Abstract
Abstract: :
Purpose: To determine (1) the location of Fas, FasL, Bcl–2, caspase in the uninjected eye during HSV–1 retinitis and (2) if treatment with caspase inhibitor affects the expression of these factors in cells from the uninoculated eye of mice with HSV–1 retinitis. Methods:Adult female BALB/c mice were injected in the AC of one eye with 2 x 104 PFU of the KOS strain of HSV–1 or with an equivalent volume of tissue culture medium (mock). Expression of Fas, FasL, Bcl–2, and caspase–3 was determined on day 8 and 15 p.i. by double immunofluorescent staining of frozen sections of the uninoculated eye. Cells from the uninoculated eye were treated with Z–VAD, FMK, a caspase inhibitor, on day 9 and 12 p.i. and expression of FasL, Bcl–2, and caspase–3 was analyzed by Western blot. Results: In the uninoculated eye of mock–injected mice, Fas, FasL and caspase–3 were expressed constitutively in the RPE and photoreceptor layer, whereas Bcl–2 was expressed constitutively in the RPE, inner nuclear layer, ganglion cell layer and plexiform layer. At the onset of acute viral retinitis in the uninjected eye (day 8 p.i.), Fas, FasL and caspase–3 were also observed in the ganglion cell layer and Bcl–2 was also seen in the outer nuclear layer. By day 15 p.i., Fas, FasL, Bcl–2 and caspase–3 positive cells were observed throughout the posterior segment, although their original location within the retina could not be determined since the architecture of the retina was destroyed by this time. Z–VAD.FMK treatment of retinal cells from the uninjected eye inhibited caspase–3 activity and processing, and the level of Bcl–2 was slightly increased in treated cells. Conclusions: Constitutive expression of Fas, FasL, Bcl–2 and caspase–3 in the RPE and in several layers of the retina of mock–inoculated mice together with the altered expression pattern of these markers during virus infection suggest that apoptosis observed in the retina of HSV–1–infected mice is a caspase–dependent process that is influenced by the timing and expression of several markers including Fas, FasL and Bcl–2. These results also suggest that destruction of the retina of the uninoculated eye requires not only virus infection of retinal cells, but also caspase–3 and expression of several apoptosis–related factors in concert during the course of virus infection. Grant EY006012
Keywords: apoptosis/cell death • immunohistochemistry • herpes simplex virus