Preconditioning of neurons to survive damaging insults by
delivering a sublethal insult of the same type was demonstrated in
various studies. It was suggested that the limited insult is sufficient
to elicit an endogenous protective response that enables the cells to
sustain a damaging insult of a higher magnitude.
11 In
light-damage experiments, rats exposed to bright light for a limited
time were less sensitive to subsequent damage by prolonged light
exposure.
7
Localized protective response in the RCS rat retina was previously
demonstrated with mechanical damage
2 4 and laser
burns,
12 13 both of which elicit limited enhancement of
photoreceptor survival at the site of injury. In the present study we
used light stress to induce a protective response in the RCS rat
retina. This approach resulted in a global effect that encompassed
large parts of the retina. A single dose of 10 to 12 hours of bright
light at P23 was sufficient to extend photoreceptor survival beyond
P42. A large number of viable photoreceptor nuclei were present in
light-treated retinas, whereas mostly pyknotic nuclei remained in
untreated RCS rat retinas of the same age.
The mechanism by which bright light enhances photoreceptor survival may
be associated with increased availability of growth factors.
Upregulation of bFGF by light was previously shown in normal rats that
were maintained for 3 weeks under a diurnal cycle with light levels of
55 to 70 fc. In situ hybridization of bFGF mRNA localized an intense
signal over the inner segments.
6 It was suggested that
light stress increases synthesis of bFGF and that this is one of the
endogenous rescue molecules that promote photoreceptor survival when
challenged with constant light.
6 Treatment of normal rats
with higher light intensities for shorter times, which protects against
subsequent constant light damage also results in elevation of bFGF gene
expression.
7 In situ hybridization experiments revealed
the presence of bFGF mRNA in the PE cell layer, the inner nuclear
layer, and the photoreceptor inner segments.
14 Müller cells might be an additional source of bFGF in
light-stressed retinas. Studies of cultured rat Müller cells
showed that the cells respond to bFGF by elevation of bFGF gene
expression.
15 Thus, release of bFGF from an endogenous
reservoir in case of injury could lead to the production of bFGF by
Müller cells, which in turn may enhance photoreceptor
survival.
15 A direct role for bFGF in promoting
photoreceptor viability was suggested by various studies. Transgenic
mice carrying mutant bFGF receptors undergo progressive retinal
degeneration.
16 Recent studies have shown that bFGF
directly stimulates the survival of mature mammalian photoreceptors in
culture.
17 In light-damage experiments, a role for bFGF in
preventing nitric oxide toxicity has been suggested.
18
In view of the significant rescue of photoreceptors in RCS rats that
were treated at P23, bFGF levels were measured after light treatment at
that age. Analysis of bFGF expression revealed a major elevation of
both mRNA and protein levels at 2.5 days after treatment with a single
dose of bright light. This observation is in agreement with the
kinetics of bFGF upregulation that was measured in light-treated normal
rat retinas,
7 although similar upregulation of CNTF was
not seen in the light-treated RCS rat retinas. It is possible that the
absence of CNTF upregulation was caused by species differences. The
effect of a single light treatment was long lasting, because a major
increase in bFGF protein level was measured 19 days after treatment.
Furthermore, the upregulation of bFGF in light-treated retinas may
extend for a longer period, because preliminary experiments revealed
considerable photoreceptor survival at P60 in treated RCS rats. The
correlation between upregulation of bFGF and photoreceptor survival in
light-treated rats was further enhanced by the observation that
illumination at P18, which did not increase photoreceptor survival,
also did not increase bFGF expression. It is possible that at P18 the
machinery responsible for upregulation of bFGF in response to light
stress is not yet in place.
Analysis of endogenous bFGF expression in RCS rat retinas revealed
lower levels of bFGF at P21 compared with normal retinas of the same
age,
19 although similar reduction in bFGF have not been
found in dystrophic mice retinas.
6 Other studies have
demonstrated that a single injection of bFGF is sufficient to promote
photoreceptor survival in RCS rats for at least 2 months.
2 Therefore, increased levels of bFGF in the RCS rat retina either by
exogenous application or by upregulation of endogenous levels by light
treatment, as shown in the present study, may provide needed
neurotrophic support to photoreceptors in the bFGF-deficient retina.
Additional alterations in the retina that could be considered as
factors in enhancing photoreceptor viability are changes in the ROS
debris layer in light-treated RCS rat retinas. The accumulated ROS
membranous debris in the subretinal space may hinder the diffusion of
oxygen and nutrients from the choroid, which could affect photoreceptor
viability. Thus, the thinning of the ROS debris layer in light-treated
rats may enhance viability by reducing a diffusion barrier. However, as
shown in
Figure 7 , there was no inverse correlation between the width
of the ROS debris layer and ONL layers. Furthermore, dual light
treatments at P18 and P23 or at P23 and P30, which further reduced the
ROS debris layer, did not increase the survival of photoreceptors. In
other studies with RCS rats, reduction in the debris zone by macrophage
transplants had little effect on photoreceptor cell
survival.
20 Therefore, thinning of the ROS debris layer
induced by bright light was probably not a significant factor in
enhancing photoreceptor survival in treated RCS rat retinas.
Damage to PE cells was observed in the posterior retina of
light-treated RCS rats. At some points in the posterior retina with
damaged PE cells, photoreceptor nuclei were found next to Bruch’s
membrane. Similar relocation of photoreceptor nuclei has been described
in type I light damage, which involves damage to PE
cells.
21 22 The damage to PE cells seen in light-stressed
RCS rat retinas was not observed in normal Sprague–Dawley rat retinas
that were exposed to an identical treatment of bright light. Although
the mechanism of retinal light damage is not yet understood, recent
studies confirm the oxidative nature of the process.
23 It
is possible that the increased susceptibility of PE cells in the RCS
rat is caused by exposure to oxidative mediators released from altered
ROS debris layer in light-treated retinas.
Differences in the level of photoreceptor rescue in the superior and
inferior retinas were observed in the treated RCS rats, with better
survival generally measured in the inferior retina. Because the rate of
photoreceptor degeneration in pink-eyed RCS rats is the same in the
superior and inferior hemispheres,
8 it can be assumed that
differences in rates of photoreceptor survival are caused by different
levels of exposure to the bright light. Analysis of normal
Sprague–Dawley rats exposed to the same level and duration of bright
light revealed localized damage in the posterior region of the superior
hemisphere. This observation is in accordance with the known
susceptibility of the posterior region in the superior retina to light
damage.
24 Thus, the free-roaming RCS rats that were
exposed to more than optimal levels of light during the 10- to 12-hour
illumination period may have had some cell loss due to light damage in
the superior hemisphere, in addition to enhancement of cell survival.
Treatments of RCS rats with bright green light produced the same
qualitative results as seen with the white light, including type I
light damage to PE cells. Therefore, the involvement of rhodopsin in
mediating the observed changes in photoreceptors and PE cells can be
assumed. The observed type I light damage to PE with green and white
light is in agreement with data that show similar damage to
photoreceptor and PE by green and short-wavelength
light.
25 In view of the damage caused by light to the PE
cells, adverse effects of light treatment should be considered. In
further studies, fine calibration of light brightness and duration of
exposure will be required to obtain optimal conditions that will be
adequate to elicit a response to light stress without causing PE cell
damage. The possibility of prolonging photoreceptor survival by
noninvasive treatment such as sublethal levels of light is an
interesting mode of therapy. Complications of exogenous applications of
bFGF such as development of cataracts in bFGF therapy
26 and other potentially harmful side effects may be avoided. The
potential of light treatment as a therapeutic mode will be explored in
other models of retinal degeneration.
The authors thank Annemarie Brown for excellent technical support.