Effective treatments for neuropathic pain are lacking due to our limited understanding of the mechanisms. The circRNAs are mainly enriched in the central nervous system. However, their function in various physiological and pathological conditions have yet to be determined. Here, we identified circFhit, an exon-intron circRNA expressed in GABAergic neurons, which reduced the inhibitory synaptic transmission in the spinal dorsal horn to mediate spared nerve injury-induced neuropathic pain. Moreover, we found that circFhit decreased the expression of GAD65 and induced hyperexcitation in NK1R⁺ neurons by promoting the expression of its parental gene Fhit in cis. Mechanistically, circFhit was directly bound to the intronic region of Fhit, and formed a circFhit/HNRNPK complex to promote Pol II phosphorylation and H2B monoubiquitination by recruiting CDK9 and RNF40 to the Fhit intron. In summary, we revealed that the exon-intron circFhit contributes to GABAergic neuron-mediated NK1R⁺ neuronal hyperexcitation and neuropathic pain via regulating Fhit in cis.
Rats ; Animals ; Posterior Horn Cells/pathology* ; Spinal Cord Dorsal Horn/metabolism* ; Neuralgia ; Synaptic Transmission

Mechanical allodynia (MA), including punctate and dynamic forms, is a common and debilitating symptom suffered by millions of chronic pain patients. Some peripheral injuries result in the development of bilateral MA, while most injuries usually led to unilateral MA. To date, the control of such laterality remains poorly understood. Here, to study the role of microglia in the control of MA laterality, we used genetic strategies to deplete microglia and tested both dynamic and punctate forms of MA in mice. Surprisingly, the depletion of central microglia did not prevent the induction of bilateral dynamic and punctate MA. Moreover, in dorsal root ganglion-dorsal root-sagittal spinal cord slice preparations we recorded the low-threshold Aβ-fiber stimulation-evoked inputs and outputs of superficial dorsal horn neurons. Consistent with behavioral results, microglial depletion did not prevent the opening of bilateral gates for Aβ pathways in the superficial dorsal horn. This study challenges the role of microglia in the control of MA laterality in mice. Future studies are needed to further understand whether the role of microglia in the control of MA laterality is etiology-or species-specific.
Mice ; Animals ; Hyperalgesia/metabolism* ; Microglia/metabolism* ; Disease Models, Animal ; Spinal Cord/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Ganglia, Spinal/metabolism*

OBJECTIVETo examine the effect of acute incisional pain on the expression of connexin 43 in rat spinal cord dorsal horn.

METHODSEighty rats were assigned into control group without any treatment and incisional pain group with incision surgery. For paw incisions, a 1-cm longitudinal incision was made through the skin and fascia of the plantar aspect of the right hind paw. After surgery, the 50% paw withdrawal threshold (PWT) was assessed in response to a tactile stimulus with calibrated von Frey monofilaments at 1, 2, 4 and 24 h, respectively. The spinal cord dorsal horn of rats was isolated at 1, 2, and 4 h after the surgery to assess the expression of connexin 43 using Western blotting and immunofluorescence assay.

RESULTSThe 50% PWT of the rats was significantly decreased after the incision surgery, and this decrement was the most obvious at 2 and 4 h. Western blotting and immunofluorescence assay showed that the expression of connexin 43 in the spinal cord dorsal horn was significantly increased in rats receiving the surgery especially at 2 and 4 h after the surgery.

CONCLUSIONIncision surgery induces an significant increase in connexin 43 expression in rat spinal cord dorsal horn, suggestting an potential role of connexin43 in postoperative incisional pain.

Animals ; Connexin 43 ; metabolism ; Pain, Postoperative ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Dorsal Horn ; metabolism

OBJECTIVETo evaluate the pain-related behavioral changes in rats with bilateral chronic constriction injury(bCCI)and identify the expressions of neuropathic pain-related proteins.

METHODSThe bCCI models were established by ligating the sciatic nerves in female Sprague Dawley rats. Both mechanical hyperalgesia and cold hyperalgesia were evaluated through electronic von Frey and acetone method. Liquid chromatography-mass spectrometry/mass spectrometry was applied to characterize the differentially expressed proteins.

RESULTSBoth mechanical withdrawal threshold and cold hyperalgesia threshold decreased significantly on the postoperative day 7 and 14, when compared with na ve or sham rats(P <0.05). Twenty five differentially expressed proteins associated with bilateral CCI were discovered, with eighteen of them were upregulated and seven of them downregulated.

CONCLUSIONSThe bCCT rats have remarkably decreased mechanical and cold hyperalgesia thresholds. Twenty five neuropathic pain-related proteins are found in the spinal cord dorsal horn.

Animals ; Constriction ; Female ; Hyperalgesia ; metabolism ; Neuralgia ; metabolism ; Pain ; metabolism ; Pain Threshold ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; Spinal Cord ; Spinal Cord Dorsal Horn ; metabolism

OBJECTIVE: To observe the analgesic effect of Tuina by pressing and kneading the Huantiao (GB30) acupoint on rats with chronic constriction injury (CCI) and to explore the analgesic mechanism of Tuina on sciatica rats. METHODS: Thirty-two SPF male SD rats weighing 180 to 220 g were randomly divided into fore groups:blank group (without any treatment), sham group (only exposed without sciatic nerve ligating), model group (sciatic nerve ligating) and Tuina group (manual intervention after lsciatic nerve ligating). The CCI model was prepared by ligating the right sciatic nerve of the rats, on the third day of modeling, the rats in the Tuina group were given pressing and kneading the Huantiao (GB30) point for 14 days, and the changes of paw withdrawal threshold(PWT), paw withdrawal latency(PWL) were measured before and on the 1st, 3rd, 7th, 10th, 14th and 17th days after modeling. The changes of sciatic functional index(SFI) were measured before and on the 1st and 17th day after modeling. The morphological changes of the sciatic nerve were observed by hematoxylin-eosin(HE) staining;and the differences in NF-κB protein expression in the right dorsal horn of the spinal cord of rats were detected. RESULTS: Following modeling, there was no significant difference in PWT, PWL and SFI between the blank group and the sham group (P>0.05), but the PWT, PWL and SFI of the model group and the Tuina group decreased significantly (P<0.01). After manual intervention, the pain threshold of rats in Tuina group increased. On the 8th day of manual intervention (the 10th day after modeling), PWT in Tuina group increased significantly compared with that in model group (P<0.01). On the 5th day of manual intervention (the 7th day after modeling), the PWL of the massage group was significantly higher than that of the model group (P<0.01). The pain threshold of rats in Tuina group continued to rise with the continuous manipulation intervention. After 14 days of manipulative intervention, the sciatic nerve function index of rats in the Tuina group increased significantly(P<0.01). Compared with the blank group and sham group, the myelinated nerve fibers of sciatic nerve in the model group were disordered and the density of axons and myelin sheath was uneven. Compared with the model group, the nerve fibers of rats in the Tuina group were gradually continuous and the axons and myelin sheath were more uniform than those in the model group. Compared with the blank group and sham group, the expression of NF-κB protein in the right spinal dorsal horn of the model group was significantly increased(P<0.01). Compared with the model group, the expression of NF-κB protein in the right spinal dorsal horn of rats in Tuina group decreased significantly(P<0.01). CONCLUSION Pressing and kneading the Huantiao (GB30) point restores nerve fiber alignment;and improves the PWT、PWL and SFI in the CCI model by decreasing NF-κB p65 protein expression in the spinal dorsal horn. There fore, Tuina demmstrates an analgesic effect and improves the gait of rats with sciatica.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Sciatica/therapy* ; NF-kappa B/metabolism* ; Acupuncture Points ; Spinal Cord Dorsal Horn/metabolism* ; Spinal Cord ; Massage

OBJECTIVEThe changes of pain threshold and expression of 5-hydroxytryptamine(5-HT) and c-Fos in spinal dorsal horn of rats were observed after targetedly damaged the cerebraspinal fluid-contacting nucleus (CSF-contacting nucleus) to provide experimental evidence for the mechanism of regulating pain CSF-contacting nucleus involved in.

METHODSMale adult SD rats were divided into control, sham, choleratoxin subunit B conjugated with horse-radish peroxidase (CB-HRP)and damage groups randomly. The pain threshold using mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded and analyzed. Immunofluorescence method was used to observe the expression of 5-HT and c-Fos in spinal dorsal horn.

RESULTSCompared with the control, sham and CB-HRP groups, the MWT and TWL of the damage group were significantly increased (P < 0.05). The results of immunofluorescence showed that 5-HT was detected in neurons of CSF-contacting nucleus. In the damage group, the number of neurons of CSF-contacting nucleus reduced gradually, and no survived neurons were observed at the 10th day. Meanwhile, both the expression of 5-HT and c-Fos in spinal dorsal horn increased gradually, and negatively correlated with the change of pain threshold.

CONCLUSIONThe method of targeted damaging CSF-contacting nucleus by cholera toxin subnit B conjugated with saporin(CB-SAP) is scientific and reliable, and it results in the changes of pain threshold and expression of 5-HT and c-Fos in spinal dorsal horn of rats. This study suggests that CSF-contacting nucleus participate in the regulation of pain, moreover, 5-HT and c-Fos play important roles in this regulation.

Animals ; Cerebrospinal Fluid ; Male ; Pain ; metabolism ; surgery ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serotonin ; metabolism ; Spinal Cord Dorsal Horn ; metabolism

OBJECTIVETo explore the relationship between the expression of brain-derived neurotrophic factor (BDNF) in the spinal dorsal horn and the increase in visceral hypersensitivity in young rats by establishing a young rat model of visceral hypersensitivity by neonatal maternal separation (NMS).

METHODSThirty-two newborn Sprague-Dawley rats were randomly and equally divided into four groups by a 2×2 factorial design: control, NMS, colorectal distension (CRD), and NMS+CRD. The newborn rats in the NMS and NMS+CRD groups were subjected to 3-hour daily maternal separation from days 2 to 14 after birth to establish a model of visceral hypersensitivity, while the rats in the control and CRD groups received no treatment after birth. At 6 weeks after birth, the CRD and CRD+NMS groups received CRD stimulation. The streptavidin-biotin complex immunohistochemical method was used to determine the expression of BDNF in the spinal dorsal horn. The immunohistochemical score (IHS) was calculated based on the percentage of BDNF-positive cells and color intensity. The percentage of BDNF-positive cells in the spinal dorsal horn and IHS were analyzed by factorial analysis of variance.

RESULTSThe expression of BDNF was detected bilaterally in the spinal dorsal horn at different levels in the four groups. The percentage of BDNF-positive cells and IHS were significantly higher in the NMS and NMS+CRD groups than in the control group (P<0.05). The results of factorial analysis of variance indicated that NMS significantly increased the percentage of BDNF-positive cells in the spinal dorsal horn and IHS; a single CRD stimulation had no effects on the IHS of BDNF-positive cells in the spinal dorsal horn; there was no interaction between NMS and a single CRD stimulation.

CONCLUSIONSThe over-expression of BDNF in the spinal dorsal horn may be involved in high visceral hypersensitivity in young rats induce by NMS.

Animals ; Brain-Derived Neurotrophic Factor ; analysis ; Female ; Hyperalgesia ; metabolism ; Immunohistochemistry ; Male ; Maternal Deprivation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Dorsal Horn ; chemistry ; Visceral Pain ; metabolism

PURPOSE: The TWIK-related spinal cord K+ channel (TRESK) has recently been discovered and plays an important role in nociceptor excitability in the pain pathway. Because there have been no reports on the TRESK expression or its function in the dorsal horn of the spinal cord in neuropathic pain, we analyzed TRESK expression in the spinal dorsal horn in a spinal nerve ligation (SNL) model. MATERIALS AND METHODS: We established a SNL mouse model by using the L5-6 spinal nerves ligation. We used real-time polymerase chain reaction and immunohistochemistry to investigate TRESK expression in the dorsal horn and L5 dorsal rot ganglion (DRG). RESULTS: The SNL group showed significantly higher expression of TRESK in the ipsilateral dorsal horn under pain, but low expression in L5 DRG. Double immunofluorescence staining revealed that immunoreactivity of TRESK was mostly restricted in neuronal cells, and that synapse markers GAD67 and VGlut2 appeared to be associated with TRESK expression. We were unable to find a significant association between TRESK and calcineurin by double immunofluorescence. CONCLUSION: TRESK in spinal cord neurons may contribute to the development of neuropathic pain following injury.
Animals ; Disease Models, Animal ; Hyperalgesia ; Ligation ; Male ; Neuralgia/*metabolism/physiopathology ; Neurons/metabolism ; Nociceptors ; Pain/metabolism/*physiopathology ; Potassium Channels/*metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Spinal Cord Dorsal Horn/*metabolism ; Spinal Nerves/*injuries

Lumbar disc herniation is commonly encountered in clinical practice and can induce sciatica due to mechanical and/or chemical irritation and the release of proinflammatory cytokines. However, symptoms are not confined to the affected spinal cord segment. The purpose of this study was to determine whether multisegmental molecular changes exist between adjacent lumbar spinal segments using a rat model of lumbar disc herniation. Twenty-nine male Sprague-Dawley rats were randomly assigned to either a sham-operated group (n=10) or a nucleus pulposus (NP)-exposed group (n=19). Rats in the NP-exposed group were further subdivided into a significant pain subgroup (n=12) and a no significant pain subgroup (n=7) using mechanical pain thresholds determined von Frey filaments. Immunohistochemical stainings of microglia (ionized calcium-binding adapter molecule 1; Iba1), astrocytes (glial fibrillary acidic protein; GFAP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid 1 (TRPV1) was performed in spinal dorsal horns and dorsal root ganglions (DRGs) at 10 days after surgery. It was found immunoreactivity for Iba1-positive microglia was higher in the L5 (P=0.004) dorsal horn and in the ipsilateral L4 (P=0.009), L6 (P=0.002), and S1 (P=0.002) dorsal horns in the NP-exposed group than in the sham-operated group. The expression of CGRP was also significantly higher in ipsilateral L3, L4, L6, and S1 segments and in L5 DRGs at 10 days after surgery in the NP-exposed group than in the sham-operated group (P<0.001). Our results indicate that lumbar disc herniation upregulates microglial activity and CGRP expression in many adjacent and ipsilateral lumbar spinal segments.
Animals ; Astrocytes/metabolism ; Calcitonin Gene-Related Peptide/*metabolism ; Calcium-Binding Proteins/*metabolism ; Disease Models, Animal ; Ganglia, Spinal/metabolism ; Humans ; Immunohistochemistry ; Intervertebral Disc Displacement/*metabolism ; Lumbar Vertebrae/*metabolism ; Male ; Microfilament Proteins/*metabolism ; Microglia/metabolism ; Neuralgia/metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Dorsal Horn/metabolism ; Up-Regulation

5-hydroxytryptamine (5-HT) released in inflammatory tissues plays a pivotal role in pain hypersensitivity. However, it is not clear whether 5-HT2A receptors in the inflamed tissues mediate this effect. The present study investigated the contribution of 5-HT2A receptors in the periphery to chronic inflammatory pain. Complete Freund's adjuvant (CFA) was injected subcutaneously in the hindpaw of rats. The selective 5-HT2A receptor antagonist ketanserin was given in the inflamed site. Paw withdrawal latency responding to heat or mechanical stimuli was measured. Expression of neuropeptide Y (NPY) in the spinal dorsal horn and dorsal root ganglia (DRG) was assayed using immunohistochemistry technique. The results showed that ketanserin administered in the inflamed site inhibited thermal hyperalgesia in a dose-dependent manner (20, 40 and 80 µg) induced by the intraplantar injection of CFA. Ketanserin given once per day at a dose of 80 µg abolished heat hyperalgesia and also attenuated mechanical allodynia on the third day. CFA injection increased the expression of NPY in superficial laminae of the spinal cord, but not in the DRG. The local treatment of ketanserin completely inhibited CFA-induced increase in NPY expression in superficial laminae of the spinal cord. These results indicated that activation of 5-HT2A receptors in the inflamed tissues was involved in the pathogenesis of inflammatory pain and the blockade of 5-HT2A receptors in the periphery could relieve pain hypersensitivity and normalize the cellular disorder in the spinal dorsal horn associated with pathological pain. The present study suggests that the peripheral 5-HT2A receptors can be a promising target for pharmaceutical therapy to treat chronic inflammatory pain without central nervous system side effects.
Animals ; Freund's Adjuvant ; adverse effects ; Ganglia, Spinal ; metabolism ; Hot Temperature ; Hyperalgesia ; chemically induced ; drug therapy ; Inflammation ; drug therapy ; Ketanserin ; pharmacology ; Neuropeptide Y ; metabolism ; Pain ; drug therapy ; Pain Measurement ; Rats ; Receptor, Serotonin, 5-HT2A ; metabolism ; Serotonin ; Serotonin 5-HT2 Receptor Antagonists ; pharmacology ; Spinal Cord Dorsal Horn ; metabolism