Light damage induced changes in mouse retinal gene expression
Introduction
Light damage in mice has long been used as a model system to study retinal degeneration (Noell et al., 1966). In this model and many others, photoreceptor death occurs through apoptosis, as determined by TUNEL and agarose gel electrophoresis demonstrating apoptosis-specific DNA laddering (Portera-Cailliau et al., 1994). Consistent with these results, in our Balb/c mouse light damage model, many photoreceptor nuclei label with TUNEL after a 7 hr bright cool white fluorescent light exposure (Chen et al., 2003).
The action spectrum of retinal light damage is similar to the absorption spectrum of rhodopsin (∼500 nm maximum), suggesting that damage may be initiated by rhodopsin bleaching (Williams and Howell, 1983). Supporting this assertion, rodents deficient in dietary vitamin A or retinal retinoid cycle proteins (Saari et al., 2001, Sieving et al., 2001, Wenzel et al., 2001b) are less susceptible to light damage than controls. Bright light induces light damage in a transducin-independent manner, while light damage from exposure to less intense light is transducin dependent (Hao et al., 2002). The bright light induced apoptosis is dependent upon activation of the transcription factor c-fos, a component of AP-1. This c-fos upregulation plays a critical pro-apoptotic role, as the c-fos knockout mouse shows marked resistance to light damage (Wenzel et al., 2000). Further, activation of the glucocorticoid receptor, which inhibits AP-1, also protects against light damage (Wenzel et al., 2001a).
Photo-oxidative stress has been implicated in light damage pathogenesis. Immunohistochemistry has demonstrated labeling for markers of oxidative damage (Tanito et al., 2002). Several antioxidant genes are upregulated following photic injury, including heme oxygenase (Kutty et al., 1995), thioredoxin (Tanito et al., 2002), glutathione peroxidase (Ohira et al., 2003) and ceruloplasmin (Chen et al., 2003). Further, exogenous antioxidants protect the rodent retina from photic injury (Li et al., 1985, Noell et al., 1987).
To increase understanding of retinal responses to photo-oxidative stress, both protective and proapoptotic, we used DNA microarray analysis to examine changes in the expression of thousands of genes simultaneously (Schena et al., 1995, DeRisi et al., 1996, Farjo et al., 2002). A similar approach has recently been used to study gene expression relevant to glaucoma (Spector et al., 2002, Lo et al., 2003, Miyahara et al., 2003).
Section snippets
Materials and methods
Mice used in the experiments presented in this study were handled in adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and experiments were approved by the University of Pennsylvania IRB.
Light damage
In order to determine whether light exposure caused retinal damage, TUNEL labeling was performed on BLE and control groups in each of the three experiments. While none of the photoreceptors from the control groups were TUNEL positive (Fig. 1(A)), all retinas from the BLE groups had TUNEL positive photoreceptor nuclei (Fig. 1(B)). In sections cut in the vertical sagittal plane through the optic nerve head, TUNEL positive photoreceptors were present in highest numbers in the superior hemisphere.
Discussion
We used microarray analysis to compare retinal gene expression in light damaged retinas to normal retinas in three independent experiments. Seventy genes were upregulated at least two-fold immediately following light damage. Several findings support the validity of these results. First, when ten genes found to be upregulated in the microarray analysis were tested by quantitative PCR, all ten were upregulated at least two-fold in each of two independent light damage experiments. Second, two
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