【作者】 汪静；

【导师】 王志平；

【作者基本信息】 兰州大学 ， 外科学， 2012， 博士

【摘要】 第一部分星形胶质细胞和A转运系统在福尔马林诱导的大鼠炎性持续性痛觉过敏中的作用目的：评价星形胶质细胞和A转运系统在福尔马林诱导的大鼠炎性持续性痛觉过敏中的作用。方法：建立炎性持续性痛动物模型,即大鼠足底注射福尔马林(5%,50μ1)。行为学检测,实验当天,使大鼠适应实验环境15-20分钟,行右后爪足底皮下注射(s.c.)5%的福尔马林50μ1,接着立即将大鼠放回笼子中,通过计量福尔马林注射后1小时内大鼠每5分钟舔咬注射后爪的持续时间和缩腿反射的次数来衡量自发性伤害性行为变化。形态学检测,应用免疫组织化学抗生物素蛋白-生物素-过氧化物酶(ABC)法,DAB显色来检测腰段脊髓背角星形胶质细胞标记物(GFAP)的表达,待所有的行为学检测结束后(即福尔马林注射1小时后),处死所有的大鼠,ABC法进行GFAP染色。结果：大鼠足底注射福尔马林(5%,50μ1),即刻引起典型的双时相伤害感受性行为,包括：大鼠舔／咬被注射爪、缩腿反射,及脊髓背角神经胶质酸性蛋白(GFAP,是星形胶质细胞被活化后的一个标志性蛋白)的表达增高,而这些效应可以被鞘内注射A转运系统的竞争性抑制剂(MeAIB,0.1,0.3,0.5,0.7mmol)所抑制,并且呈剂量依赖关系,而鞘内注射PBS的对照组对福尔马林诱导的伤害性行为和腰段脊髓背角GFAP的增高性表达无影响。结论：星形胶质细胞和A转运系统参与福尔马林诱导的大鼠炎性持续性痛的形成,抑制A转运系统能够抑制福尔马林诱导的脊髓中枢敏化和痛觉过敏。第二部分内源性大麻素通过调控星形胶质细胞参与抑制福尔马林诱导的大鼠炎性持续性痛觉过敏目的：通过阻断内源性大麻素受体,观察内源性大麻素和星形胶质细胞在福尔马林诱导的炎性持续性痛觉过敏中的作用。方法：通过大鼠足底注射福尔马林(5%,50μ1),建立炎性持续性痛动物模型,观察大麻素受体亚型(CB1和CB2)在脊髓背角的分布情况,进一步观察这两个受体亚型与神经细胞(星形胶质细胞、小胶质细胞和神经元)的共存情况,同时观察了MAPK途径与神经细胞共存情况；联合运用大麻素CB1和CB2受体拮抗剂／反向激动剂(AM281和AM630,1mg.kg-1each,腹腔注射,500μ1)混合液,完全阻断内源性大麻素受体亚型(CB1和CB2),观察对福尔马林诱导的急性期自发伤害性行为(舔咬爪和缩腿反射)的影响和慢性期诱发伤害性行为(50%的爪缩阈值,PWT)的影响,形态学观察大麻素受体亚型(CB1和CB2)和神经细胞的共存情况,同时观察MAPK途径与神经细胞共存情况。结果：完全阻断CB1和CB2受体后,易化了福尔马林诱导的急性期自发伤害性行为(舔咬爪和缩腿反射)和慢性期诱发伤害性行为(50%的爪缩阈值),与实验对照组(Formalin+12%DMSO)之间比较有统计学差异(p<0.05),并且更易出现镜像痛；形态学显示腰段脊髓背角星形胶质细胞标志物-GFAP的表达较实验对照组更明显；大麻素受体CB1随着时间延长持续性增高表达,并且主要与星形胶质细胞和小胶质细胞共表达,在第3d,有少量CB1受体与神经元共表达；CB2受体在完全空白组不表达,在实验对照组观察的7d内持续表达,在完全阻断CB1和CB2受体后,CB2受体的表达进一步增强,并主要与星形胶质细胞共表达。在实验对照组和实验组均观察到MAPK途径中的pJNK和p-p38表达,pJNK主要与星形胶质细胞共表达,而p-p38主要与小胶质细胞共表达。结论：研究结果显示,内源性大麻素通过星形胶质细胞和小胶质细胞上的CB1和CB2受体激活JNK和p-p38信号转导通路参与抑制福尔马林诱导的炎性持续性痛觉过敏,在炎性持续性痛敏情况下,可以通过提高内源性大麻素水平达到镇痛效果。更多还原

【Abstract】 AMINO ACID TRANSPORT SYSTEM A IS INVOLVED IN INFLAMMATORY NOCICEPTION IN RATS (Part1)Objective:The current study investigated the role of amino acid transport system A in central sensitization and hyperalgesia induced by intraplantar injection of formalin in rats.Methods:The rat was removed from this chamber and5%formalin (50μ1) was administered subcutaneously (s.c.) into the right hindpaw pad. The rat was immediately replaced in the chamber. Spontaneous nociceptive behaviors were assessed by measuring the duration of licking/biting and the number of flinches every5min in the injected hindpaw over a period of1h after formalin injection by two experimenters, as reported previously. After the completion of behavioral testing (i.e.,1h after formalin injection), all rats were sacrificed for GFAP staining using immunohistochemistry. The sections were used for immunohistochemical staining of GFAP using the avidin-biotin-peroxidase (ABC) method for GFAP staining.Results:Formalin (5%,50μ1) injected subcutaneously into the unilateral hindpaw pad induced typical biphase nociceptive behaviors, including licking/biting and flinching of the injected paw and an increase of glial fibrillary acid protein (GFAP, an activated astrocyte marker) expression in spinal dorsal horn, and these effects could be attenuated by intrathecal injection of the competitive inhibitor of amino acid system A transporter, methylaminoisobutyric acid (MeAIB,0.1,0.3,0.5, and0.7mmol). in a dose-dependent manner. Intrathecal injection of vehicle (PBS) had no effect on the formalin-induced nociceptive behaviors and increase of the GFAP.Conclusion:These findings suggest that amino acid transport system A is involved in inflammation-induced nociception, and inhibition of this transporter system results in inhibition of the central sensitization and hyperalgesia. INVOLVEMENT OF ENDOCANNABINOID IN THE CONTINUOUS INFLAMMATORY PAIN INDUCED BY FORMALIN THROUGH MODULATING ASTROCYTES (Part2)Objective:To investigate the roles of endocannabionoid and astrocytes in continuous inflammatory pain induced by formalin through blockade of endocannabionoid receptors (CB).Materials and methods:Continuous inflammatory pain model was established through intraplantar injection of formalin (5%,50μ1). The distribution of CB subtypes (CB1, CB2) in glial cells (microglia and astrocyte) and neurons of spinal dorsal horn and the MAPK pathway were observed through immunohistochemistry. CB antagonists (AM281, AM630,1mg/kg in500μ1, intraperitoneal) were administrated to observe the changes of formalin-evoked nociceptive responses at acute stage (licking/biting paws and twitching reflex) and allodynia at chronic stage (50%paw withdrawal threshold, PWT), and the distribution of CB1and CB2in glial cells neurons.Results:Blockade of CB1and CB2facilitated the spontaneous pain at acute stage (licking/biting paws and twitching reflex) and allodynia at chronic stage (50%paw withdrawal threshold. PWT) evoked by formalin stimulation, which was significant different from that of formalin injection alone (p<0.05). Blockade of CB1and CB2promoted the incurrence of mirror pain in formalin test. Morphological results showed that blockade of CB receptor significantly increased the expression of astrocytes in lumbar dorsal horn than formalin injection alone, that the expression of CB1continuously increased following the time elapsed and was colocalized with astrocytes while the CB2expression was not observed in blank control group but was continuously induced by formalin injection within7days and was further increased after blockade of CB1and CB2. The pJNK expression in astrocyte induced by formalin was not affected by blockade of CB1and CB2.Conclusion:The results indicated that endocannabinoid is involved in the formalin induced-continuous inflammatory hyperalgesia through JNK pathway by activating astrocytes, suggesting that enhancement of endocannabinoid level can play analgesic effect under continuous inflammatory pain.更多还原