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1 From the Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong; and 2 the Department of Ophthalmology, the Wilmer Institute, Johns Hopkins University, Baltimore, Maryland.
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Abstract |
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METHODS. Thirty-two Lewis albino rats were exposed to green fluorescent light (480–520 nm) of 300 to 320 foot-candles (3228–3443.2 lux) for 3 hours, allowed to recover in the dark, and euthanatized at 0, 1, 3, 6, 12, 24, or 96 hours after light exposure. c-Fos was detected immunohistochemically and nicked DNA by in situ TdT-dUTP terminal nick-end labeling (TUNEL). Double labeling of c-Fos and DNA nicks was also performed.
RESULTS. There was a time-dependent change in the number of c-Fos–positive photoreceptor nuclei after light injury, which paralleled the change in the number of TUNEL-positive nuclei. The temporal and spatial appearance of these nuclei also matched the appearance of pyknotic nuclei of the outer nuclear layer. Double-labeling study revealed that some c-Fos–positive nuclei were also TUNEL-positive nuclei.
CONCLUSIONS. There was an acute accumulation of c-Fos protein in photoreceptors associated with cell death. This study further supports other studies showing that c-Fos is linked to apoptotic photoreceptor cell death.
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Introduction |
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c-Fos, an immediate early gene,2 is one of the genes activated by external stimuli such as growth or differentiation factors and physical stresses like injury and heat and electric shocks. As a transcription factor alone or interacting with other transcription factors, it regulates the expression of many genes such as the c-jun family members that are associated with c-Fos within the AP-1 transcription complex.3
Accumulation of c-Fos was observed in the cytoplasm of fibroblasts undergoing apoptosis.4 Hafezi and his colleagues5 demonstrated that c-fos mRNA is necessary for light-induced apoptotic cell death of photoreceptors in mice, whereas photoreceptor degeneration in rd mice, which is not light-injury related, is c-Fos–independent.6 Hence, it is possible that mouse photoreceptors undergo c-Fos–dependent or –independent apoptotic pathways depending on the stimuli. It is not clear whether c-Fos is involved in apoptosis of photoreceptors in photic injury of rat retinas. In the present study, c-Fos after light damage was detected immunohistochemically, whereas cell death was indicated by TdT-dUTP terminal nick-end labeling (TUNEL). Double labeling of c-Fos by immunohistochemical method and DNA nicks by in situ TUNEL were also performed.
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Methods |
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Light Exposure
Seven groups of rats (four in each group) were dark-adapted for 24
hours before exposure to green light (480–520 nm) at 300 to 320
foot-candles (ft-cd) for 3 hours. After that, they were allowed to
recover in the dark and euthanatized (in groups of four animals) after
0, 1, 3, 6, 12, 24, or 96 hours. One group of rats was kept in cyclic
light and darkness for 14 days and then dark-adapted for 24 hours
before euthanasia as control. All exposed rats were light-exposed at
the same time in the morning and euthanatized in a darkroom equipped
with a red light.
Tissue Preparation
All enucleated left eyes were fixed in Davidson’s solution and
paraffin-embedded, whereas the right eyes were sampled into four strips
of the retina from superior, inferior, nasal, and temporal quadrants.
The strips were glutaraldehyde-fixed and epoxy-embedded.
Histopathology
One-micron-thick epoxy sections of the strips from the superior
retinas were cut, stained in 1% toluidine blue, and examined under
light microscopy.
In Situ TUNEL
Apop-Taq in situ Apoptosis Detection Kits from Oncor
(Gaithersburg, MD) were applied to detect nicked DNA on 4-µm-thick
paraffin sections containing the whole retina including the optic nerve
head. Diaminobenzidene (DAB; Sigma Chemical, St. Louis, MO) was used as
chromogen.
Immunohistochemistry of c-Fos
Four-micron-thick paraffin sections similar to those used for
TUNEL were deparaffinized and incubated overnight at 4°C in rabbit
antiserum to c-Fos [c-Fos(Ab-2); Calbiochem, La Jolla, CA].
Antibody binding was localized by the avidin-biotin-peroxidase method
(Vector, Burlingame, VA) using DAB as chromogen.
Colocalization of c-Fos and DNA Nicks
Retinas at twenty-four hours after photic injury were selected for
double labeling because it was the peak incidence of in situ TUNEL in
this study. After in situ TUNEL using DAB as chromogen,
immunohistochemisty for c-Fos was performed on the same section using
fluorescein (Trevigen, Gaithersburg, MD) as chromogen. The section was
excited at 495 nm, then visualized and photographed with a Leica
microscope (Leica, Germany).
Morphometry of c-Fos and TUNEL Positive Nuclei
The total number of positive nuclei per retinal section was
counted manually under a Diastar Leica microscope (Leica, Wetzler,
Germany). The length of the outer nuclear layer (ONL) was
recorded by an image analyzing system (Leica model Q500MC; Cambridge,
UK). The number of positive cells per unit length of the retina was
obtained. A pairwise multiple comparison test was applied to detect
significant differences among different groups. P <
0.05 was considered statistically significant.
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Results |
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Histopathology
Immediately after photic injury (Fig. 1)
, mild edema and isolated pyknotic nuclei were noted in the inner part
of the ONL. At 12 hours (Fig. 1C)
, more pyknotic nuclei were noted and
most of them were in the inner part of the ONL. At 24 hours (Fig. 1D) ,
the ONL was focally thinned with 8 to 10 nuclei per column compared
with 10 to 12 of the normal and showed more pyknotic nuclei, which were
scattered in the ONL. After 96 hours (Fig. 1E) , the ONL showed marked
loss of photoreceptor nuclei and few pyknotic nuclei.
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Immunohistochemistry of c-Fos
After light exposure, there was also a gradual increase in the
number of c-Fos–positive nuclei in the ONL from 0 to 24 hours, first
noted in the inner part of the ONL (Figs. 1M
and 1N)
and spread to the
whole thickness of the ONL in 24 hours (Fig. 1O) . Changes of overall
c-Fos immunoreactivity (IM) in other retinal layers were unremarkable
(data not shown).
Double-Labeling of c-Fos and DNA Nicks
Double-labeling of c-Fos and DNA nicks at 24 hours after injury
revealed that relatively strongly c-Fos–positive cells were
TUNEL-negative, that weak c-Fos IM colocalized with positive TUNEL, and
that some c-Fos–negative cells were also TUNEL-positive (Fig. 2) .
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Discussion |
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The three categories of double-labeling observed can be explained by an early appearance of c-Fos in the nuclei preceding the action of endonuclease in generating DNA nicks. As the chromosomal DNA continues to be cleaved in apoptosis, synthesis of c-Fos decreases or degradation of c-Fos increases until the cell ceases to have c-Fos but nicked DNA.
There are other possible explanations to our observation. Photoreceptors may die through both c-Fos–dependent or –independent pathways after light injury. However, the number of TUNEL-positive nuclei would have been higher than that of c-Fos–positive ones if the c-Fos–independent pathway played a significant role. Our morphometry study does not support this possibility. It is also possible that there is no causal relationship of c-Fos and photoreceptor cell death but rather a coincident induction of c-fos.10 For example, Hafezi et al.6 using c-fos-/- rd mice showed that there was extensive photoreceptor cell death without c-fos expression even though Rich and her colleagues11 observed aberrant expression of c-fos accompanying photoreceptor cell death in the rd mouse. Although this is possible, there is extensive documentation of a link between the expression of c-fos and impending cell death in a variety of neural and nonneural tissues during development or under pathologic conditions. For example, Estus and his colleagues12 revealed that c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis, leading to the hypothesis that c-fos is indeed involved in the early changes of gene expression in the apoptotic pathway. A recent study by Hafezi and his colleagues,5 in which c-fos-/- transgenic mice were used, also demonstrated the requirement of c-fos in photoreceptor cell death after photic injury. Our observation supports a causal role for c-fos in light-induced photoreceptor cell death as suggested by Hafezi et al.5 It is also possible that c-fos expression may be synchronized, whereas cell death by apoptosis as shown by TUNEL may not be synchronized, giving rise to our observed different labeling patterns.
In summary, our study suggests that there may be a narrow window between the accumulation of c-Fos and the presence of DNA nicks in photoreceptors after photic injury. Inhibition of c-fos expression may ameliorate light-induced retinal apoptosis. Further studies on c-jun and AP-1 may also help to delineate the pathways of c-fos–related photoreceptor cell death.
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Footnotes |
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Submitted for publication June 17, 1999; revised December 14, 1999 and March 14, 2000; accepted March 22, 2000.
Commercial relationships policy: N.
Corresponding author: Tim T. Lam, Department of Ophthalmology & Visual Sciences, Hong Kong Eye Hospital, 147K Argyle Street, 3/F, Kowloon, Hong Kong. timlam{at}cuhk.edu.hk
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