IL-6 from cerebrospinal fluid causes widespread pain via STAT3-mediated astrocytosis in chronic constriction injury of the infraorbital nerve.

BACKGROUND: The spinal inflammatory signal often spreads to distant segments, accompanied by widespread pain symptom under neuropathological conditions. Multiple cytokines are released into the cerebrospinal fluid (CSF), potentially inducing the activation of an inflammatory cascade at remote segments through CSF flow. However, the detailed alteration of CSF in neuropathic pain and its specific role in widespread pain remain obscure. METHODS: A chronic constriction injury of the infraorbital nerve (CCI-ION) model was constructed, and pain-related behavior was observed on the 7th, 14th, 21st, and 28th days post surgery, in both vibrissa pads and hind paws. CSF from CCI-ION rats was transplanted to naïve rats through intracisternal injection, and thermal and mechanical allodynia were measured in hind paws. The alteration of inflammatory cytokines in CCI-ION's CSF was detected using an antibody array and bioinformatic analysis. Pharmacological intervention targeting the changed cytokine in the CSF and downstream signaling was performed to evaluate its role in widespread pain. RESULTS: CCI-ION induced local pain in vibrissa pads together with widespread pain in hind paws. CCI-ION's CSF transplantation, compared with sham CSF, contributed to vibrissa pad pain and hind paw pain in recipient rats. Among the measured cytokines, interleukin-6 (IL-6) and leptin were increased in CCI-ION's CSF, while interleukin-13 (IL-13) was significantly reduced. Furthermore, the concentration of CSF IL-6 was correlated with nerve injury extent, which gated the occurrence of widespread pain. Both astrocytes and microglia were increased in remote segments of the CCI-ION model, while the inhibition of astrocytes in remote segments, but not microglia, significantly alleviated widespread pain. Mechanically, astroglial signal transducer and activator of transcription 3 (STAT3) in remote segments were activated by CSF IL-6, the inhibition of which significantly mitigated widespread pain in CCI-ION. CONCLUSION: IL-6 was induced in the CSF of the CCI-ION model, triggering widespread pain via activating astrocyte STAT3 signal in remote segments. Therapies targeting IL-6/STAT3 signaling might serve as a promising strategy for the widespread pain symptom under neuropathological conditions.

Astrocytic phosphatase and tensin homolog deleted on chromosome 10 regulates neuropathic pain by facilitating 3-hydroxy-3-methylglutaryl-CoA reductase-dependent cholesterol biosynthesis.

ABSTRACT: Recent studies have noted the role of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in developing neuropathic pain, but the underlying mechanisms are obscure. We found that PTEN was mainly expressed in astrocytes in the rat spinal cord and dramatically downregulated after chronic constriction injury (CCI). Intrathecal injection of a PTEN inhibitor induced pain-related behaviors in naive rats. By contrast, administration of a PTEN protector effectively mitigated CCI-induced pain. Adeno-associated virus-mediated overexpression of astrocytic PTEN in the spinal cord reduced glial activation and neuroinflammation and subsequently alleviated pain-related behaviors. Importantly, astrocyte-specific PTEN knockout ( Pten conditional knockout , Pten CKO) mice showed nociceptive sensitization and glial activation. Proteomic analysis revealed that PTEN overexpression upregulated at least 7 enzymes in the cholesterol biosynthesis pathway and the total cholesterol level in the spinal cord of CCI rats. Furthermore, PTEN directly interacted with enzymes, including 3-hydroxy-3-methylglutaryl-CoA reductase, in the cholesterol biosynthesis pathway. Astrocytic 3-hydroxy-3-methylglutaryl-CoA reductase overexpression alleviated both CCI-induced pain and mechanical allodynia in Pten CKO mice. Finally, cholesterol replenishment attenuated CCI-induced pain and suppressed spinal glial activation. Taken together, these findings imply that spinal astrocytic PTEN plays a beneficial role in CCI-induced pain by regulating cholesterol biosynthesis, and an increased level of PTEN may accelerate cholesterol biosynthesis and reduce glial activation, thereby alleviating neuropathic pain. Recovery of PTEN or cholesterol might be an effective therapeutic strategy for neuropathic pain.

Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat.

BACKGROUND: MyD88 is the adaptor protein of MyD88-dependent signaling pathway of TLRs and IL-1 receptor and regulates innate immune response. However, it was not clear whether and how MyD88 and related signaling pathways in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) are involved in neuropathic pain. METHODS: Chronic constriction injury (CCI) was used to induce neuropathic pain in the rat. The expression of MyD88, TRIF, IBA1, and GFAP was detected with immunofluorescent staining and Western blot. The expression of interleukin-1 beta (IL-1ß), high mobility group box 1 (HMGB1), NF-κB-p65, phosphorylated NF-κB-p65, ERK, phosphorylated ERK, and tumor necrosis factor-alpha (TNF-α) was detected with Western blot. Pain-related behavioral effects of MyD88 homodimerization inhibitory peptide (MIP) were accessed up to 3 weeks after intrathecal administration. RESULTS: Peripheral nerve injury significantly increased the protein level of MyD88 in the DRG and SDH, but had no effect on TRIF. MyD88 was found partly distributed in the nociceptive neurons in the DRGs and the astrocytes and microglia in the SDH. HMGB1 and IL-1ß were also found upregulated in nociceptive pathways of CCI rats. Intrathecal application of MIP significantly alleviated mechanical and thermal hyperalgesia in the CCI rats and also reversed CCI-induced upregulation of MyD88 in both DRG and SDH. Further investigation revealed that suppression of MyD88 protein reduced the release of TNF-α and glial activation in the SDH in the CCI rats. CONCLUSIONS: MyD88-dependent TIR pathway in the DRG and SDH may play a role in CCI-induced neuropathic pain. MyD88 might serve as a potential therapeutic target for neuropathic pain.

[Dendritic projections from meridian-related motoneurons to sympathic preganglion neurons in the spinal cord of rats].

OBJECTIVE: Previous studies indicated a close involvement of reflex activities of motoneurons in the spinal cord in the mechanism of meridian phenomena. The present study was designed to investigate the dendrite projections of meridian-related motoneurons among the motoneurons and sympathetic preganglionic neurons in the spinal cord. METHODS: A total of 41 Sprague-Dawley rats were used in the present study. Cholera toxin B-subunit conjugated horseradish peroxidase (CB-HRP) containing 1.0% HRP was respectively injected to acupoint "Chengman" (ST 20), "Liangmen" (ST 21), "Guanmen" (ST 22), "Taiyi" (ST 23), "Huaroumen" (ST 24), "Tianshu" (ST 25) and "Wailing" (ST 26) of the Stomach Meridian, and "Ganshu" (BL 18), "Danshu" (BL 19), "Pishu" (BL 20), "Weishu" (BL 21) and "Sanjiaoshu" (BL 22) of the Bladder Meridian, and "Daimai" (GB 26), "Wushu" (GB 27), "Weidao" (GB 28), "Juliao" (GB 29), "Huantiao" (GB 30), "Fengshi" (GB 31), "Zhongdu" (GB 32), "Xiyangguan" (GB 33) and "Yanglingquan" (GB 34) of the Gallbladder Meridian (for labeling preganglionic neurons), and the celiac ganglion and superior mesenteric ganglion for labeling sympathetic preganglionic neurons. Three days after injection, the animals anesthetized were transcardia-cally perfused with 1.5% paraformaldehyde, the spinal cord was removed to be fixed routinely and then cut into sections for observing the labeled cells under microscope. RESULTS: In the ipsilateral ventral horn of the spinal cord, the motoneurons retrogradely labeled by CB-HRP formed dendritic projections oriented only to those motoneurons innervating the same meridian. In the longitudinal sections of spinal cord, the labeled motoneurons formed a bead-like column with a prominent network of longitudinal dendrites connecting the motoneurons innervating acupoints from the same meridian. In the transverse sections of spinal cord, two groups of dendrites from the labeled motoneurons projected to the identified sympathetic preganglionic regions: one group extended dorsolateraly to the intermediolateral gray, another group extended intermediolateraly toward the central canal. In rats with injection of CB-HRP into both acupoint regions and ipsilateral celiac ganglion, the dendrites originated from the labeled motoneurons projected directly to the labeled sympathetic preganglionic neurons. CONCLUSION: Each of the ST, BL and GB meridians is innervated by a specific group of motoneurons in the spinal cord. The motoneurons form a column with distinct border in the ventral horn of spinal cord, and the dendritic projections from the motoneurons oriented only to those innervating the same meridian. The dendrites from the meridian-related motoneurons can specifically project to the sympathetic preganglionic neurons at the thoracolumbar level.

A new animal model of trigeminal neuralgia produced by administration of cobra venom to the infraorbital nerve in the rat.

BACKGROUND: Understanding the mechanism of trigeminal neuralgia may be elucidated by developing laboratory animal models that closely mimic the features of this specific type of neuropathic pain. We have developed an experimental animal model for trigeminal neuralgia using a technique of injecting cobra venom into the infraorbital nerve (ION) trunk. METHODS: Male Sprague-Dawley rats were subjected to the administration of cobra venom or saline into the ION trunk. Mechanical stimuli were applied to the ION territory in consecutive days after surgery. Mechanical thresholds were measured over a 90-day period on the bilateral facial region. Vascular permeability in the ION territory was measured using Evans blue dye. RESULTS: The cobra venom-treated rats developed mechanical allodynia 3 days after surgery that lasted for 60 days postoperatively at the ipsilateral side. The mechanical thresholds of the contralateral ION territory also showed a profound decrease but were sustained for only approximately 30 days. There was no change of mechanical thresholds in the control groups. The extravasation of Evans blue increased significantly in the skin after administration of cobra venom to the ION compared with control rats (P < 0.05). CONCLUSION: The cobra venom model may provide a reasonable model for investigating the mechanism of trigeminal neuropathic pain.