Morphologic evaluation of photoreceptor cell rescue was performed
by quantitative histology. One week of exposure to fluorescent light
(1200–1500 lux) reduced the thickness of the ONL of photoreceptor cell
nuclei from the normal 9 to 11 rows (35–40 μm;
Fig. 1A ) to 1 to 3 rows (5–10 μm) in the most degenerated region of the
uninjected eye
(Fig. 1B) or the PBS-injected sham control
(Fig. 1C) .
Light exposure almost completely eliminated the inner segments (ISs)
and outer segments (OSs) of most photoreceptors in this region.
However, in PEDF-injected eyes, there was significant rescue of
photoreceptors, with the ONL having six to seven rows (25–30 μm) of
nuclei, although many cells had swollen (ISs) and disorganized OS
profiles
(Fig. 1D) .
ERG was used to test the ability of PEDF to protect retinal function,
and representative data from these studies
(Fig. 2) demonstrated that constant light for 7 days completely eliminated the
ERG response, whereas PEDF, injected 2 days before constant light
exposure, provided significant functional rescue. Therefore, the
morphologic data
(Figs. 1A 1B 1C 1D) were indicative of the functional status
(Fig. 2) of the retinas. The ability of PEDF to promote the survival of
photoreceptor cells when injected at various times before, or after,
exposure to light was also investigated
(Fig. 3) . The control retinas of albino rats reared in cyclic light had a mean
ONL thickness of 38 μm, and injection of PBS or PEDF alone did not
change this. However, in the retinas of rats exposed to continuous
lighting for 7 days, the mean ONL thickness was reduced to 8 μm
(Fig. 3B) , and in PBS-injected control retinas, the mean ONL thickness was 13μ
m. Intravitreal injections of PEDF at 2 days or 1 day before
exposure to constant light were equally protective, with the ONL
thickness being 23 and 20 μm, respectively, whereas injection of PEDF
at 0, 1, or 2 days after constant light exposure did not provide any
significant protection above that seen in PBS-injected eyes. The
functional data
(Fig. 3A) were also consistent with these morphologic
findings.
To assess the damage to photoreceptor cells during exposure to constant
light for various lengths of time and to determine whether PEDF
pretreatment was equally effective in preventing the death of
photoreceptor cells during these periods, animals were exposed to
constant light for 3, 10, and 14 days, with or without PEDF injected 2
days before light exposure. The data from these experiments are
presented in
Figure 4 . After 3 days of constant light, the ONL thickness in the
posterior-to-equatorial region of the superior hemisphere was reduced
to 3 to 5 rows
(Fig. 4D) , compared with the normal 9 to 11 rows
(Fig. 1A) . PEDF preserved eight to nine rows of photoreceptor nuclei
(Fig. 4A) . Exposure to constant light for 10 days left only one to two rows
of nuclei
(Fig. 4E) , whereas pretreatment with PEDF resulted in
retention of four to five rows
(Fig. 4B) . The eyes exposed to constant
light for 14 days had the most severe degeneration with a greater loss
of photoreceptor nuclei throughout the retina, and only one row of
photoreceptor nuclei remaining in the posterior to equatorial region of
the superior hemisphere
(Fig. 4F) . PEDF pretreatment
(Fig. 4C) did not
notably protect photoreceptor cells in this group.
A summary of the ERG data from all the animals in each treatment group
can be presented as the ratio of the B-wave in the PEDF-injected eye
(right) to the B-wave in the control PBS-injected eye (left) in the
same animal
(Fig. 5) . The ERG B-wave ratio was more than three times higher in the 3-day
light-damage group, 2.5 times higher in the 7-day group, and
approximately 2 times higher in the 10-day group, and there was no
significant improvement in function in the 14-day-exposure group. It is
known that rod OS can, with time, regenerate after sublethal light
damage to photoreceptors.
24 25 To determine whether the
effects of PEDF are enhanced with time, rats were allowed to recover
for 14 days in cyclic light after 7 days of constant light. After 14
days of recovery, the ONL thickness in PEDF-treated eyes
(Fig. 6D) was similar to the ONL thickness in eyes without any recovery period
(Fig. 1D) . However, the PEDF-treated eyes showed better morphology of
the ISs and OSs after the 14 days of recovery than without any recovery
time
(Fig. 1D) .
Because bFGF has been shown to be extremely potent in protecting
photoreceptor cells from light-mediated degeneration, it was tested in
this experimental paradigm. Injection of bFGF alone
(Fig. 6E) provided
much better protection than PEDF alone
(Fig. 6D) , but when bFGF and
PEDF were applied together
(Fig. 6F) , there was a slightly better
preservation of rod OS morphology. As with morphology, functional
rescue (ERGs) improved over time with PEDF compared with animals
without a recovery period
(Fig. 2) . A graphic summary of the change in
ratios of the B-wave amplitudes of experimental and PBS-injected
control eyes is presented in
Figure 7A . These data also show some benefits of using both factors
simultaneously. Light micrographs of PBS-injected retinas at low
magnification clearly show a marked degree of photoreceptor rescue only
near the insertion sites
(Fig. 8A) , whereas the rescue in PEDF-, bFGF-, or PEDF/bFGF-injected retinas
(Figs. 8B 8C 8D) extends throughout much of the superior hemisphere in the
eyes.