Exendin-4 attenuates pain-induced cognitive impairment by alleviating hippocampal neuroinflammation in a rat model of spinal nerve ligation

Glucagon-like peptide-1 receptor has anti-apoptotic,anti-inflammatory,and neuroprotective effects.It is now recognized that the occurrence and development of chronic pain are strongly associated with anti-inflammatory responses;however,it is not clear whether glucagon-like peptide-1 receptor regulates chronic pain via anti-inflammatory mechanisms.We explored the effects of glucagon-like peptide-1 receptor on nociception,cognition,and neuroinflammation in chronic pain.A rat model of chronic pain was established using left L5 spinal nerve ligation.The glucagon-like peptide-1 receptor agonist exendin-4 was intrathecally injected into rats from 10 to 21 days after spinal nerve ligation.Electrophysiological examinations showed that,after treatment with exendin-4,paw withdrawal frequency of the left limb was significantly reduced,and pain was relieved.In addition,in the Morris water maze test,escape latency increased and the time to reach the platform decreased following exendin-4 treatment.Immunohistochemical staining and western blot assays revealed an increase in the numbers of activated microglia and astrocytes in the dentate gyrus of rat hippocampus,as well as an increase in the expression of tumor necrosis factor alpha,interleukin 1 beta,and interleukin 6.All of these effects could be reversed by exendin-4 treatment.These findings suggest that exendin-4 can alleviate pain-induced neuroinflammatory responses and promote the recovery of cognitive function via the glucagon-like peptide-1 receptor pathway.All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Renmin Hospital of Wuhan University of China(approval No.WDRM 20171214)on September 22,2017.

c-Fos expression within the L_5 spinal cord dorsal horn after spinal nerve ligation in rats Is intraplantar administration of glutamate different from intrathecal administration?

BACKGROUND： Injection of glutamate （Glu） in normal animals can cause neuronal c-Fos expression; however, whether Glu can induce spinal neuronal c-Fos expression in pain models is unclear. OBJECTIVE： To examine the effects of intraplantar and intrathecal injection of Glu on c-Fos expression in the L5 spinal cord dorsal horn Ⅰ/Ⅱ and Ⅲ/Ⅳ layers after spinal nerve ligation, and to study the effects of the N-methyI-D-aspartic acid （NMDA） receptor antagonist, D-2-amino-5-phosphonopentanoate （D-AP5）, and a selective group I mGluR antagonist, 7-hydroyiminocyclo propan[a]chromen-lacarboxylic acid ethyl ester （cpccoEt）. DESIGN, TIME AND SETTING： A randomized, controlled animal study was performed at the Department of Pharmacology, Oral Anatomy, and Neurobiology, Osaka University Graduate School of Dentistry, from December 2005 to December 2006. MATERIALS： Glu （5 μmol）, D-AP5 （50 nmot） and cpccoEt （250 nmol） were provided by Wako Pure Chemical Industries, Osaka, Japan, and diluted in saline （50 μL）. The pH of all solutions was adjusted to 7.4. METHODS： Twelve rats were randomly divided into sham operation （n = 6） and spinal nerve ligation （SNL; n = 6） groups for behavioral assessments of neuropathic pain after ligation surgery of the left L5-6 nerve segment. Another 60 rats were randomly divided into sham operation, SNL, saline-intraplantar, saline-intrathecal, Glu-intraplantar, Glu-intrathecal, D-AP5-intrathecal, Glu-D-AP5-intrathecal, cpccoEt-intrathecal, and Glu-cpccoEt-intrathecal groups, with 6 rats in each group. All groups except sham operation group received a similar SNL. On day 14, rats received a 50-μL injection of saline, Glu, D-AP5, and/or cpccoEt into the left intraplantar or intrathecal L5-4 segments. MAIN OUTCOME MEASURES： The number of c-Fos positive neurons in both Ⅰ/Ⅱ and Ⅲ/Ⅳ spinal layers at L6 was observed using immunohistochemistry 2 hours after administration. RESULTS： （1） SNL increased the level of c-Fos expression in two sides of the spinal cord, particularly on Ⅲ/Ⅳ spinal layers of the ligated side. （2） Intraplantar or intrathecal administration of saline significantly increased the c-Fos labeled neurons in Ⅰ/Ⅱ spinal layers of the ligated side, compared with SNL alone （P 〈 0.01）. （3） Intraplantar Glu （5 μmol） increased the number of c-Fos positive neurons in Ⅰ/Ⅱ spinal layers compared with intraplantar saline （P〈 0.01）. （4） The number of c-Fos neurons in Ⅰ/Ⅱ spinal layers on both the ipsilateral and contralateral side after intraplantar Glu was lower than intrathecal Glu （P〈 0.01）, with a 3-fold higher induction by intrathecal Glu. （5） Co-administration of D-AP5 or cpccoEt reduced the effects of intrathecal Glu （P 〈 0.01）. CONCLUSION： Intrathecal Glu increases c-Fos expression more than intraplantar Glu. Antagonists of NMDA and group I mGluRs block this effect.

Electrophysiological properties of spinal wide dynamic range neurons in neuropathic pain rats following spinal nerve ligation

Objective The present study aimed to investigate the electrophysiological properties of wide dynamic range （WDR） neurons in spinal dorsal horn of rats with neuropathic pain induced by lumber 5 （L5） spinal nerve ligation （SNL） in a large size of samples.Methods Adult Sprague-Dawley rats were divided into normal and SNL groups.Electrophysiological technique was used to record the characteristics of WDR neurons in the spinal dorsal horn.Results Compared with the WDR neurons in normal rats,the WDR neurons in SNL rats showed an increase in excitability,manifested by an enlargement of the receptive field size,an increase in the proportion of neurons that exhibited spontaneous activities,decreases in the Cresponse threshold and latency,and an increase in the C-response duration.In addition,the numbers of A-and C-fiberevoked discharges were smaller in SNL rats than in normal rats.Conclusion The excitability of spinal WDR neurons increased in rats with neuropathic pain induced by L5 SNL.The increase in excitability of WDR neurons may contribute to the development of neuropathic pain.

High-frequency spinal cord stimulation produces longlasting analgesic effects by restoring lysosomal function and autophagic flux in the spinal dorsal horn

High-frequency spinal cord stimulation(HF-SCS) has been established as an effective therapy for neuropathic pain. However, the analgesic mechanisms involved in HF-SCS remain to be clarified. In our study, adult rat neuropathic pain was induced by spinal nerve ligation. Two days after modeling, the rats were subjected to 4 hours of HF-SCS(motor threshold 50%, frequency 10,000 Hz, and pulse width 0.024 ms) in the dorsal horn of the spinal cord. The results revealed that the tactile allodynia of spinal nerve-injured rats was markedly alleviated by HFSCS, and the effects were sustained for 3 hours after the stimulation had ceased. HF-SCS restored lysosomal function, increased the levels of lysosome-associated membrane protein 2(LAMP2) and the mature form of cathepsin D(matu-CTSD), and alleviated the abnormally elevated levels of microtubule-associated protein 1 A/B-light chain 3(LC3)-II and sequestosome 1(P62) in spinal nerve-injured rats. HF-SCS also mostly restored the immunoreactivity of LAMP2, which was localized in neurons in the superficial layers of the spinal dorsal horn in spinal nerve-injured rats. In addition, intraperitoneal administration of 15 mg/kg chloroquine for 60 minutes reversed the expression of the aforementioned proteins and shortened the timing of the analgesic effects of HF-SCS. These findings suggest that HF-SCS may exhibit longlasting analgesic effects on neuropathic pain in rats through improving lysosomal dysfunction and alleviating autophagic flux. This study was approved by the Laboratory Animal Ethics Committee of China Medical University, Shenyang, China(approval No. 2017 PS196 K) on March 1, 2017.

CYP1B1-derived epoxides modulate the TRPA1channel in chronic pain

Pain is often debilitating,and current treatments are neither universally efficacious nor without risks.Transient receptor potential(TRP)ion channels offer alternative targets for pain relief,but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain.Here,transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation(SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid(EET)and 19,20-EpDPA post injury.Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro,and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia(DRG)neurons.TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079,and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1.This latter effect mapped to residues Y933,G939,and S921 of TRPA1.Intra-plantar injection of 19,20-EpDPA induced acute mechanical,but not thermal hypersensitivity in mice,which was also blocked by A967079.Similarly,Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury.These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Antinociceptive Effect of Najanalgesin from Naja Naja Atra in a Neuropathic Pain Model via Inhibition of c-Jun NH2-terminal Kinase

Background： Najanalgesin, a toxin isolated from the venom ofNaja nqja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect ofnajanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain. Methods： The antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase （ERK）, and c-Jun N-terminal kinase （JNK） by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry. Results： The phosphorylation levels of INK （as well as its downstream molecule c-Jun）, p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord. Conclusion： The antinociceptive effect of najanalgesin thnctions by inhibiting the JNK in a neuropathic pain model.