Roles Of CD 73 In The White Matter Lesions Induced By Chronic Cerebral Hypoperfusion

BackgroundThe term vascular cognitive impairment(VCI) covers the spectrum of all cognitive disorders from mild cognitive impairment to dementia caused by cardiovascular factors. Ischemic white matter lesions(WMLs) mediate the essential pathogenesis of of Binswanger disease and is also one of the major causes in VCI patient. Recent stu dies have demonstrated that inflammatory and immune mechanisms are involved in the pathogenesis of WMLs induced by chronic cerebral hypoperfusion(CCH).Extracellular adenosine represents a critical endogenous mechanism for regulating immune and inflammatory responses through binding to G-protein coupled adenosine receptors on the effector cells. There are two sources of extracellular adenosine. First, it may be derived from the external transport of intracellularly generated adenosine. Second, it may predominantly be formed from the extracellular conversion of adenine nucleotides(ATP, ADP and AMP) by ectonucleotidases including nucleoside triphosphate dephosphorylase(CD39) and 5’-ectonucleotidase(CD73), the latter appears to be the rate-limiting step for adenosine production. In fact, a recent study using gene knock-out technique has indicated that CD73 is the major 5’-nucleotidase in the murine brain, which accounts for 85–95% of all AMP-hydrolyzing capabilities. These results suggest that CD73 may play an important role in the development of brain diseases associated with immune disorders and inflammatory responses through its modulation of extracellular adenosine level. Braun etal reported CD73 expression significantly increased in infarcted tissue in rat middle cerebral artery occlusion model which suggested the extracellular level of adenosine in the ischemic brain might increase following the enhanced conversion of adenine nucleotides. Petrovic-Djergovicet et al found CD73-null mice were more susceptible to acute ischemic brain injury, implying CD73 expression might be a defensive mechanism to alleviate the brain damage. Our previous study indicated that adenosine A2 A receptor deficiency aggravated WMLs induced by chronic cerebral ischemia. These finding s suggested that the alteration of extracellular adenosine level might influence the outcome of the pathological process. However, there have been no research reports on the possible role of CD73 in the development of CCH-induced WMLs. To clarify this may be valuable in providing new target for the intervention of ischemic WMLs, and thereby proposing novel strategy for the treatment of VCI.In the present study, based on the hypothesis that CD73 expression might change and consequently exert important impacts on WMLs induced by CCH, we investigated the expression and activity of CD73 in the brains of mice subjected to chronic cerebral ischemia and evaluated the effects of CD73 deficiency on inflammatory responses and WMLs induced by CCH in CD73 gene knock-out(KO) mice.MethodsPart I: Chronic cerebral hypoperfusion were induced by bilateral common carotid artery stenosis surgery(BCAS)The chronic cerebral hypoperfusion in the mice were induced by BCAS according to the method of Shibata et al using 0.18 mm diameter microcoils. Cognitive function were assessed by spontaneous alternation paradigm in a Y-maze and eight-arm radial maze test at 30 day after BCAS. WMLs and proliferation of glial cells were evaluated by Klüver-Barrera(KB) and immunohistochemistry staining for GFAP and Iba-1 at 30 day after BCAS.Part II: To observe the effect of chronic cerebral hypoperfusion on CD73 expression and activity in the corpus callosumCD73 protein expression in the corpus callosum were investigated by western blot. The distribution of CD73 in the corpus callosum were observed by immunohistochemistry staining. Double-immunofluorescence staining between CD73 and GFAP/Iba-1 was carried out to detect the cell which express CD73 in the corpus callosum in BCAS mice. CD73 activity in the corpus callosum were detected by measuring the rate of AMP hydrolysisPart III: To observe the effect of CD73 deficiency on the WMLs induce by CCHC57BL/6-CD73 knockout(KO) mice and their wild-type littermates(WT) were subjected to bilateral common carotid artery stenosis surgery. Cognitive function were assessed by spontaneous alternation paradigm in a Y-maze and eight-arm radial maze test at 30 day after BCAS. WMLs and proliferation of glial cells were evaluated by Klüver-Barrera(KB) and immunohistochemistry staining for GFAP and Iba-1 at 30 day after BCAS. In addition, TNF-α, IL-1β and IL-6 protein levels were determinated by enzyme-linked immunosorbent assay(ELISA)Results:Part I: BCAS could induce working memory impairment, white matter lesions and glial proliferationan eight-arm radial maze test was adopted to assess the working memory. BCAS animals showed more errors in the test when compared with Sham gr oup, indicating CCH induced by BCAS resulted in working memory impairment. Furthermore, Y-maze test with spontaneous alternation paradigm was also performed to evaluate working memory. The test revealed BCAS mice had more working memory errors compared with their sham-WT littermates, which was identical to the result of eight-arm radial maze test. In addition, no apparent difference in locomotion was found between Sham and BCAS group as evaluated by the number of arm changes, which suggested BCAS had no obvious impact on locomotor function. Glial cell proliferation and WMLs induced by CCH were evaluated by immunohistochemistry and KB staining in this study. Anti-GFAP and anti-Iba-1 antibodies were used to mark astrocytes and microglial cells respectively. Co mpared with the sham groups, BCAS groups showed marked increase in the numeric densities of astrocytes and microglia in corpus callosum, indicating the proliferation of glial cells following CCH. White matter integrity was investigated by KB staining. The animals in BCAS groups exhibited WMLs in the corpus callosum which indicate that BCAS can induce WMLs.Part II: CD73 expression and activity upregulate in the corpus callosum after BCASThe western blot analysis indicated that CD73 protein expression significantly increased in the corpus callosum in BCAS mice when compared with that of Sham mice. Immunohistochemical staining also showed that CD73-immunopositive cells intensively distributed in the corpus callosum in the animals subjected to BCAS, but sparsely existed in Sham mice. Double-immunofluorescence staining between CD73 and GFAP/Iba-1 suggested that CD73 colocalized with GFAP but not Iba-1 which imply that astrocytes express CD73 in WMLs. The rate of AMP hydrolysis in the plasma membrane prepared from corpus callosum was tested by enzyme assay. CD73 activity significantly increased in the BCAS group compared with those in the Sham group which indicated that BCAS can induce the increment of CD73 activity in WMLs.Part III: CD73 deficiency lead to more severe white matter lesions and cognitive impaiment induced by CCH in miceCompared with BCAS-WT group, BCAS-KO animals displayed more working memory errors which implied that CD73 deficiency aggravated the working memory impairment induced by CCH. Furthermore, Y-maze test with spontaneous alternation paradigmwas also performed to evaluate working memory in the animals. The test revealed that BCAS-KO mice had more working memory errors compared with their BCAS-WT littermates, which was identical to the result of eight-arm radial maze test. In addition, no apparent difference in locomotion was found in the test between Sham-WT and Sham-KO group as evaluated by the number of arm changes which suggested CD73 d eficiency had no obvious impact on locomotor function. Compared with the sham groups, both BCAS-WT and BCAS-KO groups showed marked increase in the numeric densities of astrocytes and microglia in corpus callosum. No significant difference was found in the numeric densities of astrocyte and microglia between sham-KO group and sham-WT group, implying that CD73 deficiency had no effect on glial cell proliferation at the base line. Compared with BCAS-WT group, BCAS-KO group had significantly higher numeric densities of astrocytes and microglia in corpus callosum, indicating CD73 deficiency promoted glial cell proliferation induced by CCH. White matter integrity was investigated by KB staining. No white matter rarefaction was detected in both sham-WT and sham-KO groups, demonstrating that CD73 did not affect the WM rarefaction at the baseline. Although the animals in both BCAS-WT and BCAS-KO groups exhibited WMLs in the brain, the injury was more severe in corpus callosum in BCAS-KO group which indicated that CD73 deficiency exacerbated WMLs induced by CCH. The protein expression of pro-inflammatory cytokines IL-1β, TNF-α and IL-6 was detected by ELISA. Compared with the sham-operated control group, both BCAS-WT and BCAS-KO groups exhibited significant enhancement of IL-1β, TNF-α and IL-6 protein levels in corpus callosum. However, compared with BCAS-WT group, BCAS-KO group had much higher IL-1β, TNF-α and IL-6 protein levels, indicating CD73 deficiency increased the expression of pro-inflammatory cytokines following CCH.Conclusion:Based on the above results, we can draw the conclusions as follows: The chronic cerebral hypoperfusion in the mice were induced by bilateral common carotid artery stenosis surgery using 0.18 mm diameter microcoils. BCAS could induce sig nificant working memory impairment, white matter lesions and glial proliferation. The above findings indicate that BCAS mice is a well established mice model to investigate the white matter lesions induced by chronic cerebral hypoperfusion. In addition, CD 73 expression and activity upregulate in the corpus callosum after BCAS and CD73 deficiency leads to the exacerbation of WMLs induced by CCH which is associated with augmented glial activation and inflammatory responses. Our findings suggest CD73 play a pr otective role in CCH-induced WLMs and might be as a new target in the intervention of chronic ischemic brain injury.