The Role And Mechanism Of GLT-1/NR2B Regulate Visceral Hypersensitivity And Central Sensitivity Under PTSD Stress

Background and objective:Post-traumatic stress disorder(PTSD) is a psychological dysfunction syndrome after suffering serious physical or psychological trauma. Very recently, with the increasing of natural disasters, social violence, pressure of working life, the number of clinical cases associated with PTSD diseases significantly rises up. It is well-known that PTSD is associated with visceral hypersensitivity and central sensitivity. Previous study showed that the visceral hypersensitivity is one of the most important pathogenesis of functional gastrointestinal diseases(FGIDs) such as irritable bowel syndrome, PTSD and IBS, both of which share the similar mechanism and involve in correlated biological pathway. However, this underlying mechanism of visceral hypersensitivity driven central sensitivity at molecule level remains obscure. From what we observed, connectio n between visceral hypersensitivity and central sensitivity of spinal cord will play a pivotal role in PTSD.Glutamate was the important neurotransmitter in the transmission of nociceptive information of central nervous system, which transmitted pain’s signals by triggering glutamate receptors. N-methyl-d-aspartate receptor 2B(NR2B) involved in the formation and maintenance of the pain sensitization, when it was activated by glutamate. Some research reported that, NR2 B in spinal cord also involved in the regulation of pain hypersensitivity wiht rat models of visceral pain, inflammatory pain and neuropathic pain. Due to the lack of glutamate metabolic enzyme in the synaptic cleft nerve cells, glutamate in the central nervous system have been removed by glutamate transporters-1(GLT-1) of astrocytes. According to this hypothesis, GLT-1’s specific agonists can significantly alleviate hyperalgesia. Ceftriaxone(CTX) as a β- lactam antibiotics selectively induced GLT-1 gene transcription and translation, virtually modulating the expression of GLT-1.GLT-1 and NR2 B are two major members of glutamatergic pathway, which involved in the pain information transmission in central neuron system. We speculated that GLT-1 and NR2 B were closely correlated in participation of formation of visceral hyperalgesia-central sensitivity under PTSD stress in spinal. CTX impacted severely on GLT-1/NR2 B signaling pathway by mediating the protein expression level. This research plan to elucidate the underlying mechanism of GLT-1/NR2 B to visceral hypersensitivity disease.Experiment 1: Alterations of visceral sensitivity and effects of visceral anti-nociceptive of CTX following the exposure to PTSD-like stressMethodsThirty five adult male Sprague-Dawley rats were randomly divided into 5 groups: control group(n=7): intraperitoneal injection of saline; PTSD group(n=7): rats were given intraperitoneal injections of ovalbumin(OVA) and exposed to single-prolonged stress(SPS); CTX group(n=7): control rats were injected intraperitoneally CTX(200mg / kg) for 7 days; PTSD + CTX group(n=7): rats were given injections of OVA and exposed to SPS, then, injected intraperitoneally CTX(200mg / kg) for 7 days; PTSD + CTX + DHK groups(n=7): rats were treated as the same as PTSD + CTX groups, then, the rats were injected intraperitoneally with a dose of DHK(10 mg / kg) before CRD.The alterations of visceral sensitivity was evaluated by the visceromotor response(VMR) to graded CRD: electrodes were stitched into the external oblique musculature of rats, the grade CRD pressure was at the 20、40、60、80 mm Hg, VMR was evaluated by area under the curve(AUC) of electromyogram(EMG) following exposure to PTSD-like stress.ResultsVMR significantly increased in PTSD group compared with the control group at 20, 40, 60 and 80 mm Hg(all P<0.05); VMR significantly decreased in PTSD rats treated with CTX when compared with the PTSD rats at all graded CRD pressure(all P<0.01); however, DHK can reversed the role of anti-nociceptive by CTX at 80 mmHg(P<0.05). VMR significantly decreased in CTX group compared with the control group at 40, 60 and 80 mm Hg(all P<0.05).ConclusionsOVA and SPS can successfully establish PTSD model of visceral hypersensitivity, PTSD could induce visceral hypersensitivity; CTX can play a role of anti-nociceptive following exposure to PTSD; DHK which could selectively antagonize GLT-1 can reverse the anti-nociceptive effect of CTX.Experiment 2: Alteration of GLT-1 expression following exposure to PTSD in the spinal cord of rats and intervention effect of CTXMethodsForty two adult male Sprague-Dawley rats were randomly divided into 5 groups:: control group(n=8), PTSD group(n=9), CTX group(n=8), PTSD + CTX group(n=10), PTSD + CTX + DHK group(n=7), the rat models were established as the same as experiment 1.Spinal GLT-1 expression was evaluated by immunofluorescence using confocal laser scanning microscopy: the spinal GLT-1 expression was measured by GLT-1 mean optical density(AOD) of immunofluorescence.ResultsBy immunofluorescence analysis, AOD of the GLT-1 in the spinal cord was significantly decreased in PTSD rats compared with the control rats(P= 0.001); CTX-treated rats exhibited a increased GLT-1 expression in spinal cord compared with control group(P= 0.001); GLT-1 expression in the PTSD rats treated with CTX significantly increased compared with PTSD group(P = 0.004); there were no significant difference between PTSD + CTX group and PTSD + CTX + DHK group, control group.ConclusionsGLT-1 expression was significantly down-regulated exposure to visceral hypersensitivity of PTSD, on the contrary, CTX was up-regulated and increased in cellular system, suggesting that GLT-1 involved in visceral hypersensitivity of PTSD in spinal cord level.Experiment 3: the mechanism of CTX in NR2 B expression exposure to PTSDMethodsThirty five adult male Sprague-Dawley rats were randomly divided into 3 groups: control group(n=7), PTSD group(n=7), PTSD + CTX group(n=7), the rat models were established as the same as experiment 1.Spinal NR2 B expression was evaluated by immunofluorescence using confocal laser scanning microscopy: the spinal NR2 B expression was measured by AOD of NR2 B expression.ResultsBy immunofluorescence analysis, AOD of the NR2 B in the spinal cord was significantly increaseded in PTSD rats compared with the control rats(P=0.001); NR2 B expression in PTSD rats treated with CTX was significantly decreased compared with PTSD rats(P = 0.001); NR2 B expression in control group had no significant differences compared with PTSD + CTX group(P>0.05).ConclusionsFollowing exposure to PTSD, NR2 B expression was significantly up-regulated, in contrast, CTX can down-regulate the expression of NR2 B, which implied that NR2 B involved in the pathway regulating visceral hypersensitivity-central hyperalgesia exposure to PTSD. From what we observed, CTX may play an important in anti-nociception by affecting the expression of NR2 B.Full summary1. OVA and SPS successfully establish PTSD model of visceral hypersensitivity, and PTSD would induce visceral hypersensitivity;2. With exposure to visceral hypersensitivity of PTSD, CTX modulates anti-nociception by up-regulating the expression of GLT-1;3. GLT-1/NR2 B in spinal cord involved in the formation of visceral hypersensitivitycentral sensitivity following exposure to PTSD; CTX played anti-nociceptive role in up-regulating GLT-1 expression and indirectly down-regulating the expression of NR2 B.