Impact Of Inflammation Regulated By Adenosine A_2A Receptor In Bone Marrow Derived Cells On The White Matter Lesions Induced By Chronic Hypoperfusion And Its Possible Mechanism

BackgroundIschemic 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 AIDSOur 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.Methods1. Experimental AnimalsThe 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 modellAdenosine 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 hypoperfusionAccording 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 agonistAdenosine 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 FAfter 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.Results1.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 monitoringIn 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 barrierAfter 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 lesionsAfter 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 transplantationIn 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 behaviorThe 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 ischemiaThe 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 impairmentThe 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 hypoperfusionIn2,4and6w 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 hypoperfusionIn 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 hypoperfusionAfter 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 hypoperfusionIn 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 hypoperfusionIn 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 cellsIn2,4and6w 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 regionsIn2,4and6w 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.Conclusions1. 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.