骨髓来源细胞腺苷A_(2A)受体调控的炎性效应对慢性低灌注白质损伤的影响及其机制探讨
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摘要
背景：
缺血性白质脑病(white matter lesion)是由大脑半球深穿支小动脉(Deep perforators small artery)的广泛病变,或颈部大动脉及其颅内分支狭窄,导致持续脑血流低灌注,从而造成以半卵圆区及侧脑室旁白质疏松为其病理特点,临床以认知损害为突出表现的疾病状态,是中老年血管性认知损害的主要类型之一。目前对于缺血性白质脑病的分子病理机制认识尚不足,因而制约了治疗药物的研发,临床缺少有效药物治疗手段。因此,迫切需要更深入地探讨缺血性白质脑病的分子病理机制,以期进而提出有效的治疗策略和方法。
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,在某些疾病的临床研究和动物实验中发现,腺苷A2A受体及其介导的信号通路可能成为治疗的靶点,因此愈来愈受到基础和临床研究者的重视。目前,以腺苷A2A受体作为治疗靶点的多种药物已经进入临床试验,包括帕金森病、Huntington病、全身炎性反应综合征和艾滋病等的药物临床试验。
本课题组在前期研究中发现慢性低灌注白质损伤病理机制不同于急性缺血性脑损伤,腺苷A2A受体介导的炎性效应是影响慢性低灌注白质损伤结局的重要因素。腺苷A2A受体也存在于外周血液中骨髓来源细胞,Yu等在急性脑缺血的动物实验中发现骨髓来源细胞腺苷A2A受体的选择性缺失影响脑组织内炎性介质的产生,提示骨髓来源细胞A2A受体可能对缺血诱导的脑内炎性反应产生重要的调节作用。
为了进一步探讨腺苷A2A受体介导的效应对慢性低灌注白质损伤炎性病理过程产生影响的可能机制,我们进行了以下研究：①利用腺苷A2A受体基因敲除动物,采用骨髓移植细胞嵌合的方法建立骨髓来源细胞腺苷A2A受体选择性缺失和选择性重建的动物模型,观察对慢性低灌注白质损伤的影响,以明确腺苷作用于A2A受体并对慢性低灌注白质损伤产生影响的具体效应细胞；②检测慢性脑血流低灌注诱导的局限表达于免疫细胞和炎细胞的信号转导分子Cystatin F表达的改变,并观察干预腺苷A2A受体活性对其表达及慢性低灌注白质损伤结局的影响,探讨腺苷A2A受体对慢性低灌注白质损伤产生影响的下游分子病理途径；③检测慢性脑血流低灌注诱导的白质损伤区域前炎性细胞因子表达的变化,以及干预腺苷A2A受体活性对其表达的影响,探讨与慢性低灌注白质炎性病理损伤有关的信号转导途径。
方法：
1.实验动物
引进腺苷A2A受体基因敲除(A2A receptor gene knock-out, A2AR/KO)杂合子小鼠交配繁殖,聚合酶链反应(polymerase chain reaction, PCR)方法鉴定A2AR/KO小鼠和野生型(wild-type littermates, A2AR/WT)小鼠。
2.骨髓移植细胞嵌合模型的建立
采用骨髓移植细胞嵌合的方法建立骨髓来源细胞腺苷A2A受体选择性缺失及选择性重建模型。即放射线破坏雄性野生型或A2AR/KO(?)、鼠骨髓后,移植雌性A2AR/KO小鼠骨髓细胞至野生型小鼠构建骨髓来源细胞腺苷A2A受体选择性缺失模型(KO→WT),骨髓细胞移植WT→WT组作为其对照；移植雌性野生型小鼠骨髓细胞至A2AR/KO小鼠构建骨髓来源细胞腺苷A2A受体选择性重建模型(WT→KO),骨髓细胞移植KO→KO组作为其对照；PCR法检测雄性受体外周血性染色体及免疫荧光法检测外周血白细胞A2A受体表达,鉴定骨髓移植细胞嵌合模型是否成功建立。
3.慢性脑血流低灌注模型的建立
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,建模前及建模后2h监测脑血流以判断模型是否成功；建模前及建模后4w,对部分动物行Morris水迷宫实验,记录每只动物发现平台的平均潜伏期和穿越平台的次数,以此为指标评价空间学习和记忆能力。
4.腺苷A2A受体激动剂干预
给予慢性脑血流低灌注野生型小鼠和骨髓来源细胞腺苷A2A受体选择性重建小鼠腺苷A2A受体激动剂CGS21680(0.25mg/kg,腹膜腔注射,1次／d,直至各组动物观察终点,即脑缺血后第2w、4w和6w,比较非选择性激活与选择性激活骨髓来源细胞腺苷A2A受体对于慢性脑血流低灌注白质损伤及炎性病理反应的影响。
5.白质损伤、小胶质细胞增生、炎性因子和Cystatin F表达的检测
分别于脑缺血后2w、4w和6w取脑标本,ZO-1免疫组化染色法观察血脑屏障开放程度,Kluver-Barrera及Bielschowsky染色法评价白质损伤,髓鞘碱性蛋白(myelinbasic protein,MBP)免疫组化染色法观察髓鞘脱失,比较各组动物血脑屏障和白质损伤程度。CD11b免疫组织化学染色方法观察并计数白质区域单位面积(0.25mm2)小胶质细胞活化及增生情况,抗CD3和抗LY6G免疫组化染色方法观察白质区域外周血液来源的炎性细胞浸润情况；提取胼胝体mRNA及总蛋白,分别采用实时定量PCR(real-time quantitative PCR,rt-qPCR))和western blot方法检测致炎因子IL-1β、TNF-α、IL-6.IL-12P40以及抗炎因子IL-10的mRNA和蛋白表达；免疫荧光双标染色观察Cystatin F在脑内的表达及细胞定位,rt-qPCR方法检测其mRNA和蛋白表达；组间比较前述指标以探讨选择性干预骨髓来源和非骨髓来源细胞腺苷A2A受体活性对慢性脑血流低灌注诱导的白质炎性病理反应的影响及其可能的信号转导途径。
结果：
1.成功建立骨髓移植细胞嵌合模型
(1)骨髓细胞移植前雄性受体小鼠外周血白细胞性染色体为两条带,符合雄性染色体特征,而接受雌性小鼠骨髓细胞移植后其外周血白细胞性染色体仅为一条带,符合雌性染色体特征,表明移植后雄性受体小鼠外周血白细胞来源于雌性供体骨髓细胞。
(2)骨髓细胞移植KO→WT组小鼠外周血白细胞A2A受体阳性率(5.28%±1.05%),与KO→KO组(6.62%±2.04%)比较无显著差异(>0.05),而显著低于骨髓细胞移植WT→WT组(92.32%±15.43%)(<0.05),表明成功建立骨髓来源细胞A2A受体选择性缺失模型。
(3)骨髓细胞移植WT→KO组小鼠外周血白细胞A2A受体阳性率(96.34%±12.58%),与WT→WT组(92.32%±15.43%)比较无显著差异(>0.05),而显著高于骨髓细胞移植KO→KO组(6.62%±2.04%)(<0.05),表明成功建立骨髓来源细胞A2A受体选择性重建模型。
2.成功建立慢性脑血流低灌注动物模型
(1)脑血流监测
在双侧颈总动脉圈套前及建模后2h分别检测脑血流量,各模型组动物建模后脑血流量下降40%-50%,组间比较无统计学差异(>0.05),表明该方法造成显著的脑缺血,且各模型组动物脑缺血程度相同。
(2)血脑屏障通透性检测
分别于脑缺血后2W、4W和6W,取脑标本ZO-1免疫组化染色,各时间点与假手术组动物比较显色强度评分均显著降低(<0.05),缺血后2W评分最低,此后逐渐升高,但至缺血后6W时仍低于对照水平,表明缺血后2W血脑屏障开放最显著,缺血后6w时血脑屏障仍处于开放状态。
骨髓来源细胞腺苷A2A受体选择性缺失与选择性重建动物,以及骨髓细胞移植对照组动物,组间比较ZO-1免疫组化染色强度评分无统计学差异(P>0.05)；腺苷A2A受体激动剂处理组,包括野生型和骨髓来源细胞腺苷A2A受体选择性重建动物,以及载体溶液处理的对照组动物,组间比较ZO-1免疫组化染色强度评分无统计学差异(P>0.05)；表明腺苷A2A受体介导的效应对慢性脑血流低灌注造成的血脑屏障通透性变化无影响。
(3)白质损伤的评价
分别于脑缺血后2w、4w和6w,取脑标本Kluver-Barrera染色和Bielschowsky银染评价白质纤维损伤,MBP免疫组化染色评价髓鞘脱失,发现随着脑缺血时间延长,白质损伤逐渐加重。
3.干预腺苷A2A受体活性对慢性脑血流低灌注动物认知行为的影响
(1)骨髓细胞移植各组动物的认知行为比较
骨髓细胞移植各组动物,包括骨髓来源细胞腺苷A2A受体选择性缺失与选择性重建动物,以及骨髓细胞移植对照组动物,脑缺血前组间比较Morris水迷宫实验测定的平均潜伏期及穿越平台次数无显著差异(P>0.05),表明骨髓细胞移植对小鼠的空间学习及记忆能力无影响。
(2)慢性脑血流低灌注对认知行为的影响
慢性脑血流低灌注各组动物,缺血后第4w采集的数据分别与其缺血前的数据比较,Morris水迷宫实验测定的平均潜伏期延长及穿越平台次数减少(P<0.05),表明脑缺血后动物的空间学习和记忆能力受到损害。
(3)骨髓来源细胞腺苷A2A受体选择性缺失对脑缺血引起的认知损害的影响
骨髓细胞移植KO→WT组与WT→WT组比较,脑缺血后第4w,Morris水迷宫实验测定的平均潜伏期延长(P<0.05)，穿越平台次数显著减少(P<0.05),表明骨髓来源细胞腺苷A2A受体的选择性缺失,不但加重慢性脑血流低灌注造成的空间学习能力障碍,也加重记忆功能损害。
(4)骨髓来源细胞腺苷A2A受体选择性重建对脑缺血引起的认知损害的影响
骨髓细胞移植WT→KO组与KO→KO组比较,脑缺血后第4w,Morris水迷宫实验测定的平均潜伏期显著缩短(P<0.05),表明骨髓来源细胞腺苷A2A受体的选择性重建减轻慢性脑血流低灌注造成的空间学习能力损害。
4.干预腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
(1)干预整体腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
分别于脑缺血后第2w、4w和6w,取脑标本Kluver-Barrera染色观察胼胝体神经纤维损伤情况,比较发现腺苷A2A受体基因敲除动物胼胝体损伤较同窝野生型动物明显加重,而腺苷A2A受体激动剂CGS21680处理动物胼胝体损伤较载体溶液对照动物明显减轻,表明激活腺苷A2A受体对慢性脑血流低灌注白质损伤具有保护效应。
(2)选择性干预骨髓来源细胞腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
分别于脑缺血后第2w、4w和6w,取脑标本Kluver-Barrera和Bielschowsky染色评价白质纤维损伤情况,MBP免疫组化染色评价髓鞘脱失情况。骨髓细胞移植KO→WT组与WT组比较,白质纤维损伤和髓鞘脱失更严重；WT→KO组与KO→KO组相比,白质纤维损伤和髓鞘脱失明显较轻；表明骨髓来源细胞腺苷A2A受体的选择性缺失加重慢性脑血流低灌注导致的白质损伤,骨髓来源细胞腺苷A2A受体介导的效应减轻白质损伤。
脑缺血后各时间点,骨髓细胞移植WT组与KO组比较白质损伤更严重；脑缺血后第4w和6w,骨髓细胞移植KO组与WT组相比白质损伤较轻；提示非骨髓来源细胞的腺苷A2A受体介导的效应对慢性脑血流低灌注导致的白质损伤也产生影响,可能加重损伤,但对白质损伤结局并不发挥主导作用。
腺苷A2A受体激动剂CGS21680处理各组及其载体溶液对照组动物比较,白质损伤严重程度依次为载体溶液处理的野生型动物、载体溶液处理的骨髓来源细胞腺苷A2A受体选择性重建动物、激动剂CGS21680处理的野生型动物和激动剂CGS21680处理的骨髓来源细胞腺苷A2A受体选择性重建动物。表明选择性激活骨髓来源细胞腺苷A2A受体可以减轻慢性脑血流低灌注导致的白质损伤,也提示激活非骨髓来源细胞的腺苷A2A受体可能加重损伤。
5.干预腺苷A2A受体活性对慢性脑血流低灌注诱导的Cystatin F表达的影响
分别于脑缺血后第2w、4w和6w取脑标本,采用免疫荧光双标染色检测脑内Cystatin F的表达及其细胞定位,分别采用rt-qPCR和western blot方法定量检测其mRNA和蛋白表达的变化。
(1)慢性脑血流低灌注诱导的Cystatin F表达变化
脑缺血后白质损伤区域可见Cystatin F与活化小胶质细胞标志物CDllb共表达；胼胝体Cystatin F的mRNA和蛋白表达水平显著升高,且随着脑缺血时间的延长而逐渐增加；表明慢性脑血流低灌注诱导小胶质细胞表达Cystatin F增多。
(2)干预腺苷A2A受体活性对慢性低灌注诱导的Cystatin F表达的影响
腺苷A2A受体基因敲除动物与其同窝野生型动物相比,脑缺血后白质损伤区域单位面积Cystation F和CDllb共表达的阳性细胞数显著增多(P<0.05),胼胝体CystatinF的mRNA和蛋白表达也显著较多(P<0.05)；而腺苷A2A受体激动剂CGS21680处理的野生型小鼠与同窝野生型动物相比,Cystation F和CDllb共表达的阳性细胞计数显著较少(P<0.05),胼胝体Cystatin F的：mRNA和蛋白表达也显著较低(P<0.05)；表明腺苷A2A受体介导的效应对慢性脑血流低灌注诱导的Cystatin F表达产生影响,激活腺苷A2A受体可抑制Cystatin F表达。
6.干预腺苷A2A受体活性对慢性脑血流低灌注诱导的小胶质细胞增生和炎性因子表达的影响
(1)小胶质细胞增生和外周炎细胞浸润的观察
分别于脑缺血后第2w、4w和6w取脑标本,小胶质细胞标记物CD11b免疫组化染色发现,缺血后第4w与第2w比较,白质损伤区域小胶质细胞数量明显增多(P<0.05),突起回缩,胞体增大,前述变化与白质损伤严重程度一致。骨髓细胞移植KO→WT组与WT组比较,白质损伤区域小胶质细胞的增生更为显著(P<0.05)；骨髓细胞移植KO组与KO组相比,白质损伤区域小胶质细胞的增生显著减少(P<0.05)。表明慢性脑血流低灌注诱导小胶质细胞的增生和活化,激活骨髓来源细胞的腺苷A2A受体可抑制其增生。
淋巴细胞标记物CD3和中性粒细胞标记物LY6G免疫组化染色,在各组动物各时间点的观察,均未发现阳性显色。提示慢性脑血流低灌注过程中,白质损伤区无明显的外周血液来源的炎细胞浸润。
(2)白质损伤区炎性因子表达的观察
分别于脑缺血后第2w、4w和6w取脑标本,分离胼胝体组织,采用rt-qPCR和western blot方法定量检测炎性因子mRNA和蛋白表达的变化。
致炎因子IL-1β、IL-6、TNF-a、IL-12p40的mRNA和蛋白表达,随脑缺血时间延长逐渐升高；骨髓细胞移植KO→WT组与WT→WT组比较升高更显著；骨髓细胞移植WT→KO组与KO→KO组相比升高程度较轻；腺苷A2A受体激动剂CGS21680处理的野生型动物与载体溶液处理的对照动物相比升高程度较轻；腺苷A2A受体激动剂CGS21680处理的骨髓来源细胞腺苷A2A受体选择性重建动物与载体溶液处理的对照动物相比升高程度较轻。抗炎因子IL-10mRNA和蛋白表达的变化,以及干预腺苷A2A受体活性对其产生的影响,与致炎因子的表达变化呈相反的趋势。
提示致炎因子和抗炎因子表达变化引起的炎性病理反应可能是慢性脑血流低灌注白质损伤的重要机制,激活骨髓来源细胞的腺苷A2A受体通过抑制炎性病理反应而减轻白质损伤。
结论：
1.骨髓来源细胞腺苷A2A受体介导的效应对慢性低灌注白质损伤产生重要影响,选择性激活骨髓来源细胞腺苷A2A受体对白质损伤具有保护作用,选择性抑制骨髓来源细胞腺苷A2A受体则加重白质损伤。
2.慢性脑血流低灌注诱导小胶质细胞表达Cystatin F,激活腺苷A2A受体可抑制其表达,可能进而对下游炎性病理反应产生调控作用。
3.慢性脑血流低灌注白质损伤区致炎因子表达增加,抗炎因子表达减少；选择性激活骨髓来源细胞的腺苷A2A受体可抑制致炎因子表达和促进抗炎因子表达,是其对白质损伤发挥保护作用的重要机制。
4.进一步证明炎性病理损伤是慢性脑血流低灌注白质损伤的重要机制,揭示激活外周炎细胞腺苷A2A受体通过抑制炎性反应而减轻白质损伤。
Background
Ischemic leukoencephalopathy is a disease pathologically characterized as whitematter rarefaction in semiovale area and periventricular area, resulting from sustainedcerebral hypoperfusion due to extensive lesions of cerebral deep perforators small artery, orstenosis of cervical arteries and its intracranial branches. Its prominent clinicalmanifestation is cognitive impairment, and is one of the main types of vascular cognitiveimpairment in the elderly. The molecular mechanism of this disease remains unclear, whichrestricts the development of therapeutic drugs, and thus there is no effective drug treatmentin clinical practice. Therefore, there is an urgent need to explore the molecular mechanismof white matter lesions, in order to come up with effective treatment strategies and methods.
Adenosine is intermediate product in energy metabolism, and also importantneuromodulators in the central nervous system. Adenosine A2Areceptors are involved in theregulation of a variety of physiological activities and some pathological processes in certaindiseases. Clinical researches and animal experiments found that adenosine A2Areceptor-mediated signaling pathway might be the treatment target, and therefore, itattracted more and more attentions from basic and clinical researchers. In present, a varietyof drugs with adenosine A2Areceptor as therapeutic target for have entered clinical trials,including clinical trials for Parkinson's disease, Huntington's disease, systemicinflammatory response syndrome and AIDS
Our group has found in our previous study that chronic hypoperfusion of white matterinjury has different pathological mechanisms from acute ischemic brain injury. AdenosineA2Areceptor-mediated inflammatory effect is the important factor for outcome of chronichypoperfusion white matter injury. Adenosine A2Areceptors are found in bone marrow-derived cells in the peripheral blood. Yu et al found that the selective deletion ofadenosine A2Areceptor in bone marrow-derived cells affects the generation of inflammatorymediators in the brain tissue in animal studies of acute cerebral ischemia, suggesting thatbone marrow-derived cells A2Areceptors may play an important role in the regulation ofbrain ischemia-induced inflammatory response.
To further investigate the effect of the adenosine A2Areceptor-mediated impact on thepossible mechanism of chronic hypoperfusion of pathological process of white matter lesion,we conducted the following studies:(1) With the aid of adenosine A2Areceptor knockoutanimals, we want to establish the animal model of bone marrow-derived cells in theadenosine A2Areceptor selective deletion and selective reconstruction by bone marrowtransplanted chimeric cells in order to observe the impact of chronic hypoperfusion onwhite matter lesions, so as to clarify the role of adenosine in adenosine A2Areceptor and inchronic hypoperfusion white matter lesions, and the specific effector cells in the process;
We detected the expression change of cystatin F, a signal transduction molecule betweenimmune cells and inflammatory cells, in the chronic cerebral hypoperfusion, and observedits expression profiles after interfering adenosine A2Areceptor activity and the outcome ofhypoperfusion white matter injury, in order to explore the downstream molecular pathologyway of adenosine A2Areceptor in chronic hypoperfusion white matter lesions;(3) we alsodetected the expression changes of proinflammatory cytokines in chronic cerebralhypoperfusion-induced white matter injury area, and their changes after interferingadenosine A2Areceptor activity, and to explore the signal transduction pathways in thepathological damage related to chronic hypoperfusion and white matter inflammation.
Methods
1. Experimental Animals
The adenosine A2Areceptor gene knock-out (A2AR/KO) heterozygous mice wereemployed and bred in this study. Polymerase chain reaction (PCR) method was used toidentify A2AR/KO mice and wild-type (wild-type littermates, A2AR/WT) mice.
2. Establishment of bone marrow transplantation chimera modell
Adenosine A2Areceptor selective deletion and selective reconstruction model wasestablished by bone marrow transplantation chimera of bone marrow-derived cells. Aftermale wild-type or A2AR/KO mice were given bone marrow radiation damage, the female A2AR/KO mouse bone marrow cells were transplanted into wild-type mice to built towild-type mice bone marrow-derived cells adenosine A2Areceptor selective deficit model(KO WT), and bone marrow cells transplantation WT WT group served as its control.The female wild-type mice bone marrow cells were transplanted to A2AR/KO mouse tobuild bone marrow-derived cells adenosine A2Areceptor selective reconstruction model(WT KO), and bone marrow cells transplantation KO KO group served as its control.PCR assay was used to detect male receptor peripheral blood chromosome, andimmunofluorescence assay was used for A2Areceptor expression in peripheral bloodleukocytes in order to identify the successfully establishment of bone marrowtransplantation chimera model.
3. Mice model of chronic cerebral hypoperfusion
According to the method by Shibata et al, a micro-spring trap with inner diameter of0.18mm was used to occlude bilateral common carotid artery to establish mouse modelwith chronic cerebral hypoperfusion. Cerebral blood flow was monitored before and in2hafter operation in order to determine whether the model was successful. Before and in4wafter model establishment, Morris water maze test was carried out to record averageplatform latency and the frequency of crossing platform, which indicating the spatiallearning and memory.
4. Intervention of adenosine A2Areceptor agonist
Adenosine A2Areceptor agonist CGS21680(0.25mg/kg, intraperitoneal injection, onceper day, until the end of each time point, that is, in2,4and6w,after cerebral ischemia) wasgiven to cerebral hypoperfusion wild-type mice and bone marrow-derived cells adenosineA2Areceptor selective reconstruction mice. The effect of adenosine A2Areceptor fromnon-selective activation and selective activation of bone marrow-derived cells wascompared on chronic cerebral hypoperfusion white matter lesions and inflammatorypathological reaction.
5. Detection of white matter lesions, proliferation of glial cells, and expression ofinflammatory cytokines and cystatin F
After ischemia for2,4and6w, the mouse brain specimens were collected. ZO-1immunohistochemical staining was used to measure the openness of the blood-brain barrier.Kluver-Barrera and Bielschowsky staining was employed to evaluate white matter lesions. Immunohistochemical staining for myelin basic protein (MBP) was used to observedemyelination. The openness of blood-brain barrier and severity of white matter lesion werecompared in each group. Immunohistochemical staining for CD11b was used to observe andcount the microglia activation and proliferation in per unit area of white matter (0.25mm2).Immunohistochemical staining for CD3and Ly6G was used to observe the infiltration ofinflammatory cells from of peripheral blood in the white matter. After total mRNA andprotein of corpus callosum was extracted, respectively, real-time quantitative PCR(RT-qPCR)) and Western blotting were used to detect the expression of proinflammatorycytokines IL-1beta, TNF-alpha, IL-6, IL-12p40and anti-inflammatory factor IL-10atmRNA and protein levels. Immunofluorescence double staining was used to observe theexpression and cellular localization of Cystatin F in the brain, and rt-qPCR to detect themRNA and protein expression. The aforementioned indicators were compared to exploreselective intervention of adenosine A2Areceptor activity from bone marrow-derived andnon-bone marrow-derived cells induced by chronic low cerebral blood flow perfusion of thewhite matter inflammatory pathological response and its possible signal transductionpathways.
Results
1.Successful establishment of bone marrow transplantation chimera model
(1) Before transplantation of bone marrow cells, there are two bands of chromosome inthe peripheral blood leukocytes of male recipient mice, in line with the characteristics of themale chromosome, while, their peripheral blood leukocytes only have one band ofchromosome after accepting transplantation of female mouse bone marrow cells, in linewith the female chromosome characteristics, indicating that the peripheral blood leukocytesof male recipient mice were derived from the female donor bone marrow cells.
(2) The A2Areceptor-positive rate was (5.281.05)%in peripheral blood leukocytesin KO WT group of mice after bone marrow cell transplantation, and that was (6.622.04)%in the KO KO group, with no significant difference between2groups (P>0.05).The rate was significantly lower in KO WT group than WT WT group [(92.3215.43)%, P<0.05), indicating successful establishment of bone marrow-derived cells A2Areceptor selective deficit model.
(3) The A2Areceptor positive rate in peripheral blood leukocytes was (96.3412.58)% in WT KO group mice after bone marrow cells transplantation, which had no significantdifference compared with that of WT WT group [(92.3215.43)%, P>0.05), and wassignificantly higher than that of KO KO group [(6.622.04)%, P<0.05), indicatingsuccessful establishment of bone marrow-derived cells A2Areceptor selective reconstructionmodel.
2. Successfully establishment of mouse model of chronic cerebral hypoperfusion
(1) Cerebral blood flow monitoring
In the trap of bilateral carotid artery before and in2h after operation, the cerebralblood flow was decreased by40%to50%after the model establishment. There was nosignificant difference between the groups (P>0.05), indicating that the method causingsignificant cerebral ischemia, with same severity in each model group.
(2) Permeability of blood-brain barrier
After ischemia for2,4and6w, ZO-1immunohistochemical staining showed thatcolor intensity scores at each time point were significantly lower compared with the shamoperation group (P<0.05), that of2w rating minimum, then gradually increased, but stillbelow the level of control at6w. These indicated the openness of blood-brain barrier wasmost significant in2weeks’ ischemia, and remained after5weeks’ ischemia.
No statistically significant difference was seen in ZO-1immunohistochemical stainingintensity among bone marrow-derived cell adenosine A2Areceptor deficit and selectivereconstruction models, as well as bone marrow cell transplantation control model (P>0.05).Adenosine A2Areceptor agonist treatment groups, including the wild-type cells and bonemarrow-derived adenosine A2Areceptor selective reconstruction animals, as well as thecontrol animals had no significant difference in ZO-1immunohistochemical stainingintensity scores (P>0.05). These suggested that adenosine A2Areceptor-mediated effects hadno effect on the blood-brain barrier permeability induced by chronic cerebral blood flowhypoperfusion.
(3) Evaluation of white matter lesions
After ischemia for2,4and6w, Kluver-Barrera staining and Bielschowsky silverstaining showed white matter fiber damage, and MBP immunohistochemical stainingdisplayed demyelination. The longer cerebral ischemia time, the severe the white matterlesions
3Effect of adenosine A2Areceptor activity on cognitive behavior in mice afterchronic cerebral hypoperfusion
(1) Comparison of cognitive behavior in mice after bone marrow cells transplantation
In each animal group after bone marrow cell transplantation, including bonemarrow-derived cells adenosine A2Areceptor selective deletion and selective reconstruction,as well as bone marrow cell transplantation control mice, there was no significant differencein the average platform latency and the frequency of crossing platform in Morris watermaze before ischemia (P>0.05), indicating that bone marrow cell transplantation having noeffect on spatial learning and memory of the mice.
(2) Effect of chronic cerebral hypoperfusion on cognitive behavior
The chronic low cerebral blood flow perfusion groups, the average platform latencyand the frequency of crossing platform in Morris water maze were significantly lower in themice after4weeks’ ischemia than before ischemia (P<0.05), indicating impaired spatiallearning and memory.
(3) Effect of bone marrow-derived cells adenosine A2Areceptor selective deletion oncognitive impairment induced by cerebral ischemia
The average platform latency was longer (P<0.05) and the frequency of crossingplatform was decreased (P<0.05) in Morris water maze in bone marrow cell transplantationKO WT group compared with WT WT group after cerebral ischemia for4week,indicating that bone marrow-derived cell adenosine A2Areceptor selective deficit, not onlyenhancing the space learning disabilities caused by chronic cerebral hypoperfusion, but alsoaggravating the memory dysfunction.
(4) Effect of bone marrow-derived cells adenosine A2Areceptor selectivereconstruction on cerebral ischemia-induced cognitive impairment
The average platform latency was shorter (P<0.05) in Morris water maze in bonemarrow cell transplantation WT KO group compared with KO KO group after cerebralischemia for4week, indicating that bone marrow-derived cells adenosine A2Areceptorselective reconstruction alleviating spatial learning impairment caused by chronic cerebralhypoperfusion.
4. Effect of intervention of adenosine A2Areceptor activity on white matterlesions after chronic cerebral hypoperfusion
(1) Effect of intervention of overall adenosine A2Areceptor activity on white matterlesions after chronic cerebral hypoperfusion
In2,4and6w after ischemia, Kluver-Barrera staining showed nerve fiber injury incorpus callosum was significantly worse in the adenosine A2Areceptor gene knockout micethan the wild-type mice from same litter. And adenosine A2Areceptor agonist CGS21680significantly reduced the damage in corpus callosum damage than the control, suggestingthat activation of the adenosine A2Areceptor has a protective effect on white matter lesionsafter chronic cerebral hypoperfusion.
(2) Effect of selective intervention of bone marrow-derived cells adenosine A2Areceptor activity on white matter lesions after chronic cerebral hypoperfusion
In ischemia for2,4and6w, Kluver-Barrera and Bielschowsky staining showed thatsevere white matter fiber damage and MBP immunohistochemical staining showed severedemyelination in bone marrow cell transplantation KO WT group compared with WTWT group, but white matter fiber damage and demyelination was reduced in WT KOgroup compared with the KO KO group, indicating that the bone marrow-derived cellsadenosine A2Areceptor selective deficit aggravating white matter lesions due to chroniccerebral hypoperfusion, and bone marrow-derived cells adenosine A2Areceptor-mediatedeffects reducing the white matter lesions.
In different time points after cerebral ischemia at, white matter lesions were moreserious in bone marrow cell transplantation KO WT group than KO KO group. Incerebral ischemia for4and6week, bone marrow cell transplantation WT KO group hadmilder white matter damage than WT WT group, indicating that non-bonemarrow-derived cells adenosine A2Areceptor had effects on white matter lesions afterchronic cerebral hypoperfusion caused, and might aggravate the injury, but not in a leadingrole.
When adenosine A2Areceptor agonist CGS21680treatment groups were comparedwith their control, the severity of white matter lesions were wild-type mice after carriersolution treatment, bone marrow-derived cells adenosine A2Areceptor selectivereconstruction mice after carrier solution treatment, wild-type mice after the agonistCGS21680treatment, and bone marrow-derived cells adenosine A2Areceptor selectivereconstruction mice after the agonist CGS21680treatment in order. These showed that the selective activation of bone marrow-derived cells adenosine A2Areceptor reduced whitematter lesions due to chronic cerebral hypoperfusion, and the adenosine A2Areceptoractivation of non-bone marrow-derived cells may aggravate the injury.
5. Effect of intervention of adenosine A2Areceptor activity on expression ofcystatin F induced by chronic cerebral hypoperfusion
After ischemia for2,4and6w, double immunofluorescence staining showed cystatinF expression and cellular localization in the brain. RT-qPCR and Western blotting indicatedits quantitative expression at mRNA and protein levels.
(1) Expression of cystatin F after chronic cerebral hypoperfusion
In the injuried area of white matter after cerebral ischemia, cystatin F was co-expressedwith activated microglia marker, CD11b. Expression levels of cystatin F mRNA and proteinwere significantly increased in the corpus callosum, in a time-dependent manner, indicatingthat chronic cerebral hypoperfusion induced the expression of cystatin F in the microglia.
(2) Effect of intervention of adenosine A2Areceptor activity on expression of cystatin Finduced by chronic cerebral hypoperfusion
In comparison of adenosine A2Areceptor knockout mice and their littermate wild-typemice, the number of cystation F and CD11b co-expressed positive cells was significantlyincreased in the area of white matter lesions after cerebral ischemia (P<0.05), and cystatin Fexpression at mRNA and protein levels was significantly higher in the corpus callosum(P<0.05). For adenosine A2Areceptor agonist CGS21680treated wild-type mice and thewild-type mice from the same litter, the co-expressed cells were significantly less (P<0.05),and the mRNA and protein expression of cystatin F was also significantly lower in thecorpus callosum (P<0.05). These indicated that adenosine A2Areceptor mediated theexpression of cystatin F induced by chronic cerebral hypoperfusion, and activation ofadenosine A2Areceptor inhibited cystatin F expression.
6. Effect of intervention of adenosine A2Areceptor activity on microgliaproliferation and inflammatory cytokine expression induced by chronic cerebralhypoperfusion
(1) Proliferation of microglia cells and infiltration of peripheral inflammatory cells
In2,4and6w after ischemia, immunohistochemical staining of microglia markerCD11b found there were significantly more microglia in the area of white matter in4w than in2w (P<0.05), with processes retraction and enlarged cell body, which were inconsistence with the aforementioned changes in the white matter lesion. The microglialproliferation in the area of white matter lesion was more significant in bone marrow celltransplantation KO WT group than in WT WT group (P<0.05). But the proliferation wassignificantly reduced in bone marrow cell transplantation WT KO group compared withthe KO KO group (P<0.05). These suggested that chronic cerebral hypoperfusion inducedmicroglial proliferation and activation, and activation of bone marrow-derived cellsadenosine A2Areceptor inhibited the proliferation.
Immunohistochemical staining for lymphocyte markers CD3and neutrophil markerLy6G showed no positive cells was found in all mouse groups at all time points. It indicatedthat there was no obvious infiltration of peripheral blood inflammatory cells in injuredwhite matter regions during chronic cerebral hypoperfusion.
(2) Expression of inflammatory cytokines in injured white matter regions
In2,4and6w after ischemia, RT-qPCR and Western blotting were used to detect theexpression of inflammatory cytokines at mRNA and protein levels in the corpus callosum.
The mRNA and protein expression of proinflammatory cytokines, IL-1beta, IL-6,TNF-alpha, IL-12p40was increased in a time-dependent manner, with those of bonemarrow cell transplantation KO WT group being increased more significantly than thoseof WT WT group, and with those of bone marrow cell transplantation WT KO groupbeing slightly elevated than those of KO KO group. Adenosine A2Areceptor agonistCGS21680resulted in a mildly elevated expression of above proinflammatory cytokines inwild-type mice than in control treated with the carrier solution. adenosine It also resulted ina mildly elevated expression in the bone marrow cells derived adenosine A2Areceptorselective reconstruction than the mice treated with carrier solution. The mRNA and proteinexpression of anti-inflammatory cytokine IL-10, as well as the intervention of the adenosineA2Areceptor activity, was in an opposite trend with those of above proinflammatorycytokines.
These results indicated that the inflammatory pathological reactions caused byexpression changes of these proinflammatory cytokines and anti-inflammatory cytokinemay be an important mechanism of white matter lesions after chronic cerebral hypoperfusion,and activation of bone marrow-derived cells adenosine A2Areceptor reduced the white matter lesions by inhibiting the inflammatory pathological response.
Conclusions
1. Adenosine A2Areceptors on bone marrow-derived cells exert major effect on whitematter lesions induced by chronic cerebreal hypoperfusion. Selective activation ofadenosine A2Areceptors on bone marrow-derived cells show protective effect on whitematter injury, while selective deficency of adenosine A2Areceptors on bone marrow-derivedcells aggravate white matter lesions.
2. Chronic cerebral hypoperfusion induces the expression of cystatin F in microglia.Activation of adenosine A2Areceptor inhibits the expression and regulates the downstreaminflammatory pathological reactions in turn.
3. Expression of proinflammatory cytokines are increased, while those ofanti-inflammatory cytokines decreased in the injured white matter regions after chroniccerebral hypoperfusion. Selective activation of adenosine A2Areceptor inhibits theexpression of proinflammatory cytokines and promotes the expression of anti-inflammatorycytokines, which is the underlying important mechanism of its protective effect on whitematter lesions.
4. The inflammatory pathological damage is an important mechanism of white matterlesions after chronic cerebral hypoperfusion. Activation of adenosine A2Areceptor inperipheral inflammatory cells alleviates the white matter lesions by inhibiting theinflammatory response.
缺血性白质脑病(white matter lesion)是由大脑半球深穿支小动脉(Deep perforators small artery)的广泛病变,或颈部大动脉及其颅内分支狭窄,导致持续脑血流低灌注,从而造成以半卵圆区及侧脑室旁白质疏松为其病理特点,临床以认知损害为突出表现的疾病状态,是中老年血管性认知损害的主要类型之一。目前对于缺血性白质脑病的分子病理机制认识尚不足,因而制约了治疗药物的研发,临床缺少有效药物治疗手段。因此,迫切需要更深入地探讨缺血性白质脑病的分子病理机制,以期进而提出有效的治疗策略和方法。
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,在某些疾病的临床研究和动物实验中发现,腺苷A2A受体及其介导的信号通路可能成为治疗的靶点,因此愈来愈受到基础和临床研究者的重视。目前,以腺苷A2A受体作为治疗靶点的多种药物已经进入临床试验,包括帕金森病、Huntington病、全身炎性反应综合征和艾滋病等的药物临床试验。
本课题组在前期研究中发现慢性低灌注白质损伤病理机制不同于急性缺血性脑损伤,腺苷A2A受体介导的炎性效应是影响慢性低灌注白质损伤结局的重要因素。腺苷A2A受体也存在于外周血液中骨髓来源细胞,Yu等在急性脑缺血的动物实验中发现骨髓来源细胞腺苷A2A受体的选择性缺失影响脑组织内炎性介质的产生,提示骨髓来源细胞A2A受体可能对缺血诱导的脑内炎性反应产生重要的调节作用。
为了进一步探讨腺苷A2A受体介导的效应对慢性低灌注白质损伤炎性病理过程产生影响的可能机制,我们进行了以下研究：①利用腺苷A2A受体基因敲除动物,采用骨髓移植细胞嵌合的方法建立骨髓来源细胞腺苷A2A受体选择性缺失和选择性重建的动物模型,观察对慢性低灌注白质损伤的影响,以明确腺苷作用于A2A受体并对慢性低灌注白质损伤产生影响的具体效应细胞；②检测慢性脑血流低灌注诱导的局限表达于免疫细胞和炎细胞的信号转导分子Cystatin F表达的改变,并观察干预腺苷A2A受体活性对其表达及慢性低灌注白质损伤结局的影响,探讨腺苷A2A受体对慢性低灌注白质损伤产生影响的下游分子病理途径；③检测慢性脑血流低灌注诱导的白质损伤区域前炎性细胞因子表达的变化,以及干预腺苷A2A受体活性对其表达的影响,探讨与慢性低灌注白质炎性病理损伤有关的信号转导途径。
方法：
1.实验动物
引进腺苷A2A受体基因敲除(A2A receptor gene knock-out, A2AR/KO)杂合子小鼠交配繁殖,聚合酶链反应(polymerase chain reaction, PCR)方法鉴定A2AR/KO小鼠和野生型(wild-type littermates, A2AR/WT)小鼠。
2.骨髓移植细胞嵌合模型的建立
采用骨髓移植细胞嵌合的方法建立骨髓来源细胞腺苷A2A受体选择性缺失及选择性重建模型。即放射线破坏雄性野生型或A2AR/KO(?)、鼠骨髓后,移植雌性A2AR/KO小鼠骨髓细胞至野生型小鼠构建骨髓来源细胞腺苷A2A受体选择性缺失模型(KO→WT),骨髓细胞移植WT→WT组作为其对照；移植雌性野生型小鼠骨髓细胞至A2AR/KO小鼠构建骨髓来源细胞腺苷A2A受体选择性重建模型(WT→KO),骨髓细胞移植KO→KO组作为其对照；PCR法检测雄性受体外周血性染色体及免疫荧光法检测外周血白细胞A2A受体表达,鉴定骨髓移植细胞嵌合模型是否成功建立。
3.慢性脑血流低灌注模型的建立
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,建模前及建模后2h监测脑血流以判断模型是否成功；建模前及建模后4w,对部分动物行Morris水迷宫实验,记录每只动物发现平台的平均潜伏期和穿越平台的次数,以此为指标评价空间学习和记忆能力。
4.腺苷A2A受体激动剂干预
给予慢性脑血流低灌注野生型小鼠和骨髓来源细胞腺苷A2A受体选择性重建小鼠腺苷A2A受体激动剂CGS21680(0.25mg/kg,腹膜腔注射,1次／d,直至各组动物观察终点,即脑缺血后第2w、4w和6w,比较非选择性激活与选择性激活骨髓来源细胞腺苷A2A受体对于慢性脑血流低灌注白质损伤及炎性病理反应的影响。
5.白质损伤、小胶质细胞增生、炎性因子和Cystatin F表达的检测
分别于脑缺血后2w、4w和6w取脑标本,ZO-1免疫组化染色法观察血脑屏障开放程度,Kluver-Barrera及Bielschowsky染色法评价白质损伤,髓鞘碱性蛋白(myelinbasic protein,MBP)免疫组化染色法观察髓鞘脱失,比较各组动物血脑屏障和白质损伤程度。CD11b免疫组织化学染色方法观察并计数白质区域单位面积(0.25mm2)小胶质细胞活化及增生情况,抗CD3和抗LY6G免疫组化染色方法观察白质区域外周血液来源的炎性细胞浸润情况；提取胼胝体mRNA及总蛋白,分别采用实时定量PCR(real-time quantitative PCR,rt-qPCR))和western blot方法检测致炎因子IL-1β、TNF-α、IL-6.IL-12P40以及抗炎因子IL-10的mRNA和蛋白表达；免疫荧光双标染色观察Cystatin F在脑内的表达及细胞定位,rt-qPCR方法检测其mRNA和蛋白表达；组间比较前述指标以探讨选择性干预骨髓来源和非骨髓来源细胞腺苷A2A受体活性对慢性脑血流低灌注诱导的白质炎性病理反应的影响及其可能的信号转导途径。
结果：
1.成功建立骨髓移植细胞嵌合模型
(1)骨髓细胞移植前雄性受体小鼠外周血白细胞性染色体为两条带,符合雄性染色体特征,而接受雌性小鼠骨髓细胞移植后其外周血白细胞性染色体仅为一条带,符合雌性染色体特征,表明移植后雄性受体小鼠外周血白细胞来源于雌性供体骨髓细胞。
(2)骨髓细胞移植KO→WT组小鼠外周血白细胞A2A受体阳性率(5.28%±1.05%),与KO→KO组(6.62%±2.04%)比较无显著差异(>0.05),而显著低于骨髓细胞移植WT→WT组(92.32%±15.43%)(<0.05),表明成功建立骨髓来源细胞A2A受体选择性缺失模型。
(3)骨髓细胞移植WT→KO组小鼠外周血白细胞A2A受体阳性率(96.34%±12.58%),与WT→WT组(92.32%±15.43%)比较无显著差异(>0.05),而显著高于骨髓细胞移植KO→KO组(6.62%±2.04%)(<0.05),表明成功建立骨髓来源细胞A2A受体选择性重建模型。
2.成功建立慢性脑血流低灌注动物模型
(1)脑血流监测
在双侧颈总动脉圈套前及建模后2h分别检测脑血流量,各模型组动物建模后脑血流量下降40%-50%,组间比较无统计学差异(>0.05),表明该方法造成显著的脑缺血,且各模型组动物脑缺血程度相同。
(2)血脑屏障通透性检测
分别于脑缺血后2W、4W和6W,取脑标本ZO-1免疫组化染色,各时间点与假手术组动物比较显色强度评分均显著降低(<0.05),缺血后2W评分最低,此后逐渐升高,但至缺血后6W时仍低于对照水平,表明缺血后2W血脑屏障开放最显著,缺血后6w时血脑屏障仍处于开放状态。
骨髓来源细胞腺苷A2A受体选择性缺失与选择性重建动物,以及骨髓细胞移植对照组动物,组间比较ZO-1免疫组化染色强度评分无统计学差异(P>0.05)；腺苷A2A受体激动剂处理组,包括野生型和骨髓来源细胞腺苷A2A受体选择性重建动物,以及载体溶液处理的对照组动物,组间比较ZO-1免疫组化染色强度评分无统计学差异(P>0.05)；表明腺苷A2A受体介导的效应对慢性脑血流低灌注造成的血脑屏障通透性变化无影响。
(3)白质损伤的评价
分别于脑缺血后2w、4w和6w,取脑标本Kluver-Barrera染色和Bielschowsky银染评价白质纤维损伤,MBP免疫组化染色评价髓鞘脱失,发现随着脑缺血时间延长,白质损伤逐渐加重。
3.干预腺苷A2A受体活性对慢性脑血流低灌注动物认知行为的影响
(1)骨髓细胞移植各组动物的认知行为比较
骨髓细胞移植各组动物,包括骨髓来源细胞腺苷A2A受体选择性缺失与选择性重建动物,以及骨髓细胞移植对照组动物,脑缺血前组间比较Morris水迷宫实验测定的平均潜伏期及穿越平台次数无显著差异(P>0.05),表明骨髓细胞移植对小鼠的空间学习及记忆能力无影响。
(2)慢性脑血流低灌注对认知行为的影响
慢性脑血流低灌注各组动物,缺血后第4w采集的数据分别与其缺血前的数据比较,Morris水迷宫实验测定的平均潜伏期延长及穿越平台次数减少(P<0.05),表明脑缺血后动物的空间学习和记忆能力受到损害。
(3)骨髓来源细胞腺苷A2A受体选择性缺失对脑缺血引起的认知损害的影响
骨髓细胞移植KO→WT组与WT→WT组比较,脑缺血后第4w,Morris水迷宫实验测定的平均潜伏期延长(P<0.05)，穿越平台次数显著减少(P<0.05),表明骨髓来源细胞腺苷A2A受体的选择性缺失,不但加重慢性脑血流低灌注造成的空间学习能力障碍,也加重记忆功能损害。
(4)骨髓来源细胞腺苷A2A受体选择性重建对脑缺血引起的认知损害的影响
骨髓细胞移植WT→KO组与KO→KO组比较,脑缺血后第4w,Morris水迷宫实验测定的平均潜伏期显著缩短(P<0.05),表明骨髓来源细胞腺苷A2A受体的选择性重建减轻慢性脑血流低灌注造成的空间学习能力损害。
4.干预腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
(1)干预整体腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
分别于脑缺血后第2w、4w和6w,取脑标本Kluver-Barrera染色观察胼胝体神经纤维损伤情况,比较发现腺苷A2A受体基因敲除动物胼胝体损伤较同窝野生型动物明显加重,而腺苷A2A受体激动剂CGS21680处理动物胼胝体损伤较载体溶液对照动物明显减轻,表明激活腺苷A2A受体对慢性脑血流低灌注白质损伤具有保护效应。
(2)选择性干预骨髓来源细胞腺苷A2A受体活性对慢性脑血流低灌注白质损伤的影响
分别于脑缺血后第2w、4w和6w,取脑标本Kluver-Barrera和Bielschowsky染色评价白质纤维损伤情况,MBP免疫组化染色评价髓鞘脱失情况。骨髓细胞移植KO→WT组与WT组比较,白质纤维损伤和髓鞘脱失更严重；WT→KO组与KO→KO组相比,白质纤维损伤和髓鞘脱失明显较轻；表明骨髓来源细胞腺苷A2A受体的选择性缺失加重慢性脑血流低灌注导致的白质损伤,骨髓来源细胞腺苷A2A受体介导的效应减轻白质损伤。
脑缺血后各时间点,骨髓细胞移植WT组与KO组比较白质损伤更严重；脑缺血后第4w和6w,骨髓细胞移植KO组与WT组相比白质损伤较轻；提示非骨髓来源细胞的腺苷A2A受体介导的效应对慢性脑血流低灌注导致的白质损伤也产生影响,可能加重损伤,但对白质损伤结局并不发挥主导作用。
腺苷A2A受体激动剂CGS21680处理各组及其载体溶液对照组动物比较,白质损伤严重程度依次为载体溶液处理的野生型动物、载体溶液处理的骨髓来源细胞腺苷A2A受体选择性重建动物、激动剂CGS21680处理的野生型动物和激动剂CGS21680处理的骨髓来源细胞腺苷A2A受体选择性重建动物。表明选择性激活骨髓来源细胞腺苷A2A受体可以减轻慢性脑血流低灌注导致的白质损伤,也提示激活非骨髓来源细胞的腺苷A2A受体可能加重损伤。
5.干预腺苷A2A受体活性对慢性脑血流低灌注诱导的Cystatin F表达的影响
分别于脑缺血后第2w、4w和6w取脑标本,采用免疫荧光双标染色检测脑内Cystatin F的表达及其细胞定位,分别采用rt-qPCR和western blot方法定量检测其mRNA和蛋白表达的变化。
(1)慢性脑血流低灌注诱导的Cystatin F表达变化
脑缺血后白质损伤区域可见Cystatin F与活化小胶质细胞标志物CDllb共表达；胼胝体Cystatin F的mRNA和蛋白表达水平显著升高,且随着脑缺血时间的延长而逐渐增加；表明慢性脑血流低灌注诱导小胶质细胞表达Cystatin F增多。
(2)干预腺苷A2A受体活性对慢性低灌注诱导的Cystatin F表达的影响
腺苷A2A受体基因敲除动物与其同窝野生型动物相比,脑缺血后白质损伤区域单位面积Cystation F和CDllb共表达的阳性细胞数显著增多(P<0.05),胼胝体CystatinF的mRNA和蛋白表达也显著较多(P<0.05)；而腺苷A2A受体激动剂CGS21680处理的野生型小鼠与同窝野生型动物相比,Cystation F和CDllb共表达的阳性细胞计数显著较少(P<0.05),胼胝体Cystatin F的：mRNA和蛋白表达也显著较低(P<0.05)；表明腺苷A2A受体介导的效应对慢性脑血流低灌注诱导的Cystatin F表达产生影响,激活腺苷A2A受体可抑制Cystatin F表达。
6.干预腺苷A2A受体活性对慢性脑血流低灌注诱导的小胶质细胞增生和炎性因子表达的影响
(1)小胶质细胞增生和外周炎细胞浸润的观察
分别于脑缺血后第2w、4w和6w取脑标本,小胶质细胞标记物CD11b免疫组化染色发现,缺血后第4w与第2w比较,白质损伤区域小胶质细胞数量明显增多(P<0.05),突起回缩,胞体增大,前述变化与白质损伤严重程度一致。骨髓细胞移植KO→WT组与WT组比较,白质损伤区域小胶质细胞的增生更为显著(P<0.05)；骨髓细胞移植KO组与KO组相比,白质损伤区域小胶质细胞的增生显著减少(P<0.05)。表明慢性脑血流低灌注诱导小胶质细胞的增生和活化,激活骨髓来源细胞的腺苷A2A受体可抑制其增生。
淋巴细胞标记物CD3和中性粒细胞标记物LY6G免疫组化染色,在各组动物各时间点的观察,均未发现阳性显色。提示慢性脑血流低灌注过程中,白质损伤区无明显的外周血液来源的炎细胞浸润。
(2)白质损伤区炎性因子表达的观察
分别于脑缺血后第2w、4w和6w取脑标本,分离胼胝体组织,采用rt-qPCR和western blot方法定量检测炎性因子mRNA和蛋白表达的变化。
致炎因子IL-1β、IL-6、TNF-a、IL-12p40的mRNA和蛋白表达,随脑缺血时间延长逐渐升高；骨髓细胞移植KO→WT组与WT→WT组比较升高更显著；骨髓细胞移植WT→KO组与KO→KO组相比升高程度较轻；腺苷A2A受体激动剂CGS21680处理的野生型动物与载体溶液处理的对照动物相比升高程度较轻；腺苷A2A受体激动剂CGS21680处理的骨髓来源细胞腺苷A2A受体选择性重建动物与载体溶液处理的对照动物相比升高程度较轻。抗炎因子IL-10mRNA和蛋白表达的变化,以及干预腺苷A2A受体活性对其产生的影响,与致炎因子的表达变化呈相反的趋势。
提示致炎因子和抗炎因子表达变化引起的炎性病理反应可能是慢性脑血流低灌注白质损伤的重要机制,激活骨髓来源细胞的腺苷A2A受体通过抑制炎性病理反应而减轻白质损伤。
结论：
1.骨髓来源细胞腺苷A2A受体介导的效应对慢性低灌注白质损伤产生重要影响,选择性激活骨髓来源细胞腺苷A2A受体对白质损伤具有保护作用,选择性抑制骨髓来源细胞腺苷A2A受体则加重白质损伤。
2.慢性脑血流低灌注诱导小胶质细胞表达Cystatin F,激活腺苷A2A受体可抑制其表达,可能进而对下游炎性病理反应产生调控作用。
3.慢性脑血流低灌注白质损伤区致炎因子表达增加,抗炎因子表达减少；选择性激活骨髓来源细胞的腺苷A2A受体可抑制致炎因子表达和促进抗炎因子表达,是其对白质损伤发挥保护作用的重要机制。
4.进一步证明炎性病理损伤是慢性脑血流低灌注白质损伤的重要机制,揭示激活外周炎细胞腺苷A2A受体通过抑制炎性反应而减轻白质损伤。
Background
Ischemic leukoencephalopathy is a disease pathologically characterized as whitematter rarefaction in semiovale area and periventricular area, resulting from sustainedcerebral hypoperfusion due to extensive lesions of cerebral deep perforators small artery, orstenosis of cervical arteries and its intracranial branches. Its prominent clinicalmanifestation is cognitive impairment, and is one of the main types of vascular cognitiveimpairment in the elderly. The molecular mechanism of this disease remains unclear, whichrestricts the development of therapeutic drugs, and thus there is no effective drug treatmentin clinical practice. Therefore, there is an urgent need to explore the molecular mechanismof white matter lesions, in order to come up with effective treatment strategies and methods.
Adenosine is intermediate product in energy metabolism, and also importantneuromodulators in the central nervous system. Adenosine A2Areceptors are involved in theregulation of a variety of physiological activities and some pathological processes in certaindiseases. Clinical researches and animal experiments found that adenosine A2Areceptor-mediated signaling pathway might be the treatment target, and therefore, itattracted more and more attentions from basic and clinical researchers. In present, a varietyof drugs with adenosine A2Areceptor as therapeutic target for have entered clinical trials,including clinical trials for Parkinson's disease, Huntington's disease, systemicinflammatory response syndrome and AIDS
Our group has found in our previous study that chronic hypoperfusion of white matterinjury has different pathological mechanisms from acute ischemic brain injury. AdenosineA2Areceptor-mediated inflammatory effect is the important factor for outcome of chronichypoperfusion white matter injury. Adenosine A2Areceptors are found in bone marrow-derived cells in the peripheral blood. Yu et al found that the selective deletion ofadenosine A2Areceptor in bone marrow-derived cells affects the generation of inflammatorymediators in the brain tissue in animal studies of acute cerebral ischemia, suggesting thatbone marrow-derived cells A2Areceptors may play an important role in the regulation ofbrain ischemia-induced inflammatory response.
To further investigate the effect of the adenosine A2Areceptor-mediated impact on thepossible mechanism of chronic hypoperfusion of pathological process of white matter lesion,we conducted the following studies:(1) With the aid of adenosine A2Areceptor knockoutanimals, we want to establish the animal model of bone marrow-derived cells in theadenosine A2Areceptor selective deletion and selective reconstruction by bone marrowtransplanted chimeric cells in order to observe the impact of chronic hypoperfusion onwhite matter lesions, so as to clarify the role of adenosine in adenosine A2Areceptor and inchronic hypoperfusion white matter lesions, and the specific effector cells in the process;
We detected the expression change of cystatin F, a signal transduction molecule betweenimmune cells and inflammatory cells, in the chronic cerebral hypoperfusion, and observedits expression profiles after interfering adenosine A2Areceptor activity and the outcome ofhypoperfusion white matter injury, in order to explore the downstream molecular pathologyway of adenosine A2Areceptor in chronic hypoperfusion white matter lesions;(3) we alsodetected the expression changes of proinflammatory cytokines in chronic cerebralhypoperfusion-induced white matter injury area, and their changes after interferingadenosine A2Areceptor activity, and to explore the signal transduction pathways in thepathological damage related to chronic hypoperfusion and white matter inflammation.
Methods
1. Experimental Animals
The adenosine A2Areceptor gene knock-out (A2AR/KO) heterozygous mice wereemployed and bred in this study. Polymerase chain reaction (PCR) method was used toidentify A2AR/KO mice and wild-type (wild-type littermates, A2AR/WT) mice.
2. Establishment of bone marrow transplantation chimera modell
Adenosine A2Areceptor selective deletion and selective reconstruction model wasestablished by bone marrow transplantation chimera of bone marrow-derived cells. Aftermale wild-type or A2AR/KO mice were given bone marrow radiation damage, the female A2AR/KO mouse bone marrow cells were transplanted into wild-type mice to built towild-type mice bone marrow-derived cells adenosine A2Areceptor selective deficit model(KO WT), and bone marrow cells transplantation WT WT group served as its control.The female wild-type mice bone marrow cells were transplanted to A2AR/KO mouse tobuild bone marrow-derived cells adenosine A2Areceptor selective reconstruction model(WT KO), and bone marrow cells transplantation KO KO group served as its control.PCR assay was used to detect male receptor peripheral blood chromosome, andimmunofluorescence assay was used for A2Areceptor expression in peripheral bloodleukocytes in order to identify the successfully establishment of bone marrowtransplantation chimera model.
3. Mice model of chronic cerebral hypoperfusion
According to the method by Shibata et al, a micro-spring trap with inner diameter of0.18mm was used to occlude bilateral common carotid artery to establish mouse modelwith chronic cerebral hypoperfusion. Cerebral blood flow was monitored before and in2hafter operation in order to determine whether the model was successful. Before and in4wafter model establishment, Morris water maze test was carried out to record averageplatform latency and the frequency of crossing platform, which indicating the spatiallearning and memory.
4. Intervention of adenosine A2Areceptor agonist
Adenosine A2Areceptor agonist CGS21680(0.25mg/kg, intraperitoneal injection, onceper day, until the end of each time point, that is, in2,4and6w,after cerebral ischemia) wasgiven to cerebral hypoperfusion wild-type mice and bone marrow-derived cells adenosineA2Areceptor selective reconstruction mice. The effect of adenosine A2Areceptor fromnon-selective activation and selective activation of bone marrow-derived cells wascompared on chronic cerebral hypoperfusion white matter lesions and inflammatorypathological reaction.
5. Detection of white matter lesions, proliferation of glial cells, and expression ofinflammatory cytokines and cystatin F
After ischemia for2,4and6w, the mouse brain specimens were collected. ZO-1immunohistochemical staining was used to measure the openness of the blood-brain barrier.Kluver-Barrera and Bielschowsky staining was employed to evaluate white matter lesions. Immunohistochemical staining for myelin basic protein (MBP) was used to observedemyelination. The openness of blood-brain barrier and severity of white matter lesion werecompared in each group. Immunohistochemical staining for CD11b was used to observe andcount the microglia activation and proliferation in per unit area of white matter (0.25mm2).Immunohistochemical staining for CD3and Ly6G was used to observe the infiltration ofinflammatory cells from of peripheral blood in the white matter. After total mRNA andprotein of corpus callosum was extracted, respectively, real-time quantitative PCR(RT-qPCR)) and Western blotting were used to detect the expression of proinflammatorycytokines IL-1beta, TNF-alpha, IL-6, IL-12p40and anti-inflammatory factor IL-10atmRNA and protein levels. Immunofluorescence double staining was used to observe theexpression and cellular localization of Cystatin F in the brain, and rt-qPCR to detect themRNA and protein expression. The aforementioned indicators were compared to exploreselective intervention of adenosine A2Areceptor activity from bone marrow-derived andnon-bone marrow-derived cells induced by chronic low cerebral blood flow perfusion of thewhite matter inflammatory pathological response and its possible signal transductionpathways.
Results
1.Successful establishment of bone marrow transplantation chimera model
(1) Before transplantation of bone marrow cells, there are two bands of chromosome inthe peripheral blood leukocytes of male recipient mice, in line with the characteristics of themale chromosome, while, their peripheral blood leukocytes only have one band ofchromosome after accepting transplantation of female mouse bone marrow cells, in linewith the female chromosome characteristics, indicating that the peripheral blood leukocytesof male recipient mice were derived from the female donor bone marrow cells.
(2) The A2Areceptor-positive rate was (5.281.05)%in peripheral blood leukocytesin KO WT group of mice after bone marrow cell transplantation, and that was (6.622.04)%in the KO KO group, with no significant difference between2groups (P>0.05).The rate was significantly lower in KO WT group than WT WT group [(92.3215.43)%, P<0.05), indicating successful establishment of bone marrow-derived cells A2Areceptor selective deficit model.
(3) The A2Areceptor positive rate in peripheral blood leukocytes was (96.3412.58)% in WT KO group mice after bone marrow cells transplantation, which had no significantdifference compared with that of WT WT group [(92.3215.43)%, P>0.05), and wassignificantly higher than that of KO KO group [(6.622.04)%, P<0.05), indicatingsuccessful establishment of bone marrow-derived cells A2Areceptor selective reconstructionmodel.
2. Successfully establishment of mouse model of chronic cerebral hypoperfusion
(1) Cerebral blood flow monitoring
In the trap of bilateral carotid artery before and in2h after operation, the cerebralblood flow was decreased by40%to50%after the model establishment. There was nosignificant difference between the groups (P>0.05), indicating that the method causingsignificant cerebral ischemia, with same severity in each model group.
(2) Permeability of blood-brain barrier
After ischemia for2,4and6w, ZO-1immunohistochemical staining showed thatcolor intensity scores at each time point were significantly lower compared with the shamoperation group (P<0.05), that of2w rating minimum, then gradually increased, but stillbelow the level of control at6w. These indicated the openness of blood-brain barrier wasmost significant in2weeks’ ischemia, and remained after5weeks’ ischemia.
No statistically significant difference was seen in ZO-1immunohistochemical stainingintensity among bone marrow-derived cell adenosine A2Areceptor deficit and selectivereconstruction models, as well as bone marrow cell transplantation control model (P>0.05).Adenosine A2Areceptor agonist treatment groups, including the wild-type cells and bonemarrow-derived adenosine A2Areceptor selective reconstruction animals, as well as thecontrol animals had no significant difference in ZO-1immunohistochemical stainingintensity scores (P>0.05). These suggested that adenosine A2Areceptor-mediated effects hadno effect on the blood-brain barrier permeability induced by chronic cerebral blood flowhypoperfusion.
(3) Evaluation of white matter lesions
After ischemia for2,4and6w, Kluver-Barrera staining and Bielschowsky silverstaining showed white matter fiber damage, and MBP immunohistochemical stainingdisplayed demyelination. The longer cerebral ischemia time, the severe the white matterlesions
3Effect of adenosine A2Areceptor activity on cognitive behavior in mice afterchronic cerebral hypoperfusion
(1) Comparison of cognitive behavior in mice after bone marrow cells transplantation
In each animal group after bone marrow cell transplantation, including bonemarrow-derived cells adenosine A2Areceptor selective deletion and selective reconstruction,as well as bone marrow cell transplantation control mice, there was no significant differencein the average platform latency and the frequency of crossing platform in Morris watermaze before ischemia (P>0.05), indicating that bone marrow cell transplantation having noeffect on spatial learning and memory of the mice.
(2) Effect of chronic cerebral hypoperfusion on cognitive behavior
The chronic low cerebral blood flow perfusion groups, the average platform latencyand the frequency of crossing platform in Morris water maze were significantly lower in themice after4weeks’ ischemia than before ischemia (P<0.05), indicating impaired spatiallearning and memory.
(3) Effect of bone marrow-derived cells adenosine A2Areceptor selective deletion oncognitive impairment induced by cerebral ischemia
The average platform latency was longer (P<0.05) and the frequency of crossingplatform was decreased (P<0.05) in Morris water maze in bone marrow cell transplantationKO WT group compared with WT WT group after cerebral ischemia for4week,indicating that bone marrow-derived cell adenosine A2Areceptor selective deficit, not onlyenhancing the space learning disabilities caused by chronic cerebral hypoperfusion, but alsoaggravating the memory dysfunction.
(4) Effect of bone marrow-derived cells adenosine A2Areceptor selectivereconstruction on cerebral ischemia-induced cognitive impairment
The average platform latency was shorter (P<0.05) in Morris water maze in bonemarrow cell transplantation WT KO group compared with KO KO group after cerebralischemia for4week, indicating that bone marrow-derived cells adenosine A2Areceptorselective reconstruction alleviating spatial learning impairment caused by chronic cerebralhypoperfusion.
4. Effect of intervention of adenosine A2Areceptor activity on white matterlesions after chronic cerebral hypoperfusion
(1) Effect of intervention of overall adenosine A2Areceptor activity on white matterlesions after chronic cerebral hypoperfusion
In2,4and6w after ischemia, Kluver-Barrera staining showed nerve fiber injury incorpus callosum was significantly worse in the adenosine A2Areceptor gene knockout micethan the wild-type mice from same litter. And adenosine A2Areceptor agonist CGS21680significantly reduced the damage in corpus callosum damage than the control, suggestingthat activation of the adenosine A2Areceptor has a protective effect on white matter lesionsafter chronic cerebral hypoperfusion.
(2) Effect of selective intervention of bone marrow-derived cells adenosine A2Areceptor activity on white matter lesions after chronic cerebral hypoperfusion
In ischemia for2,4and6w, Kluver-Barrera and Bielschowsky staining showed thatsevere white matter fiber damage and MBP immunohistochemical staining showed severedemyelination in bone marrow cell transplantation KO WT group compared with WTWT group, but white matter fiber damage and demyelination was reduced in WT KOgroup compared with the KO KO group, indicating that the bone marrow-derived cellsadenosine A2Areceptor selective deficit aggravating white matter lesions due to chroniccerebral hypoperfusion, and bone marrow-derived cells adenosine A2Areceptor-mediatedeffects reducing the white matter lesions.
In different time points after cerebral ischemia at, white matter lesions were moreserious in bone marrow cell transplantation KO WT group than KO KO group. Incerebral ischemia for4and6week, bone marrow cell transplantation WT KO group hadmilder white matter damage than WT WT group, indicating that non-bonemarrow-derived cells adenosine A2Areceptor had effects on white matter lesions afterchronic cerebral hypoperfusion caused, and might aggravate the injury, but not in a leadingrole.
When adenosine A2Areceptor agonist CGS21680treatment groups were comparedwith their control, the severity of white matter lesions were wild-type mice after carriersolution treatment, bone marrow-derived cells adenosine A2Areceptor selectivereconstruction mice after carrier solution treatment, wild-type mice after the agonistCGS21680treatment, and bone marrow-derived cells adenosine A2Areceptor selectivereconstruction mice after the agonist CGS21680treatment in order. These showed that the selective activation of bone marrow-derived cells adenosine A2Areceptor reduced whitematter lesions due to chronic cerebral hypoperfusion, and the adenosine A2Areceptoractivation of non-bone marrow-derived cells may aggravate the injury.
5. Effect of intervention of adenosine A2Areceptor activity on expression ofcystatin F induced by chronic cerebral hypoperfusion
After ischemia for2,4and6w, double immunofluorescence staining showed cystatinF expression and cellular localization in the brain. RT-qPCR and Western blotting indicatedits quantitative expression at mRNA and protein levels.
(1) Expression of cystatin F after chronic cerebral hypoperfusion
In the injuried area of white matter after cerebral ischemia, cystatin F was co-expressedwith activated microglia marker, CD11b. Expression levels of cystatin F mRNA and proteinwere significantly increased in the corpus callosum, in a time-dependent manner, indicatingthat chronic cerebral hypoperfusion induced the expression of cystatin F in the microglia.
(2) Effect of intervention of adenosine A2Areceptor activity on expression of cystatin Finduced by chronic cerebral hypoperfusion
In comparison of adenosine A2Areceptor knockout mice and their littermate wild-typemice, the number of cystation F and CD11b co-expressed positive cells was significantlyincreased in the area of white matter lesions after cerebral ischemia (P<0.05), and cystatin Fexpression at mRNA and protein levels was significantly higher in the corpus callosum(P<0.05). For adenosine A2Areceptor agonist CGS21680treated wild-type mice and thewild-type mice from the same litter, the co-expressed cells were significantly less (P<0.05),and the mRNA and protein expression of cystatin F was also significantly lower in thecorpus callosum (P<0.05). These indicated that adenosine A2Areceptor mediated theexpression of cystatin F induced by chronic cerebral hypoperfusion, and activation ofadenosine A2Areceptor inhibited cystatin F expression.
6. Effect of intervention of adenosine A2Areceptor activity on microgliaproliferation and inflammatory cytokine expression induced by chronic cerebralhypoperfusion
(1) Proliferation of microglia cells and infiltration of peripheral inflammatory cells
In2,4and6w after ischemia, immunohistochemical staining of microglia markerCD11b found there were significantly more microglia in the area of white matter in4w than in2w (P<0.05), with processes retraction and enlarged cell body, which were inconsistence with the aforementioned changes in the white matter lesion. The microglialproliferation in the area of white matter lesion was more significant in bone marrow celltransplantation KO WT group than in WT WT group (P<0.05). But the proliferation wassignificantly reduced in bone marrow cell transplantation WT KO group compared withthe KO KO group (P<0.05). These suggested that chronic cerebral hypoperfusion inducedmicroglial proliferation and activation, and activation of bone marrow-derived cellsadenosine A2Areceptor inhibited the proliferation.
Immunohistochemical staining for lymphocyte markers CD3and neutrophil markerLy6G showed no positive cells was found in all mouse groups at all time points. It indicatedthat there was no obvious infiltration of peripheral blood inflammatory cells in injuredwhite matter regions during chronic cerebral hypoperfusion.
(2) Expression of inflammatory cytokines in injured white matter regions
In2,4and6w after ischemia, RT-qPCR and Western blotting were used to detect theexpression of inflammatory cytokines at mRNA and protein levels in the corpus callosum.
The mRNA and protein expression of proinflammatory cytokines, IL-1beta, IL-6,TNF-alpha, IL-12p40was increased in a time-dependent manner, with those of bonemarrow cell transplantation KO WT group being increased more significantly than thoseof WT WT group, and with those of bone marrow cell transplantation WT KO groupbeing slightly elevated than those of KO KO group. Adenosine A2Areceptor agonistCGS21680resulted in a mildly elevated expression of above proinflammatory cytokines inwild-type mice than in control treated with the carrier solution. adenosine It also resulted ina mildly elevated expression in the bone marrow cells derived adenosine A2Areceptorselective reconstruction than the mice treated with carrier solution. The mRNA and proteinexpression of anti-inflammatory cytokine IL-10, as well as the intervention of the adenosineA2Areceptor activity, was in an opposite trend with those of above proinflammatorycytokines.
These results indicated that the inflammatory pathological reactions caused byexpression changes of these proinflammatory cytokines and anti-inflammatory cytokinemay be an important mechanism of white matter lesions after chronic cerebral hypoperfusion,and activation of bone marrow-derived cells adenosine A2Areceptor reduced the white matter lesions by inhibiting the inflammatory pathological response.
Conclusions
1. Adenosine A2Areceptors on bone marrow-derived cells exert major effect on whitematter lesions induced by chronic cerebreal hypoperfusion. Selective activation ofadenosine A2Areceptors on bone marrow-derived cells show protective effect on whitematter injury, while selective deficency of adenosine A2Areceptors on bone marrow-derivedcells aggravate white matter lesions.
2. Chronic cerebral hypoperfusion induces the expression of cystatin F in microglia.Activation of adenosine A2Areceptor inhibits the expression and regulates the downstreaminflammatory pathological reactions in turn.
3. Expression of proinflammatory cytokines are increased, while those ofanti-inflammatory cytokines decreased in the injured white matter regions after chroniccerebral hypoperfusion. Selective activation of adenosine A2Areceptor inhibits theexpression of proinflammatory cytokines and promotes the expression of anti-inflammatorycytokines, which is the underlying important mechanism of its protective effect on whitematter lesions.
4. The inflammatory pathological damage is an important mechanism of white matterlesions after chronic cerebral hypoperfusion. Activation of adenosine A2Areceptor inperipheral inflammatory cells alleviates the white matter lesions by inhibiting theinflammatory response.
引文
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