HCN2 Channel Mediates Visceral Hypersensitivity And Central Sensitization Following Exposure To PTSD-like Stress In The Brain And Spinal Cord Of Rats

BackgroundPosttraumatic stress disorder(PTSD) refers to psychological disorders which was a long-term and aftermath phenotype after people suffered a serious blow. It causes pathophysiological changes to body and induces the formation of visceral sensitivity, but the underlying mechanism remained obscure. Very recently, the occurrings of PTSD-related diseases are soaring up, which seriously impacted on human life and caused the increasing of suicide rate. Unfortunately, there is no effective measure to cont rol traumatic aftermath and post-effect. Currently, more efforts have been emphasized on elucidation of mechanism of PTSD and potential drug treatment, allowing the prevention of visceral sensitivity. It assisted the drug design in near future by providing an applicable target.Hyperpolarizaion-activated cyclic nucleotide-gated channels(HCN2) widely distributed in the brain and spinal cord which associated with modulating visceral pain region. Studies showed that HCN2 were abundantly expressed in the spinal cord and brain in the chronic constriction injury rats model when screened with immunohistochemical approaches. Intriguingly, ZD7288, the suppression reagent of HCN2 channel can block the hyperpolarization-activated current(Ih) and significantly relieve the pain. Therefore, we hypothesized that HCN2 channel acted an important role in the formation of visceral hypersensitivity- central sensitization system in PTSD-like stress. Potentially, it considered to be a new target for specific drug design.N-methyl D-aspartate receptor subtype 2B(NR2B) is also widely expressed in the spinal cord and the hippocampus. HCN2 channel activated in the presynaptic neurons can cause the release of glutamate in co-cultured spinal cord dorsal horn neurons model and induce changes of NR2B-mediated in synaptic plasticity. This led to tyrosine phosphorylation of NR2 B that closely participated in the cause of pain and formation of central sensitization. In this study, a pain model encompassing HCN2 channel and Ih currents, NR2 B has been proposed to explain the formation of visceral hyperalgesia and central sensitization in the spinal cord and hippocampus in PTSD-like stress.Thus, in present study, we utilized technology ofthe intrathecal catheter implantation for drug delivery.Visceral sensitivity was quantitatively evaluated by grading and phasic CRD, and immunofluorescence staining. Confocal laser-induced fluorescence microscopy is employed to quantify the expression of HCN2 and NR2 B in the spinal cord, as well as in brain. In addition, anti-nociceptive of CTX is used to mediate the HCN2 expression, we here propose the research program aimed to elucidate the molecular mechanism of HCN2 and NR2 B in regulatingcentral sensitization and to monitor the sensitizing process under the circumstance of PTSD, providing a specific molecular mechanism for visceral hypersensitivity and central sensitization, which would pave a new way for therapeutic strategies for pain and concurrent PTSD-like disorder.Part 1: Alteration of visceral sensitivity under the circumstance of PTSDObjectiveTo illustrate the changes of visceral sensitivity in PTSD rats and the anti-nociceptive effect of visceral after treatment with by HCN2 bolcker, ZD7288.Methods1) Experimental design and animal group: The 22 of female adult rats with SD syndrome were randomly selected to represent the entire study pool, weight ranged from 150 to 200 g. All rats were separated into three independent groups, namely, control(7), PTSD(8) and PTSD + ZD7288(7). Control group without any treatment; PTSD group was treated with a single-prolonged stress model and electric stimulations, which induced the formation of visceral hypersensitivity model of PTSD; The last group was shot with HCH2 inhibitor solution(ZD7288, 100nml/L) in 7 days after PTSD.2) Assessment of visceral sensitivity under the circumstance of PTSD: The changes of visceral sensitivity was characterized by visceromotor reflex(VMR) that was induced by colorectal distension(CRD). Same strategy was applied to evaluate the effect of treatment of ZD7288, the channel blocker. The area under the curve(AUC) is integrated from electromyogram(EMG) by SPIKE2, indicating the level of visceral sensitivity.ResultsThe area under the curve of PTSD rats significantly increased relative to control rats at 40 mm Hg CRD(0.6200 ± 0.0278 vs. 0.3786 ± 0.0155, P < 0.001). The AUC of PTSD group also was significantly higher than control group at 60 mm Hg CRD(0.7663 ± 0.0262 vs. 0.5271 ± 0.0212, P < 0.001). The AUC of the PTSD rats treated with ZD7288 was significantly decreased compared with PTSD rats at 20、40 and 60 mm Hg CRD(0.2180 ± 0.0159 vs. 0.2913 ± 0.0229, P = 0.005)、(0.4618 ± 0.0157 vs. 0.6200 ± 0.0278, P < 0.001)、(0.6023 ± 0.0173 vs. 0.7663 ± 0.0262, P < 0.001).ConclusionThe sensitization modeling system was successfully established using SPS method and electric stimuli. The rats of PTSD had anti-nociceptive effect of visceral after treatment by HCN2 blocker, ZD7288.Part 2: Quantitatively determining protein expression related to HCN2 under the stimuli of PTSD.Objective: To quantitatively determine protein expression of HCN2 in the thoracic, lumbar spinal cord and cerebral cortex, hippocampus under PTSD-like stress.Methods1) Experimental animals and grouping: The 14 of female adult rats with SD syndrome were randomly selected to represent the entire study pool, weight ranged from 150 to 200 g. All rats were separated into two independent groups, namely, control(7) and PTSD(7). Control group without any treatment; PTSD group, using the method to establish PTSD model as before.2) Using immunofluorescence and confocal laser technology to study the changes of expression of HCN2 in the spinal cord and brain. Calculating the average optical density(AOD) of HCN2 expression by Image-Pro Plus6.0.Results2.1 The expression of HCN2 in the lumbar spinal cord and in visceral hypersensitivity model of PTSD rats.HCN2 neurons in visceral hypersensitivity model of PTSD group concentrated in the I-II layer of lumbar spinal cord dorsal horn, but in spinal cord anterior horn was als o a small number distributed by immunofluorescence and confocal laser technology. The AOD of HCN2 expression was significantly increased compared with the control group(121.12 ± 4.85 vs. 78.05 ± 6.49, P <0.001).2.2 The expression of HCN2 in the thoracic spinal cord in visceral hypersensitivity model of PTSD rats.HCN2 neurons in visceral hypersensitivity model of PTSD group concentrated in the I-II layer of thoracic spinal cord dorsal horn, but in spinal cord anterior horn was also a small number distributed by immunofluorescence and confocal laser technology. The AOD of HCN2 expression was significantly increased compared with the control group(124.44 ± 4.63 vs. 70.13 ± 5.46,P < 0.001).2.3 The expression of HCN2 in hippocampus in visceral hypersensitivity model of PTSD rats.There were relatively higher HCN2 expression in hippocampus under visceral hypersensitivity model of PTSD group, characterized by immunofluorescen ce and confocal laser technology. It was mapped with green fluorescence, exhibiting the 3-dimensional density plot. The AOD of its expression was significantly increased compared with the control group(95.49 ± 4.64 vs. 65.36 ± 4.34,P < 0.01).2.4 The expression of HCN2 in prefrontal cortex under visceral hypersensitivity model of PTSD rats.There were a lot of HCN2 neurons expression in prefrontal cortex in visceral hypersensitivity model of PTSD group by immunofluorescence and confocal laser technology, and the AOD of its expression was significantly increased compared with the control group(122.39 ± 4.33 vs. 96.28 ± 5.78,P < 0.001).ConclusionBased on experimental results, drastic increase of HCN2 was observed when comparing with control samples of rat tissue, brain and spinal cord, indicating that HCN2 involved into the pathway of formation of sensitization center and mediated the process.Part 3: Modulating HCN2 protein expression using CTX and determining the impact on pain formation.Objective: β- lactam antibiotics, ceftriaxone(CTX) down-regulated the expression of HCN2 in spinal cord and its anti-nociceptive effect.1) Experimental animals and grouping: The 44 of female adult rats with SD syndrome were randomly selected to represent the entire study pool, about 150~200g. All rats were separated into three independent groups, namely, control(14, seven of the rats for immunofluorescence and the others for assessment of visceral sensitivity), PTSD(15, seven of the rats for immunofluorescence and the others for assessment of visceral sensitivity) and PTSD + CTX(15, seven of the rats for immunofluorescence and the others for assessment of visceral sensitivity). Control group without any treatment; PTSD group, using the method to establish PTSD model as before; PTSD+CTX group, CTX(200 mg / kg, 7d) were intraperitoneal injected to rats after PTSD-like stress.2) Using the method of CRD-VMR to measure the changes of visceral sensitivity in the rats.3) Using immunofluorescence and confocal laser scanning technology to study the changes of HCN2 expression in the spinal cord to the PTSD rats treated with CTX.ResultsAt 20 mm Hg CRD, the AUC of PTSD+CTX group also was significantly lower than PTSD group(0.2175 ± 0.0090 vs. 0.2913 ± 0.0229, P = 0.005); at 40 mm Hg CRD, the AUC of PTSD + CTX group was significantly decreased compared with PTSD group(0.5038 ± 0.0336 vs. 0.6200 ± 0.0278, P = 0.006); Compared with PTSD group, the AUC of PTSD+CTX group was significantly decreased at 60 mm Hg CRD(0.6400 ± 0.0245 vs. 0.7663 ± 0.0262, P = 0.001). Immunofluorescence showed that the AOD of HCN2 expression in PTSD + CTX group was significantly decreased compared with the PTSD group(98.24 ± 5.86 vs. 121.12 ± 4.85,P = 0.012)by immunofluorescence and confocal laser technology.ConclusionCTX may play an anti-nociceptive effect of visceral by down-regulating the HCN2 expression.Part 4: HCN2 and NR2 B coordination regulating the mechanisms of visceral hypersensitivity in the spinal cord and the hippocampus under the stimuli of PTSD.Objective: To study the changes of HCN2 and NR2 B co-expression in the spinal cord and hippocampus under the stimuli of PTSD. Methods1) Experimental animals and grouping: The 13 of female adult rats with SD syndrome were randomly selected to represent the entire study pool, weight ranged from 150 to 200 g. All rats were separated into two independent groups, namely, control(6) and PTSD(7). Control group without any treatment; PTSD group, using the method to establish PTSD model as before.2) Using immunofluorescence and confocal laser technology to study the changes of expression of HCN2 and NR2 B in the spinal cord and hippocampus. Calculating the AOD of HCN2 and NR2 B expression by Image-Pro Plus6.0.Results1) The co-expression of HCN2 and NR2 B in the spinal cord.To study the HCN2 and NR2 B co-expression between control group and PTSD group in the spinal cord, the results of statistical analysis showed that the AOD of HCN2 and NR2 B co-expression was significantly increased compared with the control group(119.83 ± 3.13 vs. 79.33 ± 3.52, P < 0.01)与(90.83± 4.35 vs. 64.83± 3.40, P < 0.01).2) The co-expression of HCN2 and NR2 B in the hippocampus.To study the HCN2 and NR2 B co-expression between control group and PTSD group in the hippocampus, the results of statistical analysis showed that the AOD of HCN2 and NR2 B co-expression was significantly increased compared with the control group(96.17 ±3.47 vs. 63.50± 3.84,P < 0.01)与(114.17 ± 3.52 vs. 75.83± 4.12,P < 0.01).ConclusionHCN2 and NR2 B co-expression was increased in the spinal cord and hippocampus under the stimuli of PTSD. HCN2 and NR2 B co-regulation the formation of the molecular mechanisms of visceral hypersensitivity- central sensitization under the stimuli of PTSD in the central level.Methods...