腰段脊髓背角胶质细胞对内脏痛觉敏化反应的形态学研究
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摘要
目的探察和证实胶质细胞对创伤后应激障碍(PTSD)所属内脏痛觉敏化反应的时程和形态特征。方法借助单次延长应激(SPS)方法构建内脏痛觉敏化模型,32只雄性SD大鼠被随机分成正常对照组、SPS术后1 d组、SPS术后7 d组、SPS术后14 d组,应用免疫组化、免疫印迹技术探测腰段脊髓背角星形胶质细胞和小胶质细胞形态结构和蛋白水平的变化。结果免疫组化实验显示,正常脊髓灰质内明显可以探察到大量GFAP+的细胞,其形态呈星形胶质细胞的典型特征:即微小的胞体和细长的突起,其在脊髓灰质的分布不显示明显的区域特性。定量和比较资料显示,SPS不同时间组的GFAP+细胞密度均多于对照组,7 d组增加最显著。结果同时显示,GFAP~+胞体大小在7 d和14 d组均大于对照组,以7 d组的最大。正常脊髓灰质也明显可以探察到小胶质细胞(Iba1~+),其密度和胞体大小在SPS 1 d组和14 d组与对照组之间具有显著差异,且14 d组差异最为显著(P<0.05),但7 d组的变化不明显。免疫印迹实验显示,正常脊髓灰质背角可以明显探测到GFAP和Iba1蛋白。定量资料显示,SPS 7 d组和14 d组的GFAP蛋白水平均高于对照组,7 d组升高最显著。SPS不同时间点均可诱导Iba1~+蛋白的高表达,且以14 d组最显著。结论结果提示脊髓星形胶质细胞和小胶质细胞的形态结构和蛋白水平的变化可能牵涉到内脏痛觉敏化的病理过程。
Objective Glial cells are involved in the physiological and pathological mechanisms of the spinal cord.The current study aims to investigate and confirm the timing and morphological characteristics of glial cells in visceral sensitization in post-traumatic stressSD male rats were randomly divided into 4 groups:Ctrl,Day 1,Day 7,and Day 14 group(n=8),and immunohistochemistry and western blot were used to detect the morphological structure and protein levels of astrocytes and microglia in the dorsal horn of lumbar spinal cord in SPSistry showed that a large number of GFAP+cells can be clearly detected in normal spinal gray matter,which presents typical morphology of astrocytes:tiny cell bodies and thin protuberances,and there was no obvious regional characteristics of distribution in the spinal cord.Quantitative and comparative data revealed that,the density of GFAP+cells in the SPS groups at different times was higher than control group,and the SPS 7 day group were highest.At the same time,the body size of GFAP+cells in the 7 day group and 14 day group were larger than control group,the 7 day group had the largest size.The microglial cells(Iba1+)can also be detected in normal spinal gray matter.Compare with control group,there were significant increase in the density and volume for the SPS 1 day group and the 14 day group,and most notably in the SPS 14 day group(P<0.05).However,control group and SPS 7 day group showed no significant difference(P<0.05).GFAP and Iba1proteins were clearly detected by Western blot,quantitative data display that the GFAP protein levels in the SPS 7 day group and the 14 day group were higher than control group,and the 7 day group was most obviously.The expression of Iba1 protein were higher after SPS than control group,and the most notably in the 14 daypresent results suggested that changes in the morphological structure and protein levels of astrocytes and microglia in spinal cord may be involved in the pathological process of visceral sensitization.
Objective Glial cells are involved in the physiological and pathological mechanisms of the spinal cord.The current study aims to investigate and confirm the timing and morphological characteristics of glial cells in visceral sensitization in post-traumatic stressSD male rats were randomly divided into 4 groups:Ctrl,Day 1,Day 7,and Day 14 group(n=8),and immunohistochemistry and western blot were used to detect the morphological structure and protein levels of astrocytes and microglia in the dorsal horn of lumbar spinal cord in SPSistry showed that a large number of GFAP+cells can be clearly detected in normal spinal gray matter,which presents typical morphology of astrocytes:tiny cell bodies and thin protuberances,and there was no obvious regional characteristics of distribution in the spinal cord.Quantitative and comparative data revealed that,the density of GFAP+cells in the SPS groups at different times was higher than control group,and the SPS 7 day group were highest.At the same time,the body size of GFAP+cells in the 7 day group and 14 day group were larger than control group,the 7 day group had the largest size.The microglial cells(Iba1+)can also be detected in normal spinal gray matter.Compare with control group,there were significant increase in the density and volume for the SPS 1 day group and the 14 day group,and most notably in the SPS 14 day group(P<0.05).However,control group and SPS 7 day group showed no significant difference(P<0.05).GFAP and Iba1proteins were clearly detected by Western blot,quantitative data display that the GFAP protein levels in the SPS 7 day group and the 14 day group were higher than control group,and the 7 day group was most obviously.The expression of Iba1 protein were higher after SPS than control group,and the most notably in the 14 daypresent results suggested that changes in the morphological structure and protein levels of astrocytes and microglia in spinal cord may be involved in the pathological process of visceral sensitization.
引文
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[9]陈智,郑雪峰,朱耀峰,等.成年大鼠纹状体nNOS阳性神经元的形态特征及其对DA剥夺的反应[J].解剖学研究,2018(02):81-85.
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[11]Zhuang ZY,Gerner P,Woolf CJ,et al.ERK is sequentially activated in neurons,microglia,and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model[J].Pain,2005,114(1-2):149-159.
[12]Garrison CJ,Dougherty PM,Kajander KC,et al.Staining of glial fibrillary acidic protein(GFAP)in lumbar spinal cord increases following a sciatic nerve constriction injury[J].Brain Res,1991,565(1):1-7.
[13]Tichauer J,Saud K,von Bernhardi R.Modulation by astrocytes of microglial cell-mediated neuroinflammation:effect on the activation of microglial signaling pathways[J].Neuroimmunomodulation,2007,14(3-4):168-174.
[14]Miller TR,Wetter JB,Jarvis MF,et al.Spinal microglial activation in rat models of neuropathic and osteoarthritic pain:an autoradiographic study using[3H]PK11195[J].Eur J Pain,2013,17(5):692-703.
[15]Romero-Sandoval A,Chai N,Nutile-Mcmenemy N,et al.A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain[J].Brain Res,2008,1219:116-126.
[2]He YQ,Lang XQ,Lin L,et al.P2X3 receptor-mediated visceral hyperalgesia and neuronal sensitization following exposure to PTSD-like stress in the dorsal root ganglia of rats[J].Neurogastroenterol Motil,2017,29(3).
[3]Ji RR,Xu ZZ,Gao YJ.Emerging targets in neuroinflammation-driven chronic pain[J].Nat Rev Drug Discov,2014,13(7):533-548.
[4]Ellis A,Bennett DL.Neuroinflammation and the generation of neuropathic pain[J].Br J Anaesth,2013,111(1):26-37.
[5]Sun R,Zhang Z,Lei Y,et al.Hippocampal activation of microglia may underlie the shared neurobiology of comorbid posttraumatic stress disorder and chronic pain[J].Mol Pain,2016,12.
[6]张佐霞,刘玥,孙绕,等.脊髓星形胶质细胞在创伤后应激障碍诱发痛觉过敏中的作用[J].中华麻醉学杂志,2017,37(10):1230-1232.
[7]Wang HN,Peng Y,Tan QR,et al.Quetiapine ameliorates anxiety-like behavior and cognitive impairments in stressed rats:implications for the treatment of posttraumatic stress disorder[J].Physiol Res,2010,59(2):263-271.
[8]Schwetz I,Bradesi S,Mcroberts JA,et al.Delayed stress-induced colonic hypersensitivity in male Wistar rats:role of neurokinin-1 and corticotropin-releasing factor-1 receptors[J].Am J Physiol Gastrointest Liver Physiol,2004,286(4):G683-G691.
[9]陈智,郑雪峰,朱耀峰,等.成年大鼠纹状体nNOS阳性神经元的形态特征及其对DA剥夺的反应[J].解剖学研究,2018(02):81-85.
[10]Clark AK,Old EA,Malcangio M.Neuropathic pain and cytokines:current perspectives[J].J Pain Res,2013,6:803-814.
[11]Zhuang ZY,Gerner P,Woolf CJ,et al.ERK is sequentially activated in neurons,microglia,and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model[J].Pain,2005,114(1-2):149-159.
[12]Garrison CJ,Dougherty PM,Kajander KC,et al.Staining of glial fibrillary acidic protein(GFAP)in lumbar spinal cord increases following a sciatic nerve constriction injury[J].Brain Res,1991,565(1):1-7.
[13]Tichauer J,Saud K,von Bernhardi R.Modulation by astrocytes of microglial cell-mediated neuroinflammation:effect on the activation of microglial signaling pathways[J].Neuroimmunomodulation,2007,14(3-4):168-174.
[14]Miller TR,Wetter JB,Jarvis MF,et al.Spinal microglial activation in rat models of neuropathic and osteoarthritic pain:an autoradiographic study using[3H]PK11195[J].Eur J Pain,2013,17(5):692-703.
[15]Romero-Sandoval A,Chai N,Nutile-Mcmenemy N,et al.A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain[J].Brain Res,2008,1219:116-126.