• Mohammed A Mansi, Holley Hewitt, Sarah Scharber, Molly Greenshields, Sean Moen, Ciro Vasquez, Bharathi Jagadeesan, and Andrew W Grande.
• Neurosurgery. 2015 Aug 1;62 Suppl 1:196.

IntroductionDirect 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 nonneuronal cells into neurons in the brain of rodents following stroke. Human clinical trails will require an adeno-associated virus (AAV) with a greater safety profile.MethodsCanine reactive astrocyte cultures were developed by using a rodent astrocyte culture protocol modified from Heinrich et al. In brief, stroke was induced in adult Beagles by using a surgical approach adopted from Yanaka et al. Through a pterional craniotomy the circle of Willis was identified. The azygous anterior cerebral artery (ACA) was clip occluded. The middle cerebral artery (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 1-cm 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 1 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 were performed.ResultsCultures 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.ConclusionWe 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.

11 keywords...

hide…