Also, RPE phagocytosis of thetips o

Also, RPE phagocytosis of the
tips of shed ROS, and the expression of retinal c-fos and c-jun, are light entrained (LaVail,
1976b; Goldman et al., 1980; Yoshida et al., 1993; Imaki et al., 1995). In the ONL, c-fos is
normally elevated at night, while its expression in the inner retina, particularly in ganglion
cells, is transiently enhanced at light onset (Yoshida et al., 1993). In rats, RPE-65 mRNA levels
are also highest at the beginning of the day, but RPE-65 protein levels do not correlate with
circadian dependent light damage susceptibility (Beatrice et al., 2003). Other evidence
indicates that melanopsin, a protein located within a subset of ganglion cells, serves as a retinal
circadian irradiance detector (Provencio et al., 2000; Hattar et al., 2002; Berson et al., 2002).
Potential circadian phase resetting cryptochromes are also found in the inner retinal layers
(Yagita et al., 2001). Although melanopsin and cryptochromes are both photoreceptive
proteins, and even though both may trigger light-induced signals in the retina, the inner nuclear
layer (INL) is not usually damaged by visible light. This might be related to inefficient light
absorption by these photoreceptive proteins, or to high GSH levels in the INL which serves to
block oxidative damage (Winkler 2008).
Despite the relative resistance of the inner retina to light damage, rod photoreceptors are
vulnerable to light damage at night while being well protected during the day. In an attempt to
understand how changes in retinal gene expression and circadian light damage susceptibility
might be related, we performed gene array analysis and confirmed some results by real time
PCR. Figure 6 contains rt-PCR mRNA expression profiles for several retinal transcription
factors and protein markers of oxidative stress. Rat retinas were collected at 9 am and 5 pm
and compared with those obtained at 1 am, the time point of maximum light damage
susceptibility (Organisciak et al., 2000). In totally dark adapted rats, the expression of retinal
c-fos, c-jun, fra-1, GFAP, HO-1 and macrophage chemoattractant protein-1 (MCP-1) increased
1–4 fold between 1 am and 9 am (Figure 6A). Other rats were treated with intense light for 8
hours, starting at 1 am, to determine the time course of light-induced changes in transcription
factors (Figure 6B). Light exposure led to increases in mRNA expression for several
transcription factors, and most exceeded the increases found in retinas from dark adapted rats.
When rats were given the antioxidant DMTU before light treatment, c-fos and fra-1 mRNA
levels decreased 7–10 fold, suggesting that their expression was originally triggered by lightinduced
oxidative stress. At the same time, the expression of c-jun, NFkB and Atf3, an
endoplasmic reticulum factor, were largely unaffected by light.
As shown by the mRNA expression patterns for protein markers of oxidative stress, the timing
of light treatment also affects their synthesis (Figure 6C). For rats treated with 8 hours of light
starting at 1 am, the increases in retinal GFAP and MCP-1 expression were significantly greater
than for other rats treated with light starting at 9 am or 5 pm. The large increase in expression
for MCP-1 between 1 am and 9 am, suggests that a strong signal for macrophage invasion was
generated by retinal damage. Antioxidant pretreatment resulted in a dramatic reduction in
mRNA levels for MCP-1 and for the Muller cell stress protein GFAP, indicating that their expression was driven by oxidative stress. For HO-1, the light-induced increase in expression
was about 10 fold for exposures starting at 1 am and 9 am. These increases were greater than
for rats treated with light at 5 pm, the time point when light damage is minimal (Organisciak
et al., 2000). This indicates that the effect of oxidative stress in the retina is far greater for light
exposures beginning at 1 am than for light treatments at other times during the day. Our findings
also confirm some of the changes found in retinal gene array studies using bright light treated
albino mice (Chen et al., 2004; Huang et al., 2005) and further suggest that time of day can
affect the outcomes of both gene array and PCR measurements.
The synthesis of corticosterone, the rodent equivalent of human cortisol, also occurs on a 24
hour circadian cycle, and an increase in its synthesis is part of the normal stress response. In
rats, circulating corticosterone levels were found to vary with time of day, but they did not
correlate with the circadian profile of retinal light damage (Vaughan et al., 2002). In mice,
Wenzel et al. (2001) found that the stress of overnight fasting induced corticosterone synthesis
and glucocorticoid receptor translocation. Both stress induced corticosterone and
dexamethasone, a synthetic glucocorticoid, protected against light damage and both inhibited
the expression of activator protein-1 (AP-1) (Wenzel et al., 2001b). However, in the T4R
rhodopsin mutant dog, brief light exposure resulted in extensive photoreceptor cell damage
that was not inhibited by corticoids, including dexamethasone, even though AP-1 and c-fos
activation were inhibited (Gu et al., 2009). Heterodimeric complexes of c- jun and c-fos, or
fra-1 (Wenzel et al., 2002), form the transcription factor AP-1 and may promote cell death in
light exposed mice (Hafazi et al., 1997b). In the dog model this did not happen, providing yet
another example of species differences that can involve divergent pathways of light-induced
retinal cell death.
6. The Emerging Picture and Future Directions
The effects of intense or prolonged light exposure on the retina vary by species, region of the
retina, time of day, diet, prior light rearing history and genetic background. From the
perspective of sorting out mechanisms, each variable has proven useful. While the use of
different animal models has provided insights into understanding light damage, the data also
indicate that damage or cell death mechanisms are not easily extrapolated between species.
The notion that “the mouse is not a small rat” was recognized by LaVail in one of the first
comprehensive studies relating strain and species differences with the extent of retinal light
damage (LaVail et al., 1987). We now know that the L450M amino acid variation in RPE-65
correlates with resistance to light damage in many strains of mice (Danciger et al., 2000). The
oxidation of methionine 450 in RPE-65 to methionine sulfoxide is probably beneficial, because
it leads to reduced enzyme levels, slower rhodopsin regeneration, and light damage protection
(Danciger et al., 2000; Wenzel et al., 2001a). Although the regeneration of rhodopsin correlates
with light damage susceptibility in some mice, RPE-65 activity is not the rate limiting step in
regenerating rhodopsin in the rat (Iseli et al., 2002). Photobleaching of rhodopsin - and quite
possibly the initial rate of bleaching- is a key factor for light damage in rats. The same may
hold for exceedingly light sensitive dogs and mice with glycosylation defects in the amino
terminal region of rhodopsin (Gu et al., 2009; White et al., 2007). Whatever rhodopsin’s exact
role is in light damage, photoreceptors become hyperpolarized by light and remain that way
for the duration of exposure. As pointed out by Sieving et al. (2001), prolonged
hyperpolarization of rod photoreceptors is insufficient to cause retinal light damage, but lightinduced
changes in intracellular ions is still an area amenable to study in animal models.
There is also a need for additional comparative light damage studies in a variety of animal
models. For example, a comparative proteomics analysis subsequent to a standard light insult
of macaque and owl monkey retina might be very informative. The macaque shares its
diurnality with the human, and the owl monkey is only recently evolved from a diurnal ancestor (Dyer et al., 2009), so we might thereby gain insights about possible damage, repair, or cell
death mechanisms shared (or not) by traditional rodent models and humans. Diurnal rodent
studies are more feasible than primate studies and offer a complementary approach to the
existing literature, which has focused on discerning damage pathways in vulnerable nocturnal
rodents so that protective therapies can be designed. Studies of damage-resistant rodents would
provide a useful counterpoint for identifying naturally evolved protective or repair mechanisms
that appear to confer even greater bright light tolerance than is already intrinsic to the foveate
primate. The diurnal ground squirrel model has an additional feature that must be borne in
mind, which is its hibernation physiology. The emerging consensus is that animals that
hibernate exhibit neuroprotection from a variety of insults, but our understanding of
hibernation’s protective mechanisms is too incomplete to say whether they do, or do not,
contribute to light damage resistance. Hibernators spend months in continuous darkness, during
which time the metabolic activity of the predominant cones is profoundly suppressed, whereas
rods seem to retain mitochondrial activity (Figure 7). Dark-rearing potentiates light damage in
many nocturnal rodents, but it is unknown whether hibernation does so in the ground squirrel.
Finally, diurnal rodent retinas provide a means for exploring various environmental and
hereditary sensitizers to light damage, particularly those that may endanger cones. Only
cautious comparisons between species will reduce confusion and advance our understanding
of damage mechanisms.