Introduction: Direct cellular reprogramming has emerged as an innovative alternative to generating neurons in the brain after injury. We were among the first to recently demonstrate in vivo reprogramming of non-neuronal cells into neurons in the brain of rodents following stroke. Human clinical trails will require an AAV with a greater safety profile.
Methods: Canine reactive astrocyte cultures were developed using a rodent astrocyte culture protocol modified from Heinrich et. al. Briefly, stroke was induced in adult Beagles using a surgical approach adopted from Yanaka et. al. Through a pterional craniotomy the circle of willis was identified. The azygous ACA was clip occluded. The MCA distal to lenticulostriates was coagulated and cut. Collateral flow to the distal MCA was identified with Indocyanine green and coagulated. One hour later the clip was removed. In the peri-infarct region 1cm3 blocks of tissue were harvested, then dissociated and cultured using the protocol modified from Heinrich et. al.
Transduction experiments were performed on confluent cultures after a minimum of one passage. Specifically, AAV serotypes 8, 9, and 10 expressing enhanced GFP under the control of the CAG promoter were compared. Cultures were exposed to AAV for 24 hours. Cells were fixed 4 days later and immunocytochemistry studies performed.
Results: Cultures were characterized with immunocytochemistry with nearly 100% of DAPI positive cells expressing the astrocytic marker GFAP. No Tuj1 labeling was seen suggesting no neurons in our culture. All AAV serotypes successfully transduced astrocytes. Ultimately we found AAV8 followed by 9 and 10 to have the highest transduction efficiency.
Conclusions: We have demonstrated for the first time the capacity to generate reactive astrocyte cultures in vitro from adult canine neocortex after stroke. Using this culture model we have found that AAV8 has the highest transduction efficiency.
Patient Care: By activating neurogenesis at the stroke area we are hoping that patients will be able to regain at least some of the functions they have lost after their cerebrovascular accident.
Learning Objectives: We developed an in vitro culture model of adult astrocytes reactivated after stroke to then test transduction potential of various adeno-associated virus serotypes.
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