Glial Reaction To Brain Stereotactic Injection Of AAV

Stereotactic injection of AAV virus vectors into the brain is a commonly used technical method in the field of neuroscience research.At present,most researches focus on the transgene/gene editing effects mediated by AAV virus vectors.However,the AAV virus has a certain degree of immunogenicity,which may cause damage to the host tissue,and the stereotactic injection operation itself may result in physical damage as well.So far,there is little systematic study on how reduce the injury impact on research and translation,meanwhile achieving the optiamal virus infection volume around the injection site.In this study,we employed the brain stereotactic injection method,using three different injection speeds of 100 n L/min,200 n L/min and 300 n L/min to inject AAV2/9-EGFP virus or PBS into the striatum of C57 mice,21 days after the injection,the whole brain of the mouse was clarified by CUBIC method,and then the virus infection volume was measured by the lightsheet microscope.At the same time,the immunofluorescence staining method was used to detect the glial cell stress response of the injected brains,finally the western blot method was used to confirm the glial cell stress response.The results showed that CUBIC treatment can lead to 40%expansion of the mouse brain,and the infection volume acquired by the 100 n L/min injection speed group was significantly larger than the other two speed groups.Injection of PBS can also cause glial cell stress response,but injection of AAV2/9-EGFP caused glial cell stress slightly higher than that of the PBS group.The stress response of glial cells in the 100 n L/min injection speed group was significantly weaker than that of the other two speed groups.The stress of glial cells is manifested by the proliferation of astrocytes but the morphology did not changed significantly,while the morphology of microglia changed significantly without proliferation.In conclusion,injection speed is an important factor that should be considered in brain stereotactic injection.Low-speed injection can achieve the largest AAV infection volume and cause the weakest glial cell stress response.Lowspeed injection should be taken if surgical situation allow.The AAV2/9 virus per se can cause a weak glial cell stress response,the improvement of the virus preparation process may reduce the glial cell stress response caused by AAV.