The Role Of Astrocytes In The Adult Mouse Brain Angiogenesis In The Course Of The Study

ObjectiveThe process of cerebrovascular angiogenesis contains of degradation of extra-endothelial basement membrane, proliferation of the endothelial cells, migration to the degraded basement membrane, formation of the tubular structure and production of the extra-endothelial matrix membrane. Blood brain barrier comprises of endothelial cells (ECs), tight junctions, the endfeet of astrocytes, pricytes, basement membrane and extra-cellular space. Basement membrane degradation and endothelial cell migration are the key points of angiogenesis, which will necessarily cause blood brain barrier (BBB) damage. At present, there is ample evidence that, under normal circumstances, the combination of endothelial cell and its tight junction and the endfeet of astrocytes play a major role in the barrier function of the BBB. However, our recent reasearch implied that the process of angiogenesis including the opening of endothelial cell tight junction and proliferation of endothelial cell through the basement membrane does not necessarily cause serious damage to the barrier function of BBB which result in dispersion of substances in the plasma. We believe that the maintenance of the BBB permeability is probably substituted temporarily by astrocytes in the process of angiogenesis. Recent research indicated that the ephemeral opening in partial capillaries was related with astrocytes. Unfortunately, morphology in vivo has not supported the theory that the astrocytes can maintain BBB permeability. So the establishment of renascent cerebral vessels pattern in vivo has important meaning to the search of cerebral mechanism.The objective in our research is to establish the AdhVEGF165 grown-up mouse blood vessel in brain model, and research in brain blood stream kinetics. To find the morphological proof whether astrocytes take part in angiogenesis, or can temporarily maintain the BBB stability, which will provide a new route or clew for the mechanism of cerebral angiogenesis.Methods1. Taking the grown-up KM mouse as the experimental pattern and the grown-up mouse with injecting AdhVEGF165 were made as a pattern.2. The micro-vessels density (MVD), the number of the renascent vessels were counted. The blood flow volume and blood volume were measured with Laser Doppler technique, before and after the operation at 3d, 1w,2w, 3w and 4w, and draw the kinetics curves.3. Injecting Lectin, Even's blue and other dyes into the blood vessel model, astrocytes and the feet-microvessel structure were found under the light microscope.4. Using immunohistochemistry pigmentation to ascertain the categories of the cells, the structure of the endfeet of astrocytes and the proliferating ECs were observed. The morphology of the astrocytes activating in the renascent procedure and the proliferation of the ECs could be further affirmed with immunohistochemistry technique.5. Data statistics:t-test were used for group comparison and SSPS 11.0 were used for data statistics.Results1. Equivalent AdhVEGF165 and 0.9%NaCl were separately injected into the KM mouse's two hemispheres, apparent angiogenesis atmosphere were observed in the AdhVEGF165 hemisphere.2. MVD in the AdhVEGF165 hemisphere increased 2 weeks after injecting; and 3 weeks later, it tended to be stable. At 3 weeks, the percentage of the renascent vessels in the AdhVEGF165 hemisphere (19.65±3.95%), was greater than that of CK (1.91±0.41)%(t=4.5, p<0.05).The MVD were measured after 3 weeks, compared with CK, the MVD in the AdhVEGF165 hemisphere was increased by 42%(t=5.3, p<0.05),with significant discrepancy. 3. rCBF and rCBV increased with MVD, reaching a peak at 3 or 4 weeks after injection, when the rCBF of the AdhVEGF165 hemisphere were 15.1% (t=8.7, p<0.05) greater than that of the opposite hemisphere; and the rCBV of the AdhVEGF165 hemisphere were 34.6%(t=11.5, p<0.05) greater than the opposite hemisphere.4. Micro-vessels were dyed with different dye-stuffs at different times after injection of reagent. Within 1 week, all kinds of the dyes could diffused into the brain tissue close to the pin hole; after 2 weeks, no dye were observed in the brain tissue, while dye-stuffs such as Lectin, Even's blue were differentially ingested by some astrocytes nearby the micro-vessels.5. Frozen sections were treated by immunohistochemical staining, and it was found that the proliferating ECs were surrounded by the endfeet of astrocytes. Astrocyte bridges were also observed.Conclusion1. Injecting AdhVEFG165 into mature KM mouse's brain can successfully inducing in vivo brain focal angiogenesis;the renascent vessels in this model have normal function of blood transportation with the proof of hemodynamic study.2. The permeability of BBB increased apparently within 1 week after injection of reagent, while it restored 2 weeks after injection.3. There are some certain substance channels between the astrocytes and vessel-contents in the process of angiogenesis, which made the substance exchanges between the astrocytes and vessel-contents have a prosperity of high selectivity, activity and strict timeliness.4. The astrocytes temporarily maintained the BBB's permeability by the endfeet, which may be a crucial role of astrocytes in the process of cerebral angiogenesis.5. In the process of cerebral angiogenesis, astrocytes may play role in the guiding of the directivity of the neovasculars, which enables the neovasculars to form a complex network.