from the loss to the IR injury. We observed no statistically significant difference in RGC survival in CKO and global KO in IR-challenged retinas. There was even a small trend toward an increased survival in CKO vs. KO retinas. These findings suggested that Panx1 opening represents an intrinsic mechanism of neuronal death, and did not involve other cell types. To test this hypothesis, we studied survival of pan-purified primary RGCs. RGCs were purified from P7 neonatal mice using the two-step immunopanning technique, as described previously and challenged in vitro with transient 4 hour-long oxygen and glucose deprivation. This model allows for the study of neurons under well controlled conditions that mimic retinal IR injury, including intracellular glucose and ATP depletion, an increase in intercellular Ca2+, ER and oxidative stress, followed by cell death by necrosis and apoptosis. In line with our prediction, our data showed 3 Pannexin1 in Retinal Ischemia significantly higher survival rate in the KO and CKO vs. WT cells. The average RGC survival rate was 4662% in WT cultures. Survival improved by the average of 
22% and 29% in the CKO and KO cells, respectively. It is worth noting that the difference between the two genotypes was not statistically significant, indicating that the deficiency of Panx1 in RGCs is, indeed, responsible for their increased survival. The analysis of dying cells using AnnexinV and propidium iodide staining revealed that necrotic cells averaged 1660.3% in WT RGCs cultures; whereas in the CKO and KO culture, this value has dropped to 4.665% and 0.561%, respectively. Apoptotic cells averaged 4062% in WT vs. 27.463.7% in Panx1 CKO and 24.462.3% in Panx1 KO cultures challenged by OGD. Panx1 channel permeates RGCs challenged by OGD Panx1 forms a large non-specific pore, which provides the conduit to ions, dyes and small molecule metabolites with molecular weight up to 1 kDa. In isolated brain neurons, the opening of the Panx1 channel was shown to permeate the plasma membrane in response to 15 minutes of ischemia, thus allowing molecules to cross the plasma membrane. Here, we tested whether a membrane-impermeable dye calcein-488AM will leak from RGCs challenged by OGD and if the leakage can be blocked by Panx1 ablation. In the cytoplasm, this dye is de-esterified, becomes hydrophilic and stays PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189475 trapped inside the cell. Opening of a large pore or channel induces time-dependent exit of the dye from cells, which can be detected by fluorescence microscopy. We loaded whole retina explants with calcein-488 and measured the leakage after 30 minutes of OGD in an ischemic chamber. Each pair of WT and Panx1 KO retinas was prepared and challenged by OGD in parallel to avoid Cy5 NHS Ester chemical information technical deviations. After the exposure to OGD, retinal explants were immediately imaged by confocal microscopy. The total fluorescence in the 30 mm-thick optical slice of the inner nuclear layer was recorded, as detailed in Materials and Methods. We found that OGD caused an average of 33% reduction in total fluorescence in the ganglion cell layer that resulted from dye leakage in WT retinal explants. In contrast, retinas obtained from the Panx1 KO mice showed a near-complete blockade of the leakage. The ganglion cell layer contains a mixed population of RGC, amacrine and glial cells, all of which express different level of pannexins. Therefore, quantitative analysis of the dye leakage from an individual cell type is challenging when performed in retinal e
