Neuropathic pain, often featuring allodynia, imposes significant physical and psychological burdens on patients, with limited treatments due to unclear central mechanisms. Addressing this challenge remains a crucial unsolved issue in pain medicine. Our previous study, using protein kinase C gamma (PKCγ)-tdTomato mice, highlights the spinal feedforward inhibitory circuit involving PKCγ neurons in gating neuropathic allodynia. However, the regulatory mechanisms governing this circuit necessitate further elucidation. We used diverse transgenic mice and advanced techniques to uncover the regulatory role of the descending serotonin (5-HT) facilitation system on spinal PKCγ neurons. Our findings revealed that 5-HT neurons from the rostral ventromedial medulla hyperpolarize spinal inhibitory interneurons via 5-HT_2C receptors, disinhibiting the feedforward inhibitory circuit involving PKCγ neurons and exacerbating allodynia. Inhibiting spinal 5-HT_2C receptors restored the feedforward inhibitory circuit, effectively preventing neuropathic allodynia. These insights offer promising therapeutic targets for neuropathic allodynia management, emphasizing the potential of spinal 5-HT_2C receptors as a novel avenue for intervention.
Animals ; Neuralgia/physiopathology* ; Protein Kinase C/metabolism* ; Receptor, Serotonin, 5-HT2C/metabolism* ; Hyperalgesia/physiopathology* ; Mice, Transgenic ; Mice ; Spinal Cord/metabolism* ; Serotonin/metabolism* ; Male ; Neurons/metabolism* ; Mice, Inbred C57BL

Sleep deprivation has been shown to exacerbate pain sensitivity and may contribute to the onset of chronic pain, yet the precise neural mechanisms underlying this association remain elusive. In our study, we explored the contribution of cholinergic neurons within the medial habenula (MHb) to hyperalgesia induced by sleep deprivation in rats. Our findings indicate that the activity of MHb cholinergic neurons diminishes during sleep deprivation and that chemogenetic stimulation of these neurons can mitigate the results. Interestingly, we did not find a direct response of MHb cholinergic neurons to pain stimulation. Further investigation identified the interpeduncular nucleus (IPN) and the paraventricular nucleus of the thalamus (PVT) as key players in the pro-nociceptive effect of sleep deprivation. Stimulating the pathways connecting the MHb to the IPN and PVT alleviated the hyperalgesia. These results underscore the important role of MHb cholinergic neurons in modulating pain sensitivity linked to sleep deprivation, highlighting potential neural targets for mitigating sleep deprivation-induced hyperalgesia.
Animals ; Habenula/physiology* ; Sleep Deprivation/physiopathology* ; Cholinergic Neurons/physiology* ; Male ; Hyperalgesia/physiopathology* ; Rats, Sprague-Dawley ; Rats ; Interpeduncular Nucleus/physiology* ; Pain Threshold/physiology* ; Midline Thalamic Nuclei/physiology* ; Neural Pathways/physiopathology*

The pathophysiology of visceral pain in patients with irritable bowel syndrome remains largely unknown. Our previous study showed that neonatal maternal deprivation (NMD) does not induce visceral hypersensitivity at the age of 6 weeks in rats. The aim of this study was to determine whether NMD followed by adult stress at the age of 6 weeks induces visceral pain in rats and to investigate the roles of adrenergic signaling in visceral pain. Here we showed that NMD rats exhibited visceral hypersensitivity 6 h and 24 h after the termination of adult multiple stressors (AMSs). The plasma level of norepinephrine was significantly increased in NMD rats after AMSs. Whole-cell patch-clamp recording showed that the excitability of dorsal root ganglion (DRG) neurons from NMD rats with AMSs was remarkably increased. The expression of β adrenergic receptors at the protein and mRNA levels was markedly higher in NMD rats with AMSs than in rats with NMD alone. Inhibition of β adrenergic receptors with propranolol or butoxamine enhanced the colorectal distention threshold and application of butoxamine also reversed the enhanced hypersensitivity of DRG neurons. Overall, our data demonstrate that AMS induces visceral hypersensitivity in NMD rats, in part due to enhanced NE-β adrenergic signaling in DRGs.
Adrenergic Agents ; pharmacology ; Animals ; Ganglia, Spinal ; drug effects ; Hyperalgesia ; drug therapy ; physiopathology ; Hypersensitivity ; drug therapy ; Male ; Maternal Deprivation ; Neurons ; drug effects ; Patch-Clamp Techniques ; methods ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Stress, Physiological ; physiology ; Visceral Pain ; chemically induced ; metabolism

The pain peptide adrenomedullin (AM) plays a pivotal role in pathological pain. The present study was designed to investigate the effect of blockade of AM receptor on bone cancer pain (BCP) and its mechanism. BCP was developed by inoculation of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity of Sprague Dawley rats. The selective AM receptor antagonist AMwas administered intrathecally on 15 d after the inoculation. Quantitative real-time PCR was used to detect mRNA level of CC chemokine ligand 2 (CCL2) in dorsal root ganglion (DRG). Double immunofluorescence staining was used to analyze the localizations of CCL2 and AM in DRG of normal rats. The results showed that, from 6 to15 d after the inoculation, the animals showed significant reduction in the mechanical pain threshold in the ipsilateral hindpaw, companied by the decline in bone density of tibia bone. The expression of CCL2 mRNA in DRG of BCP rats was increased by 3 folds (P < 0.001 vs saline group). Intrathecal administration of AMabolished bone cancer-induced mechanical allodynia and increase of CCL2 mRNA level (P < 0.001). In normal rats, CCL2 was co-localized with AM in DRG neurons. These results suggest that AM may play a role in the pathogenesis of BCP. The increased AM bioactivity up-regulates CCL2 expression in DRG, which may contribute to the induction of pain hypersensitivity in bone cancer.
Adrenomedullin ; administration & dosage ; pharmacology ; Animals ; Bone Neoplasms ; drug therapy ; Chemokine CCL2 ; metabolism ; Ganglia, Spinal ; physiopathology ; Hyperalgesia ; drug therapy ; Pain ; drug therapy ; Pain Threshold ; Peptide Fragments ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Adrenomedullin ; antagonists & inhibitors

Trigeminal neuralgia (TN) is a kind of recurrent transient and severe pain that is limited to the trigeminal nerve in one or more branches. The clinical incidence of TN is high, which seriously affects the quality of life of the patients and is difficult to cure. The present study investigated the effects of tetramethylpyrazine (TMP) on TN induced by chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. Adult male Sprague-Dawley rats were randomly assigned to four groups: sham, sham treated with TMP (Sham+TMP), TN model (TN), and TN treated with TMP (TN+TMP). The rat TN model was established by ION-CCI and TMP (50 mg/kg) was injected intraperitoneally once a day for 2 weeks after operation. The mechanical response threshold was tested by the electronic von Frey filaments. The expression of CGRP in the trigeminal ganglia (TGs) of rats on the operative side was detected by RT-PCR, immunohistochemical staining and Western blot. In 15 days after operation, TN group showed a robust decrease in mechanical response threshold as compared with sham group. From day 9 to day 15 after operation, TMP treatment significantly suppressed the TN-induced mechanical hyperalgesia (P < 0.05). On day 15 after operation, RT-PCR, immunohistochemical staining and Western blot analysis showed an obvious increase in expression level of CGRP in TGs of TN group compared with sham group, which was downregulated by TMP treatment (P < 0.05). These results suggested that TMP might have a therapeutic potential for the treatment of TN through regulating CGRP expression in the TGs.
Animals ; Constriction ; Hyperalgesia ; drug therapy ; Male ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; physiopathology ; Trigeminal Neuralgia ; drug therapy

Nefopam has a pharmacologic profile distinct from that of opioids or other anti-inflammatory drugs. Several recent studies demonstrate that nefopam has a mechanism of action similar to those of anti-depressants and anticonvulsants for treating neuropathic pain. The present study investigates the mechanical antiallodynic effect of nefopam using immunohistochemical study and western blot analysis in a rat neuropathic pain model. Twenty-eight male Sprague-Dawley rats were subjected to left fifth lumbar (L5) spinal nerve ligation and intrathecal catheter implantation, procedures which were not performed on the 7 male Sprague-Dawley rats in the sham surgery group (group S). Nefopam, either 10 or 100 microg/kg (group N10 or N100, respectively), and normal saline (group C) were intrathecally administered into the catheter every day for 14 days. The mechanical allodynic threshold of intrathecal nefopam was measured using a dynamic plantar aesthesiometer. Immunohistochemistry targeting cluster of differentiation molecule 11b (CD11b) and glial fibrillary acidic protein (GFAP) was performed on the harvested spinal cord at the level of L5. Extracellular signal-regulated kinase 1/2 (ERK 1/2) and cyclic adenosine monophosphate response element binding protein (CREB) were measured using western blot analysis. The N10 and N100 groups showed improved mechanical allodynic threshold, reduced CD11b and GFAP expression, and attenuated ERK 1/2 and CREB in the affected L5 spinal cord. In conclusion, intrathecal nefopam reduced mechanical allodynia in a rat neuropathic pain model. Its mechanical antiallodynic effect is associated with inhibition of glial activation and suppression of the transcription factors' mitogen-activated protein kinases in the spinal cord.
Analgesics, Non-Narcotic/administration & dosage ; Animals ; Dose-Response Relationship, Drug ; Hyperalgesia/*drug therapy/etiology/*physiopathology ; Injections, Spinal ; Male ; Nefopam/*administration & dosage ; Neuralgia/complications/*drug therapy/*physiopathology ; Pain Measurement/drug effects ; Pain Perception/*drug effects ; Rats ; Rats, Sprague-Dawley ; Treatment Outcome

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

Subcutaneous injection of BmK I could be adopted to well establish a novel pain model. Moreover, 5-hydroxytryptamine (serotonin, 5-HT) receptor is involved in regulating animal pain-related behaviors. However, the underlying mechanism of 5-HT3R on BmK I-induced pain remains unclear. Animal behavioral testing, RT-PCR and Western blotting were used to yield the following results: first, intraplantar (i.pl.) injection of BmK I (10 μg) induced elevated mRNA and protein levels of 5-HT3AR in bilateral L4-L5 spinal cord; Second, intrathecal (i.t.) injection of ondansetron (a specific antagonist of 5-HT3AR) reduced spontaneous pain responses, attenuated unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I; Microglia could be activated by BmK I (i.pl.) in both sides of L4-L5 spinal cord, and this effect was reversed by intrathecal pre-treatment with 5-HT3AR antagonist. Meanwhile, the 5-HT3AR in L4-L5 spinal cord was almost co-localized with NeuN (a marker of nerve cell), but not co-expressed with Iba-1 (a marker of microglia). Finally, the expression level of CX3CL1 and CX3CR1 was reduced by intrathecal pre-treatment with ondansetron. Our results indicate that both 5-HT3AR signaling pathway and microglia are activated in the process of induction and maintenance of BmK I-induced pain nociception. Meanwhile, our results suggest that the neuronal 5-HT3AR may communicate with microglia indirectly via CX3CL1 which is involved in regulating the BmK I-induced hyperalgesia and sensitization.
Animals ; Behavior, Animal ; Chemokine CX3CL1 ; metabolism ; Hyperalgesia ; chemically induced ; Inflammation ; physiopathology ; Injections, Spinal ; Microglia ; drug effects ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin, 5-HT3 ; metabolism ; Scorpion Venoms ; adverse effects ; Spinal Cord ; metabolism ; physiopathology

OBJECTIVETo evaluate the small fiber function in patients with type 2 diabetes mellitus of the early stage by measuring the sensory threshold with the quantitative temperature testing technology.

METHODSTwenty cases of patients with type 2 diabetes with no neurological deficit (DM group) and twenty age and sex-matched healthy controls underwent the detecting of cold sensory threshold (CST), warm sensory threshold (WST), cold pain threshold (CPT), heat pain threshold (HPT) in both inside of their hands.

RESULTSThere was no significant difference in CST, WST, CPT and HPT between left and right inside of hand of the same sample among all the testers. But the four kinds of threshold showed significant difference in the right inside of hand between patients and healthy people ( P < 0.05). In addition, the CST and WST differed significantly in the left inside of hand between the patients and healthy controls while the CPT and HPT showed no significant difference in the left inside of hand between them. Patients group and control group with CST and WST on the left side of the comparison difference was statistically significant (P < 0.05).

CONCLUSIONQuantitative analysis of temperature sense threshold can not only reflect increase of the pain threshold value, also can reflect its decrease, i. e. hyperalgesia, which may help to diagnose small fibrous peripheral neuropathy recognition, especially in early diabetic peripheral neuropathy.

Case-Control Studies ; Cold Temperature ; Diabetes Mellitus, Type 2 ; physiopathology ; Diabetic Neuropathies ; diagnosis ; Hot Temperature ; Humans ; Hyperalgesia ; Pain Threshold ; Sensory Thresholds ; Thermosensing

OBJECTIVETo investigate the changes in the levels of monocarboxylate transporter-2 in spinal cord horn in a rat model of chronic inflammatory pain.

METHODSMale SD rats weighting 180 - 220 g were randomly divided into two groups(n = 48): normal saline group (NS group), complete Freund's adjuvant group (CFA group). Rats were given injections of CFA 100 µl in left hind paw in group CFA, and an equal volume of saline was given injection in group NS. Mechanical withdraw threshold(MWT) and thermal withdraw latency(TWL) were measured at before injection(T0 and 3 h, 1 d, 3 d, 7 d, 14 d, and 21 d after injection(T1-7). Four rats were chosen from each group at T0-7 and sacrificed, and L4-5 segments of the spinal cord horn were removed for measurement of the expression of monocarboxylate transporter-2 by Western blot analysis.

RESULTSIn CFA group, mechanical hyperalgesia and allodynia appeared on the 3 h after CFA injection, then until the day 14. The expression of monocarboxylate transporter-2 in the spinal dorsal horn of rats in CFA group was significantly higher than that in normal control group at T1-6(P <0.05). The protein level of monocarboxylate transporter-2 was apparently correlated with MWT and TWL(P <0.01 and P <0.05) in CFA group.

CONCLUSIONThe level of monocarboxylate transporter-2 in spinal dorsal horn is significantly increased in a rat model of chronic inflammatory pain and the change may involve in the formation and maintenance of central sensitization in spinal cord of chronic inflammatory uain.

Animals ; Disease Models, Animal ; Freund's Adjuvant ; Hyperalgesia ; chemically induced ; Inflammation ; chemically induced ; metabolism ; Male ; Monocarboxylic Acid Transporters ; metabolism ; Pain ; chemically induced ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; physiopathology