Expression of Pannexin 2 protein in healthy and ischemized brain of adult rats.

The expression pattern of the pannexin2 protein in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat pan2 was generated in chicken and purified for affinity. This antibody was used to study by Western blot, Enzyme-Linked Immunosorbent Assay, and immunohistochemistry, the expression pattern of pan2 in healthy brain of adult rats and in the hippocampus of rats submitted to bilateral clamping of carotid arteries for 20 min, followed by different times of reperfusion (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Pan2 in the healthy brain. All Pan2 positive cells were neurons which also showed no puncta on their cellular membranes. Both pyramidal cells and interneurons, the majority of which were positive to parvalbumin, were stained in healthy hippocampus. The number of Pan2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R. Interneurons which were positive for both Pan2 and parvalbumin, represented about the 85% of all parvalbumin cells stained in the hippocampus. This percentage rested grossly unmodified at different time intervals after I R in spite of the progressive neuronal depletion. Concomitantly, an intense astrogliosis occurred in the hippocampus. Most of the astroglial cells expressed de novo and for a transient time (from 24 h to 14 days . Primary co-cultures of hippocampal neurons and astrocytes were submitted to transient ischemia-like injury. This set of experiments further confirmed the in vivo results by showing that Pan2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Pan2 in the astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. In conclusion, our results showed the transient expression of Pan2 in astrocytes of reactive gliosis occurring in the hippocampus following IR injury. We hypothesize that Pan2 expression in astrocytes following an ischemic insult is principally involved in the formation of hemichannels for the release of signaling molecules devoted to influence the cellular metabolism and the redox status of the surrounding environment.