【作者】 段炜；

【导师】 郑健；

【作者基本信息】 第三军医大学 ， 神经病学， 2009， 博士

【摘要】 供应脑实质深穿支小动脉的广泛病变,或颈部大动脉及其颅内分支动脉的狭窄,导致大脑半球深部白质血流量降低,持续的低灌注状态可造成白质疏松,即缺血性白质脑病。缺血性白质脑病可以引起神经功能缺失,主要表现包括认知功能损害,是中老年血管性认知损害的主要病理类型之一。目前缺血性白质脑病的治疗方式主要是对症治疗和控制心脑血管疾病的危险因素,但治疗效果不满意,不能阻止疾病的进一步发展。因此,迫切需要探索缺血性白质脑病新的治疗策略和途径。缺血性白质脑病的分子病理机制仍不明确,对于慢性脑血流低灌注导致的脑组织能量代谢障碍和神经活性物质代谢障碍造成以白质损伤为主的病理损害的分子机制仍然缺乏清楚认识。以往研究发现慢性脑血流低灌注造成的白质损伤,同时伴有胶质细胞增生活化、白质区域炎性细胞浸润、某些炎性因子表达增加以及一氧化氮合成增加等,提示白质损伤与炎性病理过程有关,确切的机制尚需进一步研究阐明。腺苷是能量代谢的中间产物,也是中枢神经系统重要的神经调质。腺苷与其受体(A1、A2A、A2B和A3)结合后调节机体重要的生理活动,并参与多种致病因素引起的疾病过程。其中A2A受体参与机体多种生理活动的调节和某些疾病的病理过程,在某些疾病的动物实验研究中发现其治疗效应较明确,因此格外受到重视。许多研究已发现A2A受体拮抗剂或A2A受体基因敲除抑制了细胞外谷氨酸浓度的异常升高从而显著减轻急性缺血造成的脑损伤,提示对腺苷A2A受体的干预有可能成为治疗缺血性脑损伤的新途径。慢性低灌注性白质损伤的组织病理改变及病理机制与急性脑缺血性损伤有显著差异,腺苷A2A受体在慢性低灌注性白质损伤发生发展中的作用仍不清楚。此外,A2A受体也存在于外周血液中的炎性细胞和免疫细胞,研究证实A2A受体激活显著抑制外周炎性细胞和免疫细胞的黏附和迁移,减少炎性因子的分泌,从而产生抗炎效应,减轻多种致炎因子造成的炎性病理损伤,那么A2A受体是否通过对炎性效应的调控从而对慢性低灌注性白质损伤的结局产生影响?为了阐明A2A受体对慢性低灌注性白质损伤的影响及其可能机制,首先,我们参照Shibata等方法用内径为0.18mm的微型弹簧圈套双侧颈总动脉建立小鼠慢性脑血流低灌注模型,在建模第30d采用八臂迷宫实验对模型小鼠进行认知功能评价;在建模第7d、14d和30d分别采用Kluver-Barrera染色、抗GFAP、CD11b免疫组织化学染色观察模型小鼠在建模后不同时间点白质区域的神经纤维损伤程度及胶质细胞增生程度;同时,在建模后第14d采用抗CD3免疫组织化学染色及伊文氏蓝荧光显微镜观察来评价模型小鼠白质区域淋巴细胞浸润情况及血脑屏障的完整性。其次,我们采用腺苷A2A受体基因敲除技术,观察A2A受体缺失对慢性低灌注性白质损伤的影响。A2A受体基因敲除小鼠及其同窝野生型小鼠分别制作慢性脑血流低灌注模型,采用八臂迷宫实验评价A2A受体基因敲除小鼠及同窝野生型小鼠在建模第30d时认知功能的变化;在建模第7d、14d和30d分别采用Kluver-Barrera染色、抗GFAP、CD11b免疫组织化学染色观察A2A受体基因敲除小鼠及同窝野生型小鼠在建模后不同时间点白质区域的神经纤维损伤程度及胶质细胞增生程度;采用抗CD3免疫组织化学染色观察A2A受体基因敲除小鼠及同窝野生型小鼠在建模第14d白质区域淋巴细胞浸润情况;同时,采用PCR及western blot方法检测A2A受体基因敲除小鼠及同窝野生型小鼠在建模第7d、14d和30d胼胝体的TNF-α、IL-1β和IL-6 mRNA和蛋白水平。最后,我们将雌性A2A受体基因敲除小鼠的骨髓细胞通过尾静脉注射入γ射线全身照射后的雄性C57BL/6小鼠体内,从而建立选择性骨髓来源细胞A2A受体缺失模型,观察外周骨髓来源细胞A2A受体在慢性低灌注性白质损伤发生发展中的作用及其机制。骨髓细胞成功移植后的嵌合子小鼠制作慢性脑血流低灌注模型,在建模第7d、14d和30d分别采用Kluver-Barrera染色、抗GFAP、CD11b免疫组织化学染色观察嵌合子小鼠白质区域神经纤维损伤程度及胶质细胞增生程度;采用western blot方法检测嵌合子小鼠在建模第7d、14d和30d胼胝体的TNF-α和IL-1β蛋白水平。经过以上实验研究,我们得到一下结果:一、小鼠慢性脑血流低灌注模型的建立1.采用特制微型弹簧圈套小鼠双侧颈总动脉成功建立慢性脑血流低灌注小鼠模型。建模后小鼠死亡率为18.9%。建模后30d各个检测时间点模型小鼠的平均体重与假手术组比较无差异。2.在建模第30d模型小鼠八臂迷宫实验的平均工作记忆错误显著增多。3.在建模第7d时模型小鼠白质区域未见组织病理改变。在建模第14d和30d时模型小鼠白质区域可见神经纤维排列紊乱、稀疏,有空泡形成,同时星型胶质细胞和小胶质细胞增生,以胼胝体最为显著,内囊次之,视束最轻。模型小鼠随着建模时间延长,白质损伤程度分级逐渐增加,白质区域胶质细胞数目显著增多。4.在建模第14d模型小鼠胼胝体区域可见散在单个的CD3阳性细胞分布,其余白质区域则未见。并且模型小鼠胼胝体CD3阳性细胞数目显著多于相同时间点的假手术小鼠。在建模第14d模型小鼠胼胝体区域小血管边缘毛糙,成锥形突触,提示存在伊文蓝外渗现象。二、腺苷A2A受体基因敲除对慢性低灌注性白质损伤的影响及其与炎性效应调控的关系1.在建模后A2A受体基因敲除小鼠的死亡率为22.3%,野生型小鼠死亡率为16.7%。建模后30d各个检测时间点A2A受体基因敲除小鼠的平均体重与野生型小鼠无显著差异。2.在建模第30d时A2A受体基因敲除小鼠八臂迷宫实验平均工作记忆错误比相同时间点的野生型小鼠显著增多。3.在建模第7d时2组小鼠白质区域均未见显著组织病理改变。在建模第14d和30d时2组小鼠白质区域可见神经纤维稀疏、空隙增加、空泡形成,同时星型胶质细胞和小胶质细胞增生,A2A受体基因敲除小鼠在建模第30d白质区域可见胞体增大变圆,突起变短的活化小胶质细胞。在建模第14d和30d时A2A受体基因敲除小鼠白质损伤程度分级显著高于同时间点的野生型小鼠,同时A2A受体基因敲除小鼠白质区域胶质细胞数目显著多于相同时间点的野生型小鼠。4.在建模第14d时2组小鼠胼胝体区域均可见到CD3阳性细胞分布,并且A2A受体基因敲除小鼠在建模第14d胼胝体区域CD3阳性细胞数目显著多于相同时间点的野生型小鼠。5.在建模第7d、14d和30d时A2A受体基因敲除小鼠胼胝体TNF-α和IL-1β的mRNA水平与相同时间点的野生型小鼠比较显著增高,在建模第30d时A2A受体基因敲除小鼠胼胝体IL-6的mRNA水平显著高于相同时间点的野生型小鼠。在建模第30d时A2A受体基因敲除小鼠胼胝体TNF-α和IL-6蛋白水平显著高于相同时间点的野生型小鼠,在建模第14d和30d时A2A受体基因敲除小鼠胼胝体IL-1β蛋白水平显著高于相同时间点的野生型小鼠。三、骨髓来源细胞A2A受体对慢性低灌注白质损伤的影响1.选择性骨髓细胞腺苷A2A受体缺失模型的建立和评价(1)骨髓细胞移植前雄性受体小鼠性染色体相关基因PCR产物电泳结果有330bp和300bp两条条带,但骨髓细胞移植后雄性受体小鼠性染色体相关基因PCR产物电泳结果仅有330bp一条带,与雌性小鼠性染色体PCR产物电泳结果相符。(2)野生型小鼠骨髓细胞移植入C57BL/6小鼠体内的嵌合子小鼠(WT→WT组)外周血白细胞A2A受体免疫细胞荧光染色发现A2A受体阳性细胞率为(93.82±11.24)%,A2A受体基因敲除小鼠骨髓细胞移植入C57BL/6小鼠体内的嵌合子小鼠(KO→WT组)外周血白细胞A2A受体阳性细胞率下降至(9.73±2.05)%。2.骨髓来源细胞A2A受体对慢性低灌注白质损伤的影响(1) WT→WT组建模后小鼠死亡率为10.0%,KO→WT组建模后小鼠死亡率为21.05%。(2)在建模第7d时2组白质区域均未见明显的组织病理学改变,在建模第14d和30d时2组胼胝体、尾状核/壳核纤维束可见神经纤维排列紊乱、稀疏,内囊和视束均未见明显改变。在建模后第14d和30d,KO→WT组胼胝体和尾状核/壳核纤维束的神经纤维损伤级数与同时相点WT→WT组比较显著性增加。(3)在建模第7d、14d和30d时KO→WT组胼胝体和尾状核/壳核纤维束可见显著星型胶质细胞和小胶质细胞增生,WT→WT组胼胝体和尾状核/壳核纤维束仅在建模第14d和30d观察到胶质细胞增生,2组内囊和视束未见显著胶质细胞增生。在建模第7d、14d和30d时KO→WT组胼胝体和尾状核/壳核纤维束的胶质细胞数目显著多于相同时间点的WT→WT组。(4)与相同时间点的WT→WT组比较,KO→WT组胼胝体的TNF-α蛋白水平仅在建模第14d时显著升高;KO→WT组胼胝体的IL-1β蛋白水平在建模第14d和30d时均显著升高。综上所述,我们利用特制的微型弹簧成功建立了小鼠慢性脑血流低灌注模型,从行为学及组织病理学方面进行评价,证实了该模型是研究慢性低灌注白质损伤的可靠动物模型。腺苷A2A受体基因敲除加重慢性低灌注白质损伤,促进局部胶质细胞增生,促使白质区域炎性因子产生增多,提示A2A受体缺失可能是通过增强炎症反应而发挥影响。选择性骨髓来源细胞A2A受体基因敲除同样加重慢性低灌注白质损伤,促进局部胶质细胞增生和炎症因子的产生,表明骨髓来源的外周白细胞A2A受体可能在对慢性低灌注白质损伤的影响中发挥主导作用。更多还原

【Abstract】 Due to dismissed microangiopathy in the hemispheres or cerebral hypoperfusion induced by the stenosis of carotid arteries, white matter lesion is one of the major causes of vascular cognitive impairment and constitutes the core pathology of Binswanger disease. Until now, the therapies available of white matter lesions induced by chronic cerebral hypoperfusion include symptomatic treatment and controlling risk factors of cardiovascular and cerebrovascular diseases. In spite of extensive efforts and investigations, no efficient neuroprotective therapy is currently available for ischemic white matter lesions. Therefore, it is important to explore the new and effective strategies to cure ischemic white matter lesions.Much less is known about the moleculor pathomechanisms of ischemic white matter lesions. It must be elucidated the pathomechanism of white matter lesions induced by chronic cerebral hypoperfusion due to the dysmetabolism of energy and signaling moleculors. Previous studies support that there are a series of pathophysiological events including white matter lesions, proliferation of glial, periphery inflammatory cell infiltration and increased production of several inflammatory cytokines in the white matter regions after chronic cerebral hypoperfusion. These evidences indicate that inflammatory reaction is correlated to the white matter lesions induced by chronic cerebral hypoperfusion.As an intermediate product of energy metabolism, adenosine is an important neuromodulator in the central nervous systerm. Adenosine acting at it receptors regulates many physiological events and is involved in the development of the diseases. Importantly, A2A receptor is related to the provoke and development of numerous central nervous systerm diseases. Growing evidences show that A2A receptor antagonist or A2A receptor knock out attenuates the acute ischemic brain damages on account of reducing the glutamate outflow. Hence, these findings show that A2A receptor will be a new target to cure the ischemic brain injury. There are markedly differences in the neuropathological features and pathomechanisms between white matter lesions induced by chronic cerebral hypoperfusion and acute ischemic brain injury. The role of A2A receptor in the development of chronic cerebral hypoperfusion-induced white matter lesions has not yet been elucidated. In addition, the A2A receptor expression is detected in the periphery blood inflammatory cells and immune cells. Previous data show that activated A2A receptor significantly attenuates the inflammatory tissue damages by inhibiting the adhesion and migration of inflammatory cells and immune cells, reducing the production of inflammatory cytokines. Whether is the modulation of A2A receptor in the neuroinflammation involved in the role of A2A receptor in the white matter lesions induced by chronic cerebral hypoperfusion or not?In order to elucidate the effect of A2A receptor on the white matter lesions induced by chronic cerebral hypoperfusion, firstly, the mouse model of chronic cerebral hypoperfusion was established by stenosis of bilateral carotid artery using 0.18mm diameter microcoils. We evaluated the cognitive function of model mice by 8-radial maze test at 14d after the surgery. The white matter lesions and glial proliferation were examined by Kluver-Barrera staining and anti-GFAP or anti-CD11b immunohistochemistry at 7d, 14d and 30d after the surgery. We also examined the leukomonocyte infiltration in the white matter regions by anti-CD3 immunohistochemistry and integrality of blood brain barrier by Evans blue at 14d after the surgery.Secondly, A2A receptor knock out mice and their littermates were subjected to the surgery of bilateral carotid artery stenosis. We compared the cognitive function of A2A receptor knock out mice and wild type mice by 8-radial maze test at 30d after the surgery. The white matter lesions and glial proliferation of A2A receptor knock out mice and wild type mice were compared by Kluver-Barrera staining and anti-GFAP or anti-CD11b immunohistochemistry at 7d, 14d and 30d after the surgery. We also compared the leukomonocyte infiltration in the white matter regions by anti-CD3 immunohistochemistry at 14d after the surgery. Moreover, the mRNA and protein levels of TNF-α、IL-1βand IL-6 in corpus callosum were investigated by PCR and western blot at 7d, 14d and 30d after the surgery.Thirdly, the selective bone marrow cells A2A deficits model was estsblished by transplanting the female A2A receptor knock out mice bone marrow cells into the male C57BL/6 mice. At 8 weeks after successful transplantation, the chimeric mice were subjected to the surgery of bilateral carotid actery stenosis. The white matter lesions and glial proliferation of the chimeric mice were compared by Kluver-Barrera staining and anti-GFAP or anti-CD11b immunohistochemistry at 7d, 14d and 30d after the surgery. The protein levels of TNF-αand IL-1βin corpus callosum were investigated by western blot at 7d, 14d and 30d after the surgery.ⅠEstablishment of mouse model of chronic cerebral hypoperfusion1. After the surgery of bilateral carotid artery stenosis, the death rate of the model mice was 18.9%. There was no difference in the average weight between the model mice and the sham-operated mice within 30d after the surgery.2. The average working memory errors in 8-radial maze of model mice markedly increased at 30d after the surgery.3. The white matter rarefaction and proliferation of astrocytes and microglia were observed in the white matter regions of model mice at 14d and 30d after the surgery. The most significant changes were observed in the corpus callosum. Moderate changes were found in the internal capsule, and less severe changes were observed in the optic tract. More long the bilateral carotid artery stenosis, more severe the white matter lesions and glial proliferation.4. The CD3 positive cells were observed in the corpus callosum of model mice at 14d after the surgery. The number of CD3 positive cells in the corpus callosum of model mice was much more than that of sham-operated mice. We also found that Evans blue extravasation of small vessels in corpus callosum of model mice at 14d after the surgery.ⅡEffect of adenosine A2A receptor knock out on whiter matter lesions induced by chronic cerebral hypoperfusion and the role of A2A receptor in the modulation of inflammatory reaction 1. After the surgery of bilateral carotid artery stenosis, the death rate of A2A receptor knock out mice was 22.3%, the death rate of wild type mice was 16.7%. There was no difference in the average weigh between A2A receptor knock out mice and wild type mice within 30days after the surgery.2. The average working memory errors in 8-radial maze of A2A receptor knock out mice were markedly higher than that of wild type mice at 30d after the surgery.3. The white matter rarefaction and proliferation of astrocytes and microglia were observed in the white matter regions of A2A receptor knock out mice and wild type mice at 14d and 30d after the surgery. The activated microglia with swollen and hypertropic cell bodies as well as thick and short processes was found in the white matter regions of A2A receptor knock out mice at 30d after the surgery. The severity of white matter lesions and number of glial cells of A2A receptor knock out mice were much higher than that of wild type mice at 14d and 30d after the surgery.4. The CD3 positive cells were observed in the corpus callosum of both two model mice at 14d after the surgery. The number of CD3 positive cells in the corpus callosum of A2A receptor knock out mice was much more than that of wild type mice at 14d after the surgery.5. The TNF-αand IL-1βmRNA levels in the corpus callosum of A2A receptor knock out mice was much higher than that of wild type mice at 7d, 14d and 30d after the surgery. Compared with wild type mice, the IL-6 mRNA level in the corpus callosum of A2A receptor knock out mice significantly increased at 30d after the surgery. The TNF-αand IL-6 protein levels in the corpus callosum of A2A receptor knock out mice was much higher than that of wild type mice at 30d after the surgery. Compared with wild type mice, the IL-1βprotein level in the corpus callosum of A2A receptor knock out mice significantly increased at 14d and 30d after the surgery.ⅢEffect of bone marrow cells A2A receptor on the white matter lesions induced by chronic cerebral hypoperfusion1. Establishment and evaluation of selective bone marrow cells A2A receptor deficits mice ①Before the transplantation, the electrophoresis of sexual chromagene gene PCR product on periphery blood white cells of male recipient mice included 330bp and 300bp bands. After the successful transplantation, the electrophoresis of sexual chromagene gene PCR product on periphery blood white cells of male recipient mice included 330bp band.②The A2A receptor positive cells rate of wild type mice that received bone marrow cells derived from wild type mice (WT→WT group) was (93.82±11.24)%. The A2A receptor positive cells rate of wild type mice that received bone marrow cells derived from A2A receptor knock out mice (KO→WT group) decreased to (9.73±2.05)%.2. Effect of bone marrow cells A2A receptor on the white matter lesions induced by chronic cerebral hypoperfusion①The death rate of WT→WT group was 10.0% after the surgery of bilateral carotid artery stenosis. The death rate of KO→WT group was 21.05% after the surgery of bilateral carotid artery stenosis.②The white matter rarefaction was observed in the corpus callosum and fibers bund of caudoputamen of WT→WT group and KO→WT group at 14d and 30d after the surgery. The white matter lesions were not observed in internal capsule and optical tract. Compared with WT→WT group, the white matter lesions in the corpus callosum and fibers bund of caudoputamen of KO→WT group significantly increased at 14d and 30d after the surgery.③The glial proliferation was observed in the corpus callosum and fibers bund of caudoputamen of KO→WT group at 7d, 14d and 30d after the surgery. The glial proliferation was observed in the corpus callosum and fibers bund of caudoputamen of WT→WT group at 14d and 30d after the surgery. The glial proliferation was not observed in internal capsule and optical tract.The number of glial cells in the corpus callosum and fibers bund of caudoputamen of KO→WT group was much higher than that of WT→WT group at 7d, 14d and 30d after the surgery.④Compared with WT→WT group, the TNF-αprotein levels in the corpus callosum of KO→WT group markedly increased at 30d after the surgery, the IL-1βprotein levels significantly increased at 14d and 30d after the surgery. In a word, the mouse model of chronic cerebral hypoperfusion was successfully established using the 0.18mm diameter microcoils. We evaluated the cognitive function and histopathological changes of the model mice and confirmed that this model is a powerful tool to study the pathomechanisms of white matter lesions induced by chronic cerebral hypoperfusion. Adenosine A2A receptor deficits exacerbated white matter lesions and glial proliferation, promoted the production of inflammatory cytokines. These findings indicated that increased inflammation was associated with the exacerbation of white matter lesions by inactivation of A2A receptor. Selective bone marrow cells A2A receptor inactivation exacerbated white matter lesions and glial proliferation, promoted the production of inflammatory cytokines. These findings showed that A2A receptor in bone marrow cells may be an important contributor to the white matter lesions induced by chronic cerebral hypoperfusion.更多还原