorsal horn microglia and reducing the expression of BDNF.

Effect of electroacupuncture on expression of p-ERK1/2 and c-fos in spinal dorsal horn in rats with interstitial cystitis

To observe the effect of electroacupuncture (EA) at "Ciliao" (BL32) and "Huiyang" (BL35) on the expression of extracellular signal-regulated kinase 1/2 (p-ERK1/2) and cellular oncogene fos (c-fos) phosphorylated of spinal dorsal horn in rats with interstitial cystitis (IC). Eighteen female Wistar rats were randomly divided into control, model and EA groups, with 6 rats in each group. The IC model was established by intraperitoneal injection of cyclophosphamide (150 mg/kg). EA (30 Hz, 1 mA) was applied to bilateral BL32 and BL35 for 20 min, once daily for 3 consecutive days. The bladder pain was measured by using a Von Frey at 48 h after modeling and 24 h after EA. The expression levels of p-ERK1/2 and c-fos protein in L6-S1 segment of spinal cord were detected by Western blot, and the expression of p-ERK1/2 and c-fos in the right spinal dorsal horn were displayed by immunofluorescence staining. After modeling, the bladder mechanical pain threshold (PT) was significantly decreased (P<0.05), the protein expression of p-ERK1/2 and c-fos in the spinal cord was increased (P<0.05) and the immunofluorescence surface density of p-ERK1/2 and c-fos in the right dorsal horn of spinal cord was increased (P<0.05) in the model group relevant to the control group. After EA intervention, IC-induced reduction of PT, and increases of the expression of p-ERK1/2 and c-fos as well as immunofluorescence surface density of p-ERK1/2 and c-fos were reversed in the EA group relevant to the model group (P<0.05). EA at BL 32 and BL 35 has an analgesic effect in IC rats, which may be related to its effect in down-regulating the expression of p-ERK1/2 and c-fos in spinal dorsal horn.

Quercetin Alleviates Neuropathic Pain in the Rat CCI Model by Mediating AMPK/MAPK Pathway.

ContextQuercetin (que) is one abundant flavonol with a variety of biological activities. Previous studies have shown quercetin can reduce neuropathic pain in rats with chronic constriction injury (CCI).ObjectiveTo evaluate the effects of quercetin on neuropathic pain in CCI model and explore its underlying mechanism in vivo.Materials and MethodsCCI model was established by ligating the sciatic nerve of right leg on the SD rats. They were divided into ten groups: sham group, CCI model, sham+ que, CCI+ que group (30, 60, 120 mg/kg), CCI+ AICAR, CCI+ que+ compound C, CCI+etoricoxib, and the control group. They were administered for 28 days, and were performed the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) during the experiment. At the end of the experiment, sciatic nerves and spinal cord segments of rats were collected, ELISA detected the expression of inflammatory factors, detected the microglia and astrocytes with fluorescence, and Western blot detected AMPK/MAPK pathway.ResultsQue could increase the MWT of CCI rats, improve the TWL of plantar, and reduce the inflammatory cells at the ligation site of the sciatic nerve. Also, que could reduce the levels of TNF-α, IL-6, and IL-1β. Western blotting results showed that p-38 MAPK, p-ERK, and p-JNK were activated in the spinal dorsal horn of CCI model group. After treatment with que and AMPK agonists, the phosphorylation levels of related proteins were inhibited. In addition, the analgesic effect of que was abolished when the AMPK inhibitor was added.Discussion and ConclusionQuercetin alleviated the inflammatory response of sciatic nerve and spinal dorsal horn in rats induced by CCI. Quercetin alleviates neuralgia in CCI rats by activating AMPK pathway and inhibiting MAPK pathway and its downstream targets, p-38, p-ERK, and p-JNK.

Calcium-independent phospholipase A2 inhibitor produces an analgesic effect in a rat model of neuropathic pain by reducing central sensitization in the dorsal horn

ABSTRACT Objective Phospholipase A2 (PLA2) plays an important role in regulating the production of arachidonic acid and various eicosanoids. The aim of our study was to investigate the analgesic mechanisms of calcium-dependent cytosolic phospholipase A2 and calcium-independent PLA2 (iPLA2) inhibitors in the spinal cord in a rat model of neuropathic pain. Methods Lumbar 5 spinal nerve ligation was performed in male Sprague-Dawley rats to develop a peripheral neuropathic pain model. Paw withdrawal thresholds in response to von Frey filaments, brush, pressure, and pinch were measured. Lumbar wide dynamic range neuronal firing rates and iPLA2 subtype expression were measured by in vivo extracellular recording and double immunofluorescence staining, respectively. Results In our rat models, oral administration of prednisolone, a non-selective PLA2 inhibitor, and intrathecal injection of bromoenolactone, a iPLA2 inhibitor, significantly increased the ipsilateral hindpaw withdrawal thresholds in response to von Frey filament stimulation, but intrathecal injection of arachidonyl trifluoromethyl ketone, a selective cytosolic PLA2 inhibitor, did not show significant changes. In spinal dorsal horn neurons, bromoenolactone reduced neuronal firing rates in response to withdrawal stimulation and spontaneous firing rates in the ipsilateral side of the spinal dorsal horn. In addition, the expression of iPLA2 was co-localized with astrocytes and neurons on the ipsilateral side of the dorsal horn in rats that underwent spinal nerve ligation. Discussion These data suggest that selective iPLA2 inhibitor produce analgesia in neuropathic rats by reducing central sensitization in the dorsal horn.

Morphine Induces Upregulation of Neuronally Expressed CB2 Receptors in the Spinal Dorsal Horn of Rats.

Background: Cannabinoid receptors play a key role in regulating numerous physiological processes, including immune function and reward signaling. Originally, endocannabinoid contributions to central nervous system processes were attributed to CB1 receptors, but technological advances have confirmed the expression of CB2 receptors in both neurons and glia throughout the brain. Mapping of these receptors is less extensive than for CB1 receptors, and it is still not clear how CB2 receptors contribute to processes that involve endocannabinoid signaling. Objectives: The goal of our study was to assess the effects of peripheral nerve injury and chronic morphine administration, two manipulations that alter endocannabinoid system function, on CB2 receptor expression in the spinal dorsal horn of rats. Methods: Twenty-four male Sprague Dawley rats were assigned to chronic constriction injury (CCI), sham surgery, or pain naïve groups, with half of each group receiving once daily injections of morphine (5 mg/kg) for 10 days. On day 11, spinal cords were isolated and prepared for fluorescent immunohistochemistry. Separate sections from the deep and superficial dorsal horn were stained for neuronal nuclei (NeuN), CD11b, or 4',6-diamidino-2-phenylindole (DAPI) to mark neurons, microglia, and cell nuclei, respectively. Double labeling was used to assess colocalization of CB2 receptors with NeuN or microglial markers. Quantification of mean pixel intensity for each antibody was assessed using a fluorescent microscope, and CB2 receptor expressing cells were also counted manually. Results: Surgery increased DAPI cell counts in the deep and superficial dorsal horn, with CCI rats displaying increased CD11b labeling ipsilateral to the nerve injury. Surgery also decreased NeuN labeling in both regions, an effect that was blocked by morphine administration. CB2 receptors were expressed, predominantly, on NeuN-labeled cells with significant increases in CB2 receptor labeling across all surgery groups in both deep and superficial areas following morphine administration. Conclusions: Our findings provide supporting evidence for the expression of CB2 receptors on neurons and reveal upregulation of receptor expression in the dorsal spinal cord following surgery and chronic morphine administration, with the latter producing a larger effect. Synergistic effects of morphine-cannabinoid treatments, therefore, may involve CB2-mu opioid receptor interactions, pointing to novel therapeutic treatments for a variety of medical conditions.

Spinophilin modulates pain through suppressing dendritic spine morphogenesis via negative control of Rac1-ERK signaling in rat spinal dorsal horn

Both spinophilin (SPN, also known as neurabin 2) and Rac1 (a member of Rho GTPase family) are believed to play key roles in dendritic spine (DS) remodeling and spinal nociception. However, how SPN interacts with Rac1 in the above process is unknown. Here, we first demonstrated natural existence of SPN-protein phosphatase 1-Rac1 complex in the spinal dorsal horn (DH) neurons by both double immunofluorescent labeling and co-immunoprecipitation, then the effects of SPN over-expression and down-regulation on mechanical and thermal pain sensitivity, GTP-bound Rac1-ERK signaling activity, and spinal DS density were studied. Over-expression of SPN in spinal neurons by intra-DH pAAV-CMV-SPN-3FLAG could block both mechanical and thermal pain hypersensitivity induced by intraplantar bee venom injection, however it had no effect on the basal pain sensitivity. Over-expression of SPN also resulted in a significant decrease in GTP-Rac1-ERK activities, relative to naive and irrelevant control (pAAV-MCS). In sharp contrast, knockdown of SPN in spinal neurons by intra-DH pAAV-CAG-eGFP-U6-shRNA[SPN] produced both pain hypersensitivity and dramatic elevation of GTP-Rac1-ERK activities, relative to naive and irrelevant control (pAAV-shRNA [NC]). Moreover, knockdown of SPN resulted in increase in DS density while over-expression of it had no such effect. Collectively, SPN is likely to serve as a regulator of Rac1 signaling to suppress DS morphogenesis via negative control of GTP-bound Rac1-ERK activities at postsynaptic component in rat DH neurons wherein both mechanical and thermal pain sensitivity are controlled.