Effect Of Adenosine A_2A Receptor Knock Out On Whiter Matter Lesions Induced By Chronic Cerebral Hypoperfusion And The Role Of A_2A Receptor In The Modulation Of Inflammatory Reaction

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.