Endoplasmic Reticulum Stress In Spinal Dorsal Horn Mediates Neuropathic Pain And Disinhibition Of The Spinal Nociceptive Circuitry

The underlying mechanism of spinal hypersenstivity in neuropathic pain is very complicated, studies based on neuropathic animals showed that spinal neurons and glial cells contribute to the process of spinal hypersensitivity. The signal pathway involved inclued: inflammatory signal pathway, changes of ligand and receptor functions, reactive oxygen species signal pathway. Endoplasmic reticulum stress(ER stress) is involved in many nerve system diseases, recent studies showed that ER stress contribued to the peripheral and central mechanism of neuropathic pain.In mammal cells, many factors can induce ER stress: inflammatory respond, hypoxia and oxidative stress can lead ER stress. ER stress triggers three unfolded protein respond(UPR) pathways: IER1 pathway, PERK pathway and ATF6 pathway. Activation of IRE1 axis initiates cell autophagy, which make cell capable of cleaning damaged organelles. Activation of PERK suspends translation, most mRNA translations are stopped to alleviate ER stress. Activation of ATF6 initiates proteolysis, regulates the process of proteolysis and lipid synthesis. Protein disulfide isomerase(PDI) is a multifunctional oxidoreductase and chaperone, PDI contributes to the process of oxidative protein folding and the production of ROS. During the process of ER stress, the expression of PDI is upregulated. So inhibiton of the function of PDI can protect the cell from injury.In this study, adult male wistar rats were used to establish neuropathic pain model. Changes in mechanical pain thresholds were observed, we also detected the expressions of ER stress signaling molecules. We tried to disclose the interaction between ER stress and pain behavior. We next observed changes in mechanical pain thresholds in neuropathic rats treated with PDI inhibitor, expressions of of ER stress signaling molecules were also detected. And we intrathecal injected ER stress inducer to normal rats, Changes in mechanical pain thresholds were observed, we also detected the expressions of ER stress signaling molecules. And we treated neuropathic rats with ROS scavanger, changes in mechanical thresholds and expressions of spinal ER stress signaling molecules were observed. Also, we found most spinal inhibitory interneuron showed ER stress, we observed the effect of ER stress on IPSCs of neurons of spinal dorsal horn using electrophysiological recording.1. Expressions of ER stress pathway molecules of neuropathic rat spinal dorsal horn were upregulatedAs compared with sham rat, mechanical threshold of spinal nerve ligated rat began to decline on day 3 post operation, and was significant lower than sham rat on day 7, 14, 21 post operation. Expressions of ER stress pathway molecules of neuropathic rat spinal dorsal horn were slightly upregulated 3 post operation, and was significant higher than sham rat on day 7, 14, 21 post operation.2. Intrathecal injection of PDI inhitibitor blocked spinal nerve liagation induced ER stress and alleviated pain behavior; intrathecal injection of ER stress inducer led to upregulated expression of ER stress pathway molecules and pain behavior in normal ratAs compared with saline-treated rat, mechanical threshold of bacitracin treated neuropathic rat was significant higher on day 3, 7, 14, 21 post operation. Expressions of ER stress pathway molecules in spinal dorsal horn of bacitracin treated neuropatihc rat were significant lower compared with saline treated rat.In normal rat, intrathecal injection of ER stress inducer significantly increased expressions of spinal ER stress pathway molecules and signicantly decreased mechanical pain threshold.3. Intrathecal injection of ROS scavenger could alleviate pain behavior in neuropathic rat and decrease the expressions of spinal ER stress pathway moleculesOn day 3, 7, 14, 21 post spinal nerve ligation, intrathecal injection of ROS scavenger significantly alleviated the mechanical pain behavior of rat. Compared with rat treated with saline, expression of spinal tyrosine-nitrated MnSOD in neuropathic rat treated with ROS scavenger was significantly lower. Expressions of ER stress pathway molecules in spinal dorsal horn of rat treated with ROS scanvenger were significant lower compared with saline treated rat.4. Most inhibitory interneurons in spinal dorsal horn of neuropathic rat showed ER stress, the frequency and the altitude of inhibitory interneurons were significantly decreased by ER stressWe showed in our immunofluorescence results that most GAD67 postitive cell showed expressions of ER stress pathway molecules. Only a few PKC? postive cell showed expressions of ER stress pathway molecules. We concluded that most spinal inhibitory interneurons in neuropathic rat showed ER stress.We showed in our electrophysiological results that the frequency and the altitude of IPSC in neuron of ER stress spinal slices were significantly lower compared with normal spinal slices. Capsacin could increased the frequency and the altitude of sIPSC in neurons of spinal slices, because spinal inhbitory interneurons expressed TRPV1. However, capsacin could significantly decreased the frequency and the altitude of m IPSC in neurons of spinal slices. Because the central fiber of primary afferent neuron expressed TRPV1.When delivering capsacin to ER stress spinal slices, the frequency and altitude of m IPSC recorded from dorsal horn neurons were decreased. The frequency and altitude of m IPSC recorded from ER stress slices were significantly lower compared with normal spinal slices.In summary, our study showed that spinal nerve liagation induced upregulated expression of ER stress pathway molecules in spinal doral horn, the expression levels of ER stress pathway molecules were tightly interrelated with pain behavior. In spinal dorsal horn of neuropathic rat, ROS expression and ER stress pathway molecules expressions interacted as both cause and effect, leading to spinal hypersensitivity. We also showed in our study that most inhibitory interneurons in spinal dorsal horn of neuropathic rats showed ER stress, which could decrease the activity of the cells, and contributed to spinal hypersensitivity.