Introduction: Recent studies have shown that astrocytes can be directly converted into neurons by transcription factors such as Ascl1 and Neurog2 for the treatment of stroke. However, most effective transcription factors have not been clearly defined. In addition, while various viral vectors have been used translation to human clinical trials will necessitate safer vectors such as adeno-associated virus. In our study we compare transduction efficiency of various AAV serotypes in vivo in a mouse model of stroke. Using the most efficient transcription factor in future studies we will compare reprogramming efficacy of Ngn2, Ascl1, Sox2 and NeuroD1 in vivo in our mouse stroke model.
Methods: Permanent distal MCA occlusion was generated in C57/B6 wild type mice through a small craniectomy and coagulation of a distal MCA branch over the frontal lobe. Transduction efficiency was then compared 5 days later between AAV serotypes 8, 9, and rh10. Specifically, 1ul of virus was injected each into two sites around the stroke. One week later brains were compared using immunohistochemistry for astrocytes (GFAP+) and transduced cells (GFP+).
Results: All mice had a clinically silent stroke. After surgery, they were able to resume normal activities. Infection rates of AAV 8, 9, and 10 for astrocytes were 0.5%, 6.9% and 9.1%, respectively and for neurons were 5.0%, 11.6%, and 7.6%, respectively.
Conclusions: We were able to create a stroke model in wild type mice that gave clinically silent strokes. AAV 10 had the highest infection rate for astrocytes and a relatively lower infection rate for neurons, when compared to AAVs 8 and 9. Therefore, we conclude that AAV 10 is the best vehicle for delivering transcription factors to glial cells in stroke mice. Future studies using AAV rh10 will be used for comparing reprogramming efficacy with the neurogenic transcription factors Ngn2, Ascl1, Sox2, and NeuroD1.
Patient Care: This will lead to better understanding of reactive glial cells after stroke and possibility of converting reactive glial cells into neurons. Eventually better patient care.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) understand that AAV 10 has highest infection rate for reactive glial cells when compared to AAV 8 and 9. 2) understand that transcription factors can successfully convert glial cells into neuron in cortical stroke area in brain
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