Expression of pannexin2 protein in healthy and ischemized brain of adult rats

The expression pattern of the pannexin2 protein (Px2) in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat Px2 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 Px2 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 (I/R) (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Px2 in the healthy brain. All Px2(+) 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 Px2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R, with a negative peak of about -40% after 72 h from I/R. Interneurons which were positive for both Px2 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 from I/R), Px2. 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 Px2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Px2 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 Px2 in astrocytes of reactive gliosis occurring in the hippocampus following I/R injury. We hypothesize that Px2 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.