FIGURE 2.

Microinjection of HIV-infected cells induces bystander apoptosis in mice cortex. Human U87-CD4-CCR5 cells infected with HIV_ADA (50 ng/ml) were microinjected into the cortices of Cx43^fl/fl as well as hGFAP-cre Cx43^fl/fl mice, and after 48 h, animals were sacrificed and brain sections were analyzed for apoptosis by TUNEL assay. (A) Representative images showing apoptosis (TUNEL, red) upon microinjection of HIV-infected astrocytes in Cx43^fl/fl as well as hGFAP-cre Cx43^fl/fl mice. HIV-p24 has been labeled with FITC (green), and cell nuclei with DAPI (blue). White lines in the merged images show the direction of microinjection. Scale bar denotes 75 μm. (B) Graphical representation of the extent of bystander apoptosis observed in a circular area of 200 μm around the site of microinjection in Cx43^fl/fl and hGFAP-cre Cx43^fl/fl mice. Microinjection of HIV-infected cells led to significant apoptosis in Cx43^fl/fl mice as GJs were formed between the microinjected human cells and murine host cells, which resulted in the diffusion of apoptotic factors generated in a few HIV-infected cells to host cells leading to their apoptosis. However, hGFAP-cre Cx43^fl/fl mice displayed minimal apoptosis even after microinjection of HIV-infected cells, possibly due to the absence of Cx43 GJs and HCs in these animals. (C) Represents the radius of bystander apoptosis which is the maximum distance of apoptotic cells from the site of microinjection of either HIV-infected or uninfected cells. The Cx43^fl/fl mice where HIV-infected cells were microinjected exhibit significantly larger radius of bystander apoptosis as compared to Cx43^fl/fl mice that received uninfected cells or hGFAP-cre Cx43^fl/fl mice that received either HIV-infected or uninfected cells. Each dot in the graphs (B,C) represents a microinjection. Each animal was microinjected at four sites; hence, in total, there were 16 microinjections for Cx43^fl/fl mice and 20 microinjections for hGFAP-cre Cx43^fl/fl mice. ^∗∗p < 0.005; n.s. – not significant.