We recently initiated several pilot studies to investigate whether Nrf2 signaling influences the phenotype of the fundus from chronic cigarette smoke exposure by evaluating mice deficient in Nrf2 signaling (Nrf2−/−). We exposed Nrf2−/− and Nrf2+/+ mice for 5 h/day, as described above, to either cigarette smoke or filtered air. We first evaluated the RPE/choroid for an Nrf2 signaling response by determining whether Nrf2 responsive anti-oxidant genes, NADPH quinine oxidoreductase (NQO-1) and Glutamate-cysteine ligase regulatory unit (GCLM), are induced by acute cigarette smoke exposure in wild type mice. shows that GCLM and NQO-1 both are induced by cigarette smoke exposure in the RPE/choroid of Nrf2+/+ mice after one day, and more strongly after 21 days. On the other hand, as expected Nrf2−/− mice do not show any induction of these enzymes.
RT-qPCR analysis showing acute induction of Nrf2 responsive anti-oxidant genes (NQO-1 and GCLM) by the RPE of Nrf2+/+ mice (n = 4 each group) after 1 and 21 day exposure to cigarette smoke. Expression was normalized to β-actin.
We next evaluated whether there is evidence of oxidative damage in the fundus with chronic cigarette smoke exposure in Nrf2−/− mice. Using the same protocol as described above, Nrf2−/− mice exposed for 6 months in air showed a slight increase (9.5%; n = 3) in immunolabeling of RPE nuclei for 8-OHdG compared to Nrf2+/+ mice in air (0%; p < 0.001; n = 3). When Nrf2−/− mice were exposed to cigarette smoke for 6 months, 85% of RPE cell nuclei were labeled with anti-8-OHdG (p < 0.0001; n = 3). Far more sections per Nrf2−/− mouse exposed to smoke were needed for evaluation since the number of RPE cell nuclei for counting was reduced, presumably from increased cell death.
We subsequently evaluated whether there was evidence of ultrastructural injury to the RPE, Bruch membrane and choriocapillaris in Nrf2+/+ and Nrf2−/− mice exposed to cigarette smoke or air for 6 months. At this age, the ultrastructure of the RPE, Bruch membrane, and choriocapillaris in Nrf2+/+ mice raised in air (n
= 3) are healthy (). Bruch membrane maintained a pentalaminar structure composed of the RPE basement membrane, inner collagenous layer, middle elastic layer, outer collagenous layer, and choriocapillaris basement membrane. The choriocapillaris endothelium appeared healthy with fenestrations. We chose to evaluate RPE basolateral infoldings and cytoplasmic vacuoles as indicators of RPE cell degeneration because loss of basal infoldings is a marker of epithelial injury (Drueke, Hennessen, et al., 1990
; Olsen, Burdick, et al., 1989
; Olsen, Wassef, et al., 1986
) and cytoplasmic vacuoles have been identified in RPE that overlie drusen deposits in AMD (Anderson et al., 2002
). Like previously, Nrf2+/+ mice raised in cigarette smoke (n
= 3) showed ultrastructural injury with loss of basolateral infoldings and cytoplasmic vacuole formation (). The RPE of 8 mo old Nrf2−/− mice raised in air (n
= 3) also appear relatively healthy, but displayed mild focal loss of normal basolateral infoldings (). In contrast, Nrf2−/− mice raised in cigarette smoke (n
= 3) exhibited profound ultrastructural injury to the RPE-Bruch membrane (). The RPE basolateral infoldings are dilated and fewer in number, and contain large cytoplasmic vacuoles. These changes appear more severe than Nrf2+/+ mice exposed to smoke. While preliminary, our assessment is that the RPE demonstrated more injury than Bruch membrane or the choriocapillaris.
Fig. 6 Transmission electron microscopy of the RPE-Bruch membrane-choriocapillaris of Nrf2+/+ (A and B) and Nrf2−/− mice (C and D) exposed to air or cigarette smoke. (A) 8 month old Nrf2+/+ mouse raised in air for 6 months has healthy RPE basolateral (more ...)
The main function of Nrf2 signaling is to induce an anti-oxidant response upon an oxidative stress, as the baseline expression of many anti-oxidant genes are not appreciably regulated by Nrf2 (Thimmulappa et al., 2002
). If the anti-oxidant response from reactive oxygen species is insufficient, and ROS depletes cellular glutathione, cells die from oxidatively mediated apoptosis (Rahman et al., 2005
; Walsh et al., 1995
; Will et al., 1999
). We therefore, evaluated whether RPE cell apoptosis is influenced by the Nrf2 signaling response after chronic cigarette smoke exposure. Nrf2−/− mice raised in air for 6 months (n
= 3) did not show any RPE cell apoptosis by TUNEL labeling. On the other hand, Nrf2−/− mice exposed to cigarette smoke for 6 months (n
= 3), had a 9% RPE cell apoptosis (p
< 0.001). Like with the 8-OHdG immunolabeling evaluation, many more sections of RPE/choroid from Nrf2−/− mice exposed to cigarette smoke were needed to identify RPE nuclei than mice raised in air. The need to evaluate more sections suggested to us that many dead cell remnants had already been removed at the time we tested for apoptosis, and hence, in the Nrf2−/− mice, this value is an under-representation of the true degree of apoptosis. These results suggest that there is minimal oxidative stress leading to oxidative damage when mice are raised in air, but that with cigarette smoke exposure, there is significant oxidative DNA damage and increased RPE cell apoptosis.