August 2000
Volume 41, Issue 9
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Retinal Cell Biology  |   August 2000
c-Fos Protein in Photoreceptor Cell Death after Photic Injury in Rats
Author Affiliations
  • Hong–Kit Poon
    From the Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong; and
  • Mark O. M. Tso
    From the Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong; and
    the Department of Ophthalmology, the Wilmer Institute, Johns Hopkins University, Baltimore, Maryland.
  • Tim T. Lam
    From the Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong; and
Investigative Ophthalmology & Visual Science August 2000, Vol.41, 2755-2758. doi:
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      Hong–Kit Poon, Mark O. M. Tso, Tim T. Lam; c-Fos Protein in Photoreceptor Cell Death after Photic Injury in Rats. Invest. Ophthalmol. Vis. Sci. 2000;41(9):2755-2758.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To examine the involvement of c-Fos protein in light-induced photoreceptor cell death in rats.

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.

Apoptosis in light-induced photoreceptor cell death has been demonstrated by several groups since 1996. 1 However, the underlying mechanism of photoreceptor cell death remains poorly understood. 
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 colleagues 5 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. 
Methods
Animals
Thirty-two 35-day-old Lewis albino rats were equally divided into eight groups. They were reared in cyclic light and darkness for 14 days before the experiment. All animal handling and experimentation adhered to the guidelines established in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by Institutional Animal Use and Ethics Committee. 
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. 
Results
In this series of experiments we used a light exposure schedule (i.e., 300–320 ft-cd for 3 hours) to generate a mild injurious insult to rat retinas. Because the tissue responses of the retina varied according to the region, the results we present here were restricted to the most sensitive area (i.e., the equatorial region of the superior quadrant of the retina). 7  
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. 
In Situ TUNEL
There was a gradual increase in the number of TUNEL-positive photoreceptor nuclei from 0 to 24 hours after light exposure, starting in the inner part of the ONL (Figs. 1G and 1H) and gradually spreading to the outer part of the ONL at 24 hours (Fig. 1I) . There were few TUNEL-positive nuclei at 96 hours (Fig. 1J)
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)
Morphometry of c-Fos–Positive Nuclei and TUNEL-Positive Nuclei in the ONL
There was a concomitant increase in the number of c-Fos–positive nuclei and TUNEL-positive nuclei in the ONL from 0 and 24 hours after light exposure, followed by a concomitant drop at 96 hours (Fig. 3)
Discussion
The present study showed temporal and spatial appearance of histologically degenerating cells that paralleled the appearance of DNA nicks and c-Fos accumulation after photic injury in rat retinas. Double-labeling showed intense c-Fos IM without DNA nicks, coexistence of weak c-Fos IM and extensive DNA nicks, and no c-Fos IM but extensive DNA nicks in scattered photoreceptor nuclei. Morphometry of c-Fos–positive nuclei and TUNEL-positive nuclei showed parallel trends. These observations are consistent with an active role for c-Fos in light-induced apoptosis of photoreceptor cells preceding DNA nicks in rats. 
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 colleagues 11 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 colleagues 12 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. 
 
Figure 1.
 
Histopathologic features (A through E), in situ TUNEL (F through J), and c-Fos IM (L through P) of the superior retina after photic injury. Note the appearance of pyknotic nuclei (solid arrows). There is a focal thinning of the ONL at 24 (D) and 96 (E) hours. Also note the increase of TUNEL-positive nuclei (arrowheads) from 0 to 24 hours (G through I) then subsidence at 96 hours (J). Positive control for TUNEL: 35-day-old RCS rat retinas 8 (K). An increase in the number of c-Fos–positive nuclei (open arrows) is also shown in the ONL from 0 to 24 hours (M through O), followed by a decrease (P). Positive control: rat Purkinje cells 9 (Q). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 1.
 
Histopathologic features (A through E), in situ TUNEL (F through J), and c-Fos IM (L through P) of the superior retina after photic injury. Note the appearance of pyknotic nuclei (solid arrows). There is a focal thinning of the ONL at 24 (D) and 96 (E) hours. Also note the increase of TUNEL-positive nuclei (arrowheads) from 0 to 24 hours (G through I) then subsidence at 96 hours (J). Positive control for TUNEL: 35-day-old RCS rat retinas 8 (K). An increase in the number of c-Fos–positive nuclei (open arrows) is also shown in the ONL from 0 to 24 hours (M through O), followed by a decrease (P). Positive control: rat Purkinje cells 9 (Q). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
Double-labeling of c-Fos (A) and in situ TUNEL (B) in the same superior rat retina at 24 hours after photic injury. Note that some nuclei were moderately c-Fos–positive but TUNEL-negative (arrows), whereas some relatively weak c-Fos–positive nuclei were TUNEL-positive (arrowheads). Note that some intense TUNEL-positive nuclei (open arrows) were c-Fos–negative (open arrows). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
Double-labeling of c-Fos (A) and in situ TUNEL (B) in the same superior rat retina at 24 hours after photic injury. Note that some nuclei were moderately c-Fos–positive but TUNEL-negative (arrows), whereas some relatively weak c-Fos–positive nuclei were TUNEL-positive (arrowheads). Note that some intense TUNEL-positive nuclei (open arrows) were c-Fos–negative (open arrows). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 3.
 
Morphometry of c-Fos– or TUNEL-positive nuclei in the superior rat retina after photic injury. Note that the change in the number of c-Fos–positive nuclei paralleled that of TUNEL-positive nuclei showing a time-dependent increase after light exposure with maximal values at 24 hours. *P < 0.05 when compared with normal. #P < 0.05 when compared with their respective preceding groups. All pairwise multiple comparison procedures (Student–Newman–Keuls method).
Figure 3.
 
Morphometry of c-Fos– or TUNEL-positive nuclei in the superior rat retina after photic injury. Note that the change in the number of c-Fos–positive nuclei paralleled that of TUNEL-positive nuclei showing a time-dependent increase after light exposure with maximal values at 24 hours. *P < 0.05 when compared with normal. #P < 0.05 when compared with their respective preceding groups. All pairwise multiple comparison procedures (Student–Newman–Keuls method).
Abler AS, Chang CJ, Ful J, Tso MOM, Lam TT. Photic injury triggers apoptosis of photoreceptor cells. Res Commun Mol Pathol Pharmacol. 1996;92:177–189. [PubMed]
Sheng M, Greenberg ME. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron. 1990;4:477–485. [PubMed]
Franza BR, Jr, Rauscher FJ, III, Josephs SF, Curran T. The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 sites. Science. 1988;239:1150–1153. [CrossRef] [PubMed]
Smeyne RJ, Schilling K, Oberdick J, et al. Continuous c-fos expression precedes programmed cell death in vivo. Nature. 1993;363:166–169. [CrossRef] [PubMed]
Hafezi F, Steinbach JP, Marti A, et al. The absence of c-fos prevents light-induced apoptotic cell death of photoreceptors in retinal degeneration in vivo. Nat Med. 1997;3:346–349. [CrossRef] [PubMed]
Hafezi F, Abegg M, Crimm C, et al. Retinal degeneration in the rd mouse in the absence of c-fos. Invest Ophthalmol Vis Sci. 1998;39:2239–2244. [PubMed]
Rapp LM, Williams TP. The role of ocular pigmentation in protecting against retinal light damage. Vision Res. 1980;20:1127–1131. [CrossRef] [PubMed]
Tso MOM, Zhang C, Abler AS, et al. Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy of RCS rats. Invest Ophthalmol Vis Sci. 1994;35:2693–2699. [PubMed]
Ruan Y, Li WWY, Lam DWL, Yew DT. The c-Fos immunoreactivities in the developing and adult rat cerebella. Cell Mol Biol Res. 1995;41:111–115. [PubMed]
Gajate C, Alonso MT, Schimmang T, Mollinedo F. c-Fos is not essential for apoptosis. Biochem Biophys Res Commun. 1996;218:267–272. [CrossRef] [PubMed]
Rich KA, Zhan Y, Blanks JC. Aberrant expression of c-Fos accompanies photoreceptor cell death in the rd mouse. J Neurobiol. 1997;32:593–612. [CrossRef] [PubMed]
Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EM, Jr. Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J Cell Biol. 1994;127:1717–1727. [CrossRef] [PubMed]
Figure 1.
 
Histopathologic features (A through E), in situ TUNEL (F through J), and c-Fos IM (L through P) of the superior retina after photic injury. Note the appearance of pyknotic nuclei (solid arrows). There is a focal thinning of the ONL at 24 (D) and 96 (E) hours. Also note the increase of TUNEL-positive nuclei (arrowheads) from 0 to 24 hours (G through I) then subsidence at 96 hours (J). Positive control for TUNEL: 35-day-old RCS rat retinas 8 (K). An increase in the number of c-Fos–positive nuclei (open arrows) is also shown in the ONL from 0 to 24 hours (M through O), followed by a decrease (P). Positive control: rat Purkinje cells 9 (Q). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 1.
 
Histopathologic features (A through E), in situ TUNEL (F through J), and c-Fos IM (L through P) of the superior retina after photic injury. Note the appearance of pyknotic nuclei (solid arrows). There is a focal thinning of the ONL at 24 (D) and 96 (E) hours. Also note the increase of TUNEL-positive nuclei (arrowheads) from 0 to 24 hours (G through I) then subsidence at 96 hours (J). Positive control for TUNEL: 35-day-old RCS rat retinas 8 (K). An increase in the number of c-Fos–positive nuclei (open arrows) is also shown in the ONL from 0 to 24 hours (M through O), followed by a decrease (P). Positive control: rat Purkinje cells 9 (Q). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
Double-labeling of c-Fos (A) and in situ TUNEL (B) in the same superior rat retina at 24 hours after photic injury. Note that some nuclei were moderately c-Fos–positive but TUNEL-negative (arrows), whereas some relatively weak c-Fos–positive nuclei were TUNEL-positive (arrowheads). Note that some intense TUNEL-positive nuclei (open arrows) were c-Fos–negative (open arrows). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
Double-labeling of c-Fos (A) and in situ TUNEL (B) in the same superior rat retina at 24 hours after photic injury. Note that some nuclei were moderately c-Fos–positive but TUNEL-negative (arrows), whereas some relatively weak c-Fos–positive nuclei were TUNEL-positive (arrowheads). Note that some intense TUNEL-positive nuclei (open arrows) were c-Fos–negative (open arrows). INL, inner nuclear layer. Scale bar, 20 μm.
Figure 3.
 
Morphometry of c-Fos– or TUNEL-positive nuclei in the superior rat retina after photic injury. Note that the change in the number of c-Fos–positive nuclei paralleled that of TUNEL-positive nuclei showing a time-dependent increase after light exposure with maximal values at 24 hours. *P < 0.05 when compared with normal. #P < 0.05 when compared with their respective preceding groups. All pairwise multiple comparison procedures (Student–Newman–Keuls method).
Figure 3.
 
Morphometry of c-Fos– or TUNEL-positive nuclei in the superior rat retina after photic injury. Note that the change in the number of c-Fos–positive nuclei paralleled that of TUNEL-positive nuclei showing a time-dependent increase after light exposure with maximal values at 24 hours. *P < 0.05 when compared with normal. #P < 0.05 when compared with their respective preceding groups. All pairwise multiple comparison procedures (Student–Newman–Keuls method).
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