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Retinal Cell Biology  |   June 2017
Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model
Author Affiliations & Notes
  • Chih-Cheng Chien
    School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
    Department of Anesthesiology, Cathay General Hospital, Taipei, Taiwan
  • Chi-Jung Huang
    Department of Medical Research, Cathay General Hospital, Taipei, Taiwan
    Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
  • Lu-Tai Tien
    School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
  • Yu-Che Cheng
    School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
    Department of Medical Research, Cathay General Hospital, Taipei, Taiwan
  • Chia-Ying Ke
    School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
  • Yih-Jing Lee
    School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
  • Correspondence: Yih-Jing Lee, School of Medicine, Fu-Jen Catholic University, 510 Chungcheng Road, Hsinchuang, New Taipei City 24205, Taiwan; yjlee@mail.fju.edu.tw
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3107-3117. doi:10.1167/iovs.16-21007
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      Chih-Cheng Chien, Chi-Jung Huang, Lu-Tai Tien, Yu-Che Cheng, Chia-Ying Ke, Yih-Jing Lee; Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model. Invest. Ophthalmol. Vis. Sci. 2017;58(7):3107-3117. doi: 10.1167/iovs.16-21007.

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

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Abstract

Purpose: We used a light-induced retinal degeneration animal model to investigate possible roles of heat shock protein 27 (HSP27) in retinal/photoreceptor protection.

Methods: Sprague-Dawley rats were used for the light-induced retinal degeneration animal model. The histology of eye sections was observed for morphologic changes in the retina. Cell apoptosis was examined in each group using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electroretinography was used to evaluate retinal function. Protein and mRNA expression levels of different retinal cell markers were also detected through immunofluorescence staining, Western blotting, and real-time PCR.

Results: The thickness of the outer nuclear layer significantly decreased after 7-day light exposure. Moreover, we injected a viral vector for silencing HSP27 expression into the eyes and observed that photoreceptors were better preserved in the HSP27-suppressed (sHSP27) retina 2 weeks after injection. HSP27 suppression also reduced retinal cell apoptosis caused by light exposure. In addition, the loss of retinal function caused by light exposure was reversed on suppressing HSP27 expression. We subsequently found that the expression of the Rho gene and immunofluorescence staining of rhodopsin and arrestin (cell markers for photoreceptors) increased in sHSP27-treated retinas. HSP27 suppression did not affect the survival of ganglion and amacrine cells.

Conclusions: Retinal cell apoptosis and functional loss were observed after 7-day light exposure. However, in the following 2 weeks after light exposure, HSP27 suppression may initiate a protective effect for retinal cells, particularly photoreceptors, from light-induced retinal degeneration.

Many people lose their sight because of degenerative retinal diseases, and these diseases often cause irreversible damage to retinal cells and lead to blindness. Among degenerative retinal diseases, AMD has been one of the major causes of blindness in developed countries in recent years. Therefore, the development of an effective therapeutic method for restoring vision loss caused by retinal degenerative diseases is crucial. 
Many animal models of retinal degeneration have been developed, and such models include the rodless mouse,1 the Royal College of Surgeons rat,2,3 and the retinal degeneration mouse (rd mouse).4 Since Noell et al.5 demonstrated light-induced retinal damage in rats in 1966, numerous studies have been conducted that mimicked retinal degeneration by exposing animal models to luminous environments. Retinal damage may be induced using different light intensities and exposure durations to control the degree of degeneration.6 In most animal models of light-induced retinal degeneration, only the outer nuclear layer (ONL) is affected, whereas the other retinal layers remain unaffected.6,7 In addition to structural changes, light also affects the physiologic function of the retina.8,9 Therefore, we used an animal model of light-induced retinal degeneration to study retinal degenerative diseases. 
Heat shock proteins (HSPs) are induced in response to various physiologic and environmental stressors in humans. Among them, HSP70 and HSP27 are highly inducible in glial cells and neurons by ischemia, epileptic seizure, and hyperthermia.10 According to a previous report, HSP27 was expressed in many sensory and motor neurons in the central nervous system, with HSP70 having little or no constitutive expression.10 Another HSP, namely HSP90, was described to play multiple roles in the retina, with HSP90 inhibitors showing potential for use in preventing retinal degeneration in animal models of retinitis pigmentosa and AMD.11 HSP27, also known as HSPB1, is most commonly involved in stress. Stress-induced HSP27 expression in the central nervous system has been observed in the rat hippocampus12 and the rat visual system.13 The differentiating cells accumulated a high level of HSP27; by contrast, HSP27 underexpression in other cells led to cell apoptosis.14,15 In an acute intraocular pressure–induced retinal ischemia animal model, both HSP27 and HSP72 were found to be upregulated in retinal ganglion cells (GCs) after 90-minute ischemia.16 Moreover, HSP27 expression in GCs plays a crucial role during postnatal development.17 HSP27 was markedly expressed in retinal glial cells including in Müller cell processes in the ONL.18,19 The protective effect of HSP27 on GCs was also observed in a retinal ischemia rat model when HSP27 was applied to the vitreous through electroinjection.20 Furthermore, an HSP27 antibody administered to retinal cells in vivo and ex vivo revealed DNA fragmentation and caspase activation and facilitated cell apoptosis.21 However, in our previous study, knocking down of HSP27 guided the differentiation of glutamatergic neurons from a cultured mesenchymal cell system.22 This finding suggests that HSP27 may play different roles in different situations. Because Müller cells are highly associated with the secretion of neurotrophic factors, this study concluded that HSP27 might be involved in the developmental and neuroprotective effects on the retina. 
In the present study, we examined the process of retinal degeneration after light exposure and the physiologic function of the retina by using electroretinography (ERG). In addition, we measured the retinal thickness and determined the gene and protein expression levels of several retinal cell markers to investigate retinal changes caused by long-term light exposure with and without HSP27 expression. 
Materials and Methods
Animal Model of Light-Induced Retinal Degeneration
Sprague-Dawley rats (male; weight, 200 to 225 g) were obtained from BioLASCO Technology (Taipei, Taiwan) and were kept under a normal light environment of 100 lux. Animal protocols were approved by the Institutional Animal Care and Use Committee of Fu-Jen Catholic University, and the experiments were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal model of retinal degeneration was constructed according to Joly et al.9 and Cheng et al.23 with minor modification. In brief, retinal degeneration was induced through 7 days of exposure to a cyclic, bright, and luminous light (5000 lux; 12 hours in dark/12 hours in light). After the bright light exposure, rats were returned to a normal light environment (100 to 200 lux) in an animal care facility. 
Subretinal Transplantation of Viral Vector
A lentiviral-silencing sequence for the rat HSP27 (sHSP27) gene (NCBI Reference Sequence: NM_031970.4) vector (constructed at the National RNAi Core Facility of Academia Sinica, Taipei, Taiwan) was subretinally injected after 7-day light exposure. A blank lentiviral vector was subretinally injected to the contralateral eye of the same animal for lentiviral sHSP27-vector injection about the same time as the sham control. The effect of suppressing HSP27 gene expression from the transfected vectors was 21% on day 7 after transfection. All animals were anesthetized using an intraperitoneal injection of zoletil (40 mg/kg; Virbac, Carros, France). Subretinal injections were administered under a stereomicroscope using a Hamilton syringe fitted with a 33-gauge blunt-end needle, and 1 μL (20,000 IU) vector solution was injected into the subretinal space near the ora serrata on the lateral side of each eye. The animals were kept on a heating pad during recovery. 
Histologic Study and Retinal Layer Thickness
To study retinal cell loss, the histology of the retinas and the thickness of the ONL of the retinas were examined. The thickness of the ONL was examined using a method modified from LaVail et al.24 and Cheng et al.23 In each nasal and temporal hemisphere, the thickness of the ONL was measured in nine defined areas. The first measurement was made 250 μm from the optic nerve head, and the subsequent 250-μm areas were more peripherally defined. Five defined points were measured within each 250-μm area, and the mean thickness was calculated. An upright microscope (Leica DM2500; Leica Microsystems GmbH, Wetzlar, Germany) and a digital camera system (Leica DFC420; Leica Microsystems GmbH) were used for the morphologic study and measurement. 
Apoptotic Cell Detection
A TUNEL assay was performed using an apoptosis detection kit (In Situ Cell Death Detection Kit; Fluorescein, Roche, Germany) to detect the degradation of DNA strands in apoptotic cells through fluorescence microscopy. TUNEL-positive cells were enumerated in the entire area of each retinal section.25 Images were observed using a fluorescent microscope (Leica DM2500; Leica Microsystems GmbH) and a digital camera system (CoolSNAP EZ; Roper Scientific, Tucson, AZ, USA). 
Electroretinography
Retinal function was evaluated using a scotopic electroretinogram. A RETIport ERG system (AcriVet, Berlin, Germany) was used, and ERG signals were recorded using the method described by Bayer et al.26 In brief, after 2 hours of dark adaptation, animals were anesthetized, and a gold foil corneal electrode (CH Electronics, Bromley, UK) was placed on the corneal surface; in this process, super gel was used as the active electrode, a genuine grass platinum subdermal needle electrode (Grass Technologies, West Warwick, RI, USA) was used as the reference electrode, and an ear-clip electrode was used as the ground electrode. The flash interval was 1 second with a flash duration of 3 ms, and the average of 10 stimulations was calculated using a computer program to produce final patterns. For ERG waveform analysis, the maximum a-wave amplitude was measured from the prestimulus baseline to the first negative peak of the ERG response, whereas the b-wave amplitude was measured from the a-wave peak to the most positive peak of the evoked response. Each rat served as its own internal control, with ERG signals recorded before the experiment from both eyes. The ERG signals of each eye were examined before and after the light exposure and weekly after the viral injection for at least 2 weeks (Fig. 1). In brief, the a- and b-wave amplitudes before the light exposure of each eye were used as the denominator (100%), and those of experimental groups following light exposure and every week after light exposure were divided by their own denominator and presented as the “ratio to normal.” 
Figure 1
 
Schematic representation of the experimental design. ERG signals were obtained weekly before and after 7 days of light exposure. Retinal degeneration was induced through 7 days of exposure to a cyclic, bright, and luminous light (5000 lux; 12 hours in dark/12 hours in light). Viruses carrying the HSP27-silenced gene or sham-control nucleotides were subretinally injected after 7-day light exposure. The eyes were collected for apoptotic, histologic, protein expression, or PCR studies 2 weeks after the viral injection.
Figure 1
 
Schematic representation of the experimental design. ERG signals were obtained weekly before and after 7 days of light exposure. Retinal degeneration was induced through 7 days of exposure to a cyclic, bright, and luminous light (5000 lux; 12 hours in dark/12 hours in light). Viruses carrying the HSP27-silenced gene or sham-control nucleotides were subretinally injected after 7-day light exposure. The eyes were collected for apoptotic, histologic, protein expression, or PCR studies 2 weeks after the viral injection.
Immunofluorescence Staining
HSP27, rhodopsin, Thy1, and arrestin expression levels were examined through immunohistochemistry. The retinal cryostat sections were mounted on a slide and fixed again with 4% paraformaldehyde in PBS (pH 7.4). Antibodies of different cytokines and specific retinal cell markers were used to detect different retinal cell types on the contralateral control and experimental eyes. Primary antibodies used are outlined as follows: anti-HSP27 (#2442; Cell Signaling Technology, Danvers, MA, USA), anti-rhodopsin (#8710; Cell Signaling Technology), anti–β-actin (#4970; Cell Signaling Technology), anti-syntaxin (sc-13994; Santa Cruz Biotechnology, Dallas, TX, USA), anti–Thy-1 (sc-19614; Santa Cruz Biotechnology), and anti-arrestin (sc-34547; Santa Cruz Biotechnology). Different fluorescent colors were used to detect cell marker expression. Alexa 488 (A11055; Molecular Probes, Life Technologies, Eugene, OR, USA) and Alexa 555 (A31572; Molecular Probes, Life Technologies) were used as secondary antibodies. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; sc-3598; Santa Cruz Biotechnology). An upright fluorescent microscope (Leica DM2500; Leica Microsystems GmbH) with a CCD camera system (CoolSNAP EZ; Roper Scientific) was used to observe the epi-fluorescence expression of the retinal sections. For the negative control, primary antibodies were omitted and replaced with nonimmune IgG. 
Quantitative Real-Time PCR
After euthanasia, retinas were isolated and processed immediately following the method described by Rocca et al.27 In brief, the retinas were homogenized using a precellys 24-tissue homogenizer (Bertin Technology, Siège, France). Their total RNA was prepared using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions and was then quantified using a NanoDrop ND 1000 spectrophotometer (Thermo Fisher Scientific). Changes in target gene expression were determined using quantitative real-time PCR (LightCycler; Roche Diagnostics, Mannheim, Germany). In summary, all complementary DNAs were generated using an oligo(dT)20 primer from 4 μg total RNA using the SuperScript III First-Strand Synthesis System for real-time PCR (Thermo Fisher Scientific), and they were relatively quantified in the following quantitative PCR analyses. The mRNA levels of target genes in the rat eyes, rhodopsin (Rho, NM_033441), arrestin (Sag, NM_013023), Thy1 cell surface antigen (Thy1, NM_012673), syntaxin 1A (Stx1a, NM_053788), and HSP27 (Hsp27, NM_031970), were quantified relative to their own glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_017008). Furthermore, except for Gapdh (Rn01775763_g1; Life Technologies, Grand Island, NY, USA), the target genes were measured using TaqMan probes from the Universal Probe Library and TaqMan Master Mix (Roche Diagnostics). The primers and universal probe numbers are listed in the Table. PCR kinetics to calculate the quantitative data were analyzed using LightCycler software, Version 4.05 (Roche Diagnostics. 
Table
 
Primer Sequences and Universal Probe Numbers for Quantitative PCR Analysis
Table
 
Primer Sequences and Universal Probe Numbers for Quantitative PCR Analysis
Western Blot Analysis
The posterior part (containing retina, RPE, choroid, and sclera) of each eye from different preparations was collected in a protein extraction buffer. The protein concentrations of each sample were quantified before blotting. Equal amounts of proteins were loaded into a polyacrylamide gel, separated through gel electrophoresis, and transferred to a membrane. The membrane was blocked and then probed with primary antibodies: anti-rhodopsin and anti–β-actin (#8710 and #4970; Cell Signaling Technology). The reacted proteins were detected and quantified using the electrochemiluminescence method. 
Statistical Analysis
Data were analyzed using SPSS software, Version 20 (IBM, New York, NY, USA). Statistical analyses were performed to determine differences in the relative mRNA levels of target genes among the examined groups by using the Mann-Whitney U test. One-way ANOVA with least significant difference (LSD) multiple comparisons was used to determine the significance of differences between the groups in other experiments. 
Results
A schematic representation of the procedure is presented in Figure 1. Retinal sections were selected and stained for morphologic examination. Histologic examination of normal (naïve group) retinas revealed the RPE and three distinct layers of retinal cell bodies: the ONL (nuclei of photoreceptors), INL (nuclei of bipolar, amacrine, and horizontal cells), and GC (nuclei of GCs) (Fig. 2A). Three other images (Figs. 2B–2D) also show retinal histologic investigations conducted 2 weeks after 7-day light exposure. Two weeks after light exposure, the ONL was markedly degenerated in the light-only (Fig. 2B) and sham-control virus vector-injected (light + sham) groups (Fig. 2D). However, the ONL was better preserved in the HSP27-silenced virus vector-injected (light + sHSP27) group than in the light + sham and light-only groups (Fig. 2C). The mean ONL thicknesses of the different groups of retinas are presented in Figure 2E. The ONL thickness decreased significantly by approximately 40% to 50% 2 weeks after 7-day light exposure (Fig. 2E), in which the suppression of HSP27 expression significantly reduced the ONL cell loss in the light + sHSP27 retinas compared with the light-only and sham-control retinas. Figure 2F shows the ONL thickness in different areas of different retinal preparations. In some areas of the retina, a greater ONL thickness was preserved in the light + sHSP27 retinas than in the light + sham retinas 2 weeks after 7-day light exposure (Fig. 2F), indicating that HSP27 suppression reduces photoreceptor cell loss after long-term light exposure. The statistical details of ONL thickness studies are presented in the Supplementary Table
Figure 2
 
Histologic investigations of the retina after different treatments. Retinal sections of the naïve retina (A), light-only retina (B), light + sHSP27 retina (C), and light + sham control retina (D) were observed 2 weeks after light exposure. Cells in the ONL significantly decreased in the light-only retinas (B) compared with the naïve retinas (A). The ONLs were thicker in the light + sHSP27 retinas (C) than in the light + sham retinas (D), in which the ONLs were considerably decreased. The mean ONL thicknesses of the different groups are also presented (E). The ONL thickness was markedly decreased in all three groups that underwent light exposure; however, the retinas that received an sHSP27 injection after light exposure exhibited significantly better ONL thickness than the light-only and sham control retinas. The ONL thicknesses in different areas of the retina are also presented (F), suggesting that this preservation effect was only observed in some areas of the retina. The values are presented as the mean ± SEM, and the sample size was nine from each different eye preparation in E and F. One-way ANOVA with LSD multiple comparisons was performed for determining the significance of differences between the groups in E and F. *P < 0.05 compared with the normal retina, #P < 0.05 compared with the light-only retina, and $P < 0.05 compared with the light + sham retina. Scale bars denote 50 μm.
Figure 2
 
Histologic investigations of the retina after different treatments. Retinal sections of the naïve retina (A), light-only retina (B), light + sHSP27 retina (C), and light + sham control retina (D) were observed 2 weeks after light exposure. Cells in the ONL significantly decreased in the light-only retinas (B) compared with the naïve retinas (A). The ONLs were thicker in the light + sHSP27 retinas (C) than in the light + sham retinas (D), in which the ONLs were considerably decreased. The mean ONL thicknesses of the different groups are also presented (E). The ONL thickness was markedly decreased in all three groups that underwent light exposure; however, the retinas that received an sHSP27 injection after light exposure exhibited significantly better ONL thickness than the light-only and sham control retinas. The ONL thicknesses in different areas of the retina are also presented (F), suggesting that this preservation effect was only observed in some areas of the retina. The values are presented as the mean ± SEM, and the sample size was nine from each different eye preparation in E and F. One-way ANOVA with LSD multiple comparisons was performed for determining the significance of differences between the groups in E and F. *P < 0.05 compared with the normal retina, #P < 0.05 compared with the light-only retina, and $P < 0.05 compared with the light + sham retina. Scale bars denote 50 μm.
Retinal cell apoptosis was detected using the TUNEL assay. The apoptotic cells were labeled with green fluorescence in the naïve group (normal) (Fig. 3A), light-only group immediately after 7-day light exposure (light only [1 W]; Fig. 3B), light-only group 2 weeks after light exposure (light only [3 W]; Fig. 3C), light + sHSP27 group 2 weeks after light exposure (Fig. 3D), and light + sham group 2 weeks after light exposure (Fig. 3E). Apoptotic cells were observed mainly in the ONL in the light-only 1 W (Fig. 3B), light-only 3 W (Fig. 3C), and light + sham (Fig. 3E) groups. However, few apoptotic cells were observed in the light + sHSP27 group (Fig. 3D). The number of apoptotic cells was counted for the entire retinal section for each group (Fig. 3F), and nine retinas were examined in each group. The statistical results revealed an increase in the apoptotic cell number in the light-only and light + sham groups, and the suppression of HSP27 reduced the number of apoptotic cells in the retina 2 weeks after light exposure. 
Figure 3
 
Apoptotic cells in retinas were observed and counted for all groups. The apoptotic cells were labeled with a fluorescence TUNEL kit (green), and all cell nuclei were labeled with DAPI (blue). Microscopic images of the naïve (normal) retina (A), light-only retina immediately after 7-day light exposure (B, light only 1 W), light-only retina 2 weeks after light exposure (C, light only), light + sHSP27 retina 2 weeks after light exposure (D), and light + sham control retina 2 weeks after light exposure (E) are shown. The apoptotic cells were counted for the entire retinal slide section of each group (F). The values are presented as the mean ± SEM. One-way ANOVA with LSD multiple comparisons was used for statistical analysis; n = 9 in each group. *P = 0.008 for the light-only group and P = 0.004 for the light + sham group compared with the naïve group. #P = 0.003 for the light + sHSP27 group compared with the light + sham (3 W) group. Scale bars denote 50 μm.
Figure 3
 
Apoptotic cells in retinas were observed and counted for all groups. The apoptotic cells were labeled with a fluorescence TUNEL kit (green), and all cell nuclei were labeled with DAPI (blue). Microscopic images of the naïve (normal) retina (A), light-only retina immediately after 7-day light exposure (B, light only 1 W), light-only retina 2 weeks after light exposure (C, light only), light + sHSP27 retina 2 weeks after light exposure (D), and light + sham control retina 2 weeks after light exposure (E) are shown. The apoptotic cells were counted for the entire retinal slide section of each group (F). The values are presented as the mean ± SEM. One-way ANOVA with LSD multiple comparisons was used for statistical analysis; n = 9 in each group. *P = 0.008 for the light-only group and P = 0.004 for the light + sham group compared with the naïve group. #P = 0.003 for the light + sHSP27 group compared with the light + sham (3 W) group. Scale bars denote 50 μm.
The physiologic function of the retinas of all the groups was investigated through ERG (Fig. 4). Figure 4 presents examples of the ERG signals of the naïve (Fig. 4A), light-only (Fig. 4B), light + sHSP27 (Fig. 4C), and light + sham control retinas (Fig. 4D), as well as the changes in a- and b-wave amplitudes. The traces of the a- and b-wave signals in the light-only, light + sHSP27, and light + sham groups were measured weekly and are presented in Figures 4E and 4F. Both a- and b-wave ERG signals decreased after 7-day light exposure, and no significant change was observed in these signals 1 week after light exposure. However, there was a significant increase in the b-wave amplitude of the light+sHSP27 group 2 weeks after light exposure (up to 60%; see Fig. 4F). Therefore, we concluded that suppressing HSP27 expression can reduce retinal cell apoptosis and reverse retinal function loss caused by long-term light exposure. 
Figure 4
 
Examples and quantification of ERG signals. Representative ERG signals from a naïve (normal) adult rat eye (A), an adult rat eye exposed to light for 7 days (B), a light + sHSP27 eye 2 weeks after light exposure (C), and the contralateral light + sham eye 2 weeks after light exposure (D). These images suggest that suppressing HSP27 expression reversed the loss of the physiologic function of the retina after light exposure. The results of the quantification of ERG a-wave and b-wave recordings are presented in E and F, and the values are shown as the mean ± SEM. One-way ANOVA with LSD comparisons was used for statistical analysis, with *P = 0.019.
Figure 4
 
Examples and quantification of ERG signals. Representative ERG signals from a naïve (normal) adult rat eye (A), an adult rat eye exposed to light for 7 days (B), a light + sHSP27 eye 2 weeks after light exposure (C), and the contralateral light + sham eye 2 weeks after light exposure (D). These images suggest that suppressing HSP27 expression reversed the loss of the physiologic function of the retina after light exposure. The results of the quantification of ERG a-wave and b-wave recordings are presented in E and F, and the values are shown as the mean ± SEM. One-way ANOVA with LSD comparisons was used for statistical analysis, with *P = 0.019.
The expression levels of the retinal markers rhodopsin, arrestin, Thy1, and syntaxin were quantified through real-time PCR (Fig. 5). The real-time PCR data are presented only for the light-only and relative sHSP27 groups. In the light-only group, a change was observed in the ratio of gene expression of the light-only eyes to the naïve (normal) eyes, whereas in the relative sHSP27 group, a change was observed in the ratio of the sHSP27 vector-injected eyes to the contralateral sham-control vector-injected eyes. The gene expression of these retinal cell markers was almost unchanged after the suppression of HSP27 expression, except for that of Rho, which represented the gene expression of rhodopsin (a cell marker of photoreceptors). This result is in agreement with our previous finding regarding retinal histology because sHSP27 transfection preserves ONL loss after light exposure and ONL represents photoreceptor nuclei. Furthermore, the expression of retinal cell markers and HSP27 was confirmed immunohistochemically (Fig. 6). HSP27 was highly expressed over the entire retina, including the GC, IPL, OPL, outer segment of the photoreceptor, and RPE, in the light-only group (Figs. 6E–6H). However, in the light + sHSP27 group, no HSP27-positive cells were observed (Figs. 6I–6L). HSP27 expression was observed only in the subretinal and GC areas in the light + sham retinas (Figs. 6M–6P). The outer segment areas of photoreceptors, which were labeled with rhodopsin and arrestin, became thinner in the light-only (Figs. 6E, 6F) and light + sham retinas (Figs. 6M, 6N) than in the naïve retinas (Figs. 6A, 6B). In comparison with the naïve retinas, the areas of rhodopsin and arrestin expression decreased in the light + sHSP27 retinas (Figs. 6I, 6J), which were still thicker than the light-only and light + sham retinas. There was little change in the Thy1 and syntaxin expression levels in the light-only, light + sham, and light + sHSP27 groups. These results confirmed our previous finding of suppression of HSP27 preventing photoreceptor cell loss caused by long-term light exposure. The protein expression of rhodopsin was also quantified through Western blotting (Fig. 7). The expression of rhodopsin in each preparation is presented as the ratio to its protein expression in the figure. The expression of rhodopsin was downregulated in the light-only and light + sham groups. However, the expression of rhodopsin was significantly increased in the light + sHSP27 group compared with the light-only group, although it was still less than that in the naïve group. These results suggest that transfected sHSP27 vectors may protect the photoreceptors from cell loss. 
Figure 5
 
Quantitative analysis of the gene expression of different retinal cell markers using real-time PCR. The gene expression levels of rhodopsin (Rho, A), arrestin (Arrestin, B), Thy1 (C), Hsp27 (D), and syntaxin 1a (Stx1a, E) are presented using box and whisker plot charts. The differences in P values between the light-only and relative sHSP27 groups determined using the Mann-Whitney U test are shown at the bottom right. The boxes indicate the interval between the 25th and 75th percentiles, the whiskers denote the interval between the 10th and 90th percentiles, the horizontal lines in the rectangles denote the medians, and the circle denotes an outlier. According to the results, Rho expression significantly increased after HSP27 suppression.
Figure 5
 
Quantitative analysis of the gene expression of different retinal cell markers using real-time PCR. The gene expression levels of rhodopsin (Rho, A), arrestin (Arrestin, B), Thy1 (C), Hsp27 (D), and syntaxin 1a (Stx1a, E) are presented using box and whisker plot charts. The differences in P values between the light-only and relative sHSP27 groups determined using the Mann-Whitney U test are shown at the bottom right. The boxes indicate the interval between the 25th and 75th percentiles, the whiskers denote the interval between the 10th and 90th percentiles, the horizontal lines in the rectangles denote the medians, and the circle denotes an outlier. According to the results, Rho expression significantly increased after HSP27 suppression.
Figure 6
 
Immunofluorescence staining of HSP27 and retinal cell markers. Histologic sections of naive retinas (AD), light-only retinas (EH), light+sHSP27 retinas (I–L), and light + sham retinas (M–P) are presented. The cell nuclei were stained with DAPI (blue), and the slides were double-stained with anti-HSP27 (red), anti-rhodopsin (green, in A, E, I, and M), anti-arrestin (green, in B, F, J, and N), anti-Thy1 (green, in C, G, K, and O), or anti-syntaxin (green, in D, H, L, and P). Moreover, the slides of the light-only and light + sham groups exhibited upregulated HSP27 in both the outer and inner retinal layers and the outer segment of photoreceptors (E–H and M–P); however, rhodopsin and arrestin expression decreased in the outer segment area (E, F and M, N) in these two groups. In the light + sHSP27 retinas, HSP27 expression was not detected (hollow arrows in I–L), and rhodopsin and arrestin were still highly expressed 2 weeks after light exposure (I, J). Scale bars denote 50 μm.
Figure 6
 
Immunofluorescence staining of HSP27 and retinal cell markers. Histologic sections of naive retinas (AD), light-only retinas (EH), light+sHSP27 retinas (I–L), and light + sham retinas (M–P) are presented. The cell nuclei were stained with DAPI (blue), and the slides were double-stained with anti-HSP27 (red), anti-rhodopsin (green, in A, E, I, and M), anti-arrestin (green, in B, F, J, and N), anti-Thy1 (green, in C, G, K, and O), or anti-syntaxin (green, in D, H, L, and P). Moreover, the slides of the light-only and light + sham groups exhibited upregulated HSP27 in both the outer and inner retinal layers and the outer segment of photoreceptors (E–H and M–P); however, rhodopsin and arrestin expression decreased in the outer segment area (E, F and M, N) in these two groups. In the light + sHSP27 retinas, HSP27 expression was not detected (hollow arrows in I–L), and rhodopsin and arrestin were still highly expressed 2 weeks after light exposure (I, J). Scale bars denote 50 μm.
Figure 7
 
Western blot analysis for rhodopsin protein expression in different preparations. Examples of Western blot images of β-actin and rhodopsin are shown in A, and the statistical analysis of the protein expressions of rhodopsin is shown in B. One-way ANOVA with LSD multiple comparison was used for statistical analysis; n = 9 in each group. *P = 0.000, 0.030, and 0.001 in the light-only, light + sHSP27, and light + sham groups, respectively, compared with the naïve retina; #P = 0.016 compared with the light-only group.
Figure 7
 
Western blot analysis for rhodopsin protein expression in different preparations. Examples of Western blot images of β-actin and rhodopsin are shown in A, and the statistical analysis of the protein expressions of rhodopsin is shown in B. One-way ANOVA with LSD multiple comparison was used for statistical analysis; n = 9 in each group. *P = 0.000, 0.030, and 0.001 in the light-only, light + sHSP27, and light + sham groups, respectively, compared with the naïve retina; #P = 0.016 compared with the light-only group.
Discussion
Several studies have reported that light-induced retinal damage induces photoreceptor apoptosis.28,29 Two major apoptotic pathways have been described, namely the extrinsic and intrinsic pathways, and various cysteine aspartate-specific proteases involved in these pathways have been identified.30,31 However, the molecular events involved in the model of light-induced retinal degeneration depend on the animal strain and light intensity.32 Some antioxidant agents, such as dimethylthiourea, protect against retinal light damage.33 Moreover, naloxone, a classic opioid receptor antagonist, reduces light-induced photoreceptor degeneration by inhibiting microglial activation.25 However, no evidence supports the use of these compounds to rescue photoreceptor cells or to cure retinal degeneration. Therefore, it is crucial to determine a suitable mechanism for reducing light-induced retinal damage for treating retinal degeneration. 
HSPs are stress proteins that play a complex defense role under critical environmental conditions. Among these proteins, the functions of HSP27, HSP70, and HSP90 are mostly studied in the eyes.11,14,34,35 Inhibition of HSP90 showed a protective effect on animal models of retinitis pigmentosa, AMD, and uveal melanoma.11 HSP70 was reported to enhance the survival of GCs in glaucoma and other optic neuropathies.34 Expressions of HSP70 and HSP27 also observed in many areas of the brain and retina; the expression of these HSPs was assumed to be associated with cellular resistance to various insults.10 HSP27 is considered a crucial regulator of the switch between cell differentiation and apoptosis. Mehlen et al. reported that transient HSP27 expression was essential for preventing embryonic stem cells from undergoing apoptosis.14 HSP27 was shown to be released after RPE injury, and it induces the differentiation of RPE cells. These differentiated RPE cells are associated with increased viability and resist membrane degradation, which leads to cell apoptosis.35 This observation suggests that the level of HSP27 plays a crucial role in RPE-mediated retinal degenerative diseases such as AMD. In our study, we observed that HSP27 expression was upregulated in the retina after long-term light exposure. Previous studies have reported that HSP27 upregulation may either prevent retinal cell apoptosis or lead to retinal cell differentiation.36 Light-induced HSP27 expression was observed in almost the entire retina, including the RPE and choroid, at the end of light exposure. Our findings demonstrate that the ONL thickness decreased 2 weeks after light exposure in the light-only, light + sHSP27, and light + sham groups compared with naïve retinas. Meanwhile, HSP27 upregulation caused by long-term light exposure was observed in the light-only and light + sham control retinas; however, HSP27 expression was effectively suppressed in the light + sHSP27 retinas at the same time point. HSP27 suppression also recovered retinal function and reduced retinal cell apoptosis during this period. We found that there was no significant change in a-wave of different groups, but there was a significant increase of b-wave of light + sHSP27 group compared with the light-only group. It was unexpected to see that b-wave increase instead of a-wave, because sHSP27 reduced cell loss in ONL; however, a higher b-wave amplitude may be produced by a better signal transmission between remaining photoreceptors and the inner retina. Our previous study suggested that suppressing HSP27 might induce glutamatergic neuron differentiation in a cultured mesenchymal cell system.22 It is highly possible that the suppression of HSP27 after light exposure would protect photoreceptors, which are also glutamatergic, and preserve retinal function against damage induced by long-term light exposure. 
Nahomi et al. reported that the downregulation of HSP27, which was promoted by the formation of reactive oxygen species and nitric oxide, promoted apoptosis in epithelial–endothelial cells of the retina and further reduced HSP27 expression.37 Similar findings were reported immediately after retinal injury. In our animal model of light-induced retinal degeneration, photoreceptor apoptosis was clearly observed in both histologic images and immunofluorescent TUNEL staining images. The progress of photoreceptor apoptosis persisted even 2 weeks after light exposure. By contrast, the photoreceptor cell loss was clearly decreased in the sHSP27-injected retinas 2 weeks after light exposure. These results suggest that HSP27 plays a critical role in inhibiting apoptosis in the later time points after the initial light exposure but might then lead to retinal differentiation. 
Apart from a histologic observation of photoreceptor cell loss, we used the animal model of light-induced retinal degeneration to examine HSP27-regulated gene expression after light exposure. Rho expression was significantly increased after HSP27 silencing. Rho is only expressed in the photoreceptor and in no other retinal cells. This finding suggests that light-induced HSP27 is involved in triggering photoreceptor apoptosis 2 weeks after light exposure. This phenomenon was not observed in other retinal cell marker genes. Therefore, we concluded that HSP27 silencing may facilitate the prevention of photoreceptor cell loss caused by long-term light exposure. 
The protein expression of retinal cell markers observed using immunofluorescent staining suggests that only arrestin and rhodopsin, cell markers for photoreceptors, were upregulated after HSP27 suppression. The expression of the amacrine cell marker syntaxin 1a (Stx1a) and the GC marker Thy1 remained unchanged after HSP27 suppression, indicating that the light-induced apoptosis of photoreceptors is inhibited by HSP27 downregulation. In addition, the protein expression of rhodopsin was quantified through immunoblotting. Our data reveal that the expression of rhodopsin was downregulated in the light-only and light + sham groups but significantly reversed in the light + sHSP27 group, although we did not see significant recovery of a-wave in the light + sHSP27 group in the ERG experiment. It might be because that opsin, as well as rhodopsin, could also affect the amplitude of a-wave in ERG. The result suggests that transfected sHSP27 vectors prevent the apoptosis of photoreceptors after light exposure. This observation is consistent with the discussed findings on photoreceptor gene expression. 
We conclude that long-term light exposure upregulates HSP27, which may subsequently regulate light-induced photoreceptor apoptosis. However, during the later time points after light exposure, HSP27 suppression may protect against photoreceptor cell loss. We assume that HSP27 is necessary to protect retinal cells during light exposure but should be suppressed after light exposure to promote cell recovery. 
Acknowledgments
Supported by grants from the National Science Council (NSC102-2320-B-030-007), Taipei, Taiwan; Ministry of Science and Technology (MOST103-2628-B-030-001-MY3), Taipei, Taiwan; and Cathy General Hospital (102-CGH-FJU-10), Taipei, Taiwan. The authors alone are responsible for the content and writing of the paper. 
Disclosure: Chih-Cheng Chien, None; Chi-Jung Huang, None; Lu-Tai Tien, None; Yu-Che Cheng, None; Chia-Ying Ke, None; Yih-Jing Lee, None 
References
Keeler CE. The inheritance of a retinal abnormality in white mice. Proc Natl Acad Sci U S A. 1924; 10: 329–333.
Bourne MC, Campbell DA, Tansley K. Hereditary degeneration of the rat retina. Br J Ophthalmol. 1938; 22: 613–623.
Dowling JE, Sidman RL. Inherited retinal dystrophy in the rat. J Cell Biol. 1962; 14: 73–109.
Pittler SJ, Keeler CE, Sidman RL, Baehr W. PCR analysis of DNA from 70-year-old sections of rodless retina demonstrates identity with the mouse rd defect. Proc Natl Acad Sci U S A 1993; 90: 9616–9619.
Noell WK, Walker VS, Kang BS, Berman S. Retinal damage by light in rats. Invest Ophthalmol Vis Sci. 1966; 5: 450–473.
Wenzel A, Grimm C, Samardzija M, Reme CE. Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration. Prog Retinal Eye Res. 2005; 24: 275–306.
Zhang C, Lei B, Lam TT, Yang F, Sinha D, Tso MO. Neuroprotection of photoreceptors by minocycline in light-induced retinal degeneration. Invest Ophthalmol Vis Sci. 2004; 45: 2753–2759.
Li F, Cao W, Anderson RE. Alleviation of constant-light-induced photoreceptor degeneration by adaptation of adult albino rat to bright cyclic light. Invest Ophthalmol Vis Sci. 2003; 44: 4968–4975.
Joly S, Pernet V, Dorfman AL, Chemtob S, Lachapelle P. Light-induced retinopathy: comparing adult and juvenile rats. Invest Ophthalmol Vis Sci. 2006; 47: 3202–3212.
Franklin TB, Krueger-Naug AM, Clarke DB, Arrigo AP, Currie RW. The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system. Int J Hyperthermia. 2005; 21: 379–392.
Aguila M, Cheetham ME. Hsp90 as a potential therapeutic target in retinal disease. Adv Exp Med Biol. 2016; 854: 161–167.
Bechtold DA, Brown IR. Induction of Hsp27 and Hsp32 stress proteins and vimentin in glial cells of the rat hippocampus following hyperthermia. Neurochem Res. 2003; 28: 1163–1173.
Krueger-Naug AM, Emsley JG, Myers TL, Currie RW, Clarke DB. Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system. Neuroscience. 2002; 110: 653–665.
Mehlen P, Mehlen A, Godet J, Arrigo AP. hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells. J Biol Chem. 1997; 272: 31657–31665.
Mehlen P, Coronas V, Ljubic-Thibal V, et al. Small stress protein Hsp27 accumulation during dopamine-mediated differentiation of rat olfactory neurons counteracts apoptosis. Cell Death Different. 1999; 6: 227–233.
Windisch BK, LeVatte TL, Archibald ML, Chauhan BC. Induction of heat shock proteins 27 and 72 in retinal ganglion cells after acute pressure-induced ischaemia. Clin Exp Ophthalmol. 2009; 37: 299–307.
Hawkes EL, Krueger-Naug AM, Nickerson PE, Myers TL, Currie RW, Clarke DB. Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development. J Comp Neurol. 2004; 478: 143–148.
Kalesnykas G, Niittykoski M, Rantala J, et al. The expression of heat shock protein 27 in retinal ganglion and glial cells in a rat glaucoma model. Neuroscience. 2007; 150: 692–704.
Kalesnykas G, Tuulos T, Uusitalo H, Jolkkonen J. Neurodegeneration and cellular stress in the retina and optic nerve in rat cerebral ischemia and hypoperfusion models. Neuroscience. 2008; 155: 937–947.
Yokoyama A, Oshitari T, Negishi H, Dezawa M, Mizota A, Adachi-Usami E. Protection of retinal ganglion cells from ischemia-reperfusion injury by electrically applied Hsp27. Invest Ophthalmol Vis Sci. 2001; 42: 3283–3286.
Tezel G, Wax MB. The mechanisms of hsp27 antibody-mediated apoptosis in retinal neuronal cells. J Neurosci. 2000; 20: 3552–3562.
Cheng YC, Huang CJ, Lee YJ, et al. Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells. Scientific Rep. 2016; 6: 30314.
Cheng YP, Ke CY, Kuo CC, Lee YJ. Effect of a complex lutein formula in an animal model for light-induced retinal degeneration. Chin J Physiol. 2016; 59: 202–209.
LaVail MM, Gorrin GM, Repaci MA. Strain differences in sensitivity to light-induced photoreceptor degeneration in albino mice. Curr Eye Res. 1987; 6: 825–834.
Ni YQ, Xu GZ, Hu WZ, Shi L, Qin YW, Da CD. Neuroprotective effects of naloxone against light-induced photoreceptor degeneration through inhibiting retinal microglial activation. Invest Ophthalmol Vis Sci. 2008; 49: 2589–2598.
Bayer AU, Mittag T, Cook P, Brodie SE, Podos SM, Maag KP. Comparisons of the amplitude size and the reproducibility of three different electrodes to record the corneal flash electroretinogram in rodents. Doc Ophthalmol. 2000; 98: 233–246.
Rocca CJ, Kreymerman A, Ur SN, et al. Treatment of inherited eye defects by systemic hematopoietic stem cell transplantation. Invest Ophthalmol Vis Sci. 2015; 56: 7214–7223.
Hafezi F, Marti A, Munz K, Reme CE. Light-induced apoptosis: differential timing in the retina and pigment epithelium. Exp Eye Res. 1997; 64: 963–970.
Abler AS, Chang CJ, Ful J, Tso MO, Lam TT. Photic injury triggers apoptosis of photoreceptor cells. Res Commun Molec Pathol Pharmacol. 1996; 92: 177–189.
Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999; 274: 20049–20052.
Cohen GM. Caspases: the executioners of apoptosis. Biochem J. 1997; 326 (Pt 1): 1–16.
Perche O, Doly M, Ranchon-Cole I. Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci. 2007; 48: 2753–2759.
Kutty RK, Kutty G, Wiggert B, Chader GJ, Darrow RM, Organisciak DT. Induction of heme oxygenase 1 in the retina by intense visible light: suppression by the antioxidant dimethylthiourea. Proc Natl Acad Sci U S A. 1995; 92: 1177–1181.
Piri N, Kwong JM, Gu L, Caprioli J. Heat shock proteins in the retina: focus on HSP70 and alpha crystallins in ganglion cell survival. Prog Retinal Eye Res. 2016; 52: 22–46.
Strunnikova N, Baffi J, Gonzalez A, Silk W, Cousins SW, Csaky KG. Regulated heat shock protein 27 expression in human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2001; 42: 2130–2138.
O'Reilly AM, Currie RW, Clarke DB. HspB1 (Hsp 27) expression and neuroprotection in the retina. Mol Neurobiol. 2010; 42: 124–132.
Nahomi RB, Palmer A, Green KM, Fort PE, Nagaraj RH. Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells. Biochim Biophys Acta. 2014; 1842: 164–174.
Figure 1
 
Schematic representation of the experimental design. ERG signals were obtained weekly before and after 7 days of light exposure. Retinal degeneration was induced through 7 days of exposure to a cyclic, bright, and luminous light (5000 lux; 12 hours in dark/12 hours in light). Viruses carrying the HSP27-silenced gene or sham-control nucleotides were subretinally injected after 7-day light exposure. The eyes were collected for apoptotic, histologic, protein expression, or PCR studies 2 weeks after the viral injection.
Figure 1
 
Schematic representation of the experimental design. ERG signals were obtained weekly before and after 7 days of light exposure. Retinal degeneration was induced through 7 days of exposure to a cyclic, bright, and luminous light (5000 lux; 12 hours in dark/12 hours in light). Viruses carrying the HSP27-silenced gene or sham-control nucleotides were subretinally injected after 7-day light exposure. The eyes were collected for apoptotic, histologic, protein expression, or PCR studies 2 weeks after the viral injection.
Figure 2
 
Histologic investigations of the retina after different treatments. Retinal sections of the naïve retina (A), light-only retina (B), light + sHSP27 retina (C), and light + sham control retina (D) were observed 2 weeks after light exposure. Cells in the ONL significantly decreased in the light-only retinas (B) compared with the naïve retinas (A). The ONLs were thicker in the light + sHSP27 retinas (C) than in the light + sham retinas (D), in which the ONLs were considerably decreased. The mean ONL thicknesses of the different groups are also presented (E). The ONL thickness was markedly decreased in all three groups that underwent light exposure; however, the retinas that received an sHSP27 injection after light exposure exhibited significantly better ONL thickness than the light-only and sham control retinas. The ONL thicknesses in different areas of the retina are also presented (F), suggesting that this preservation effect was only observed in some areas of the retina. The values are presented as the mean ± SEM, and the sample size was nine from each different eye preparation in E and F. One-way ANOVA with LSD multiple comparisons was performed for determining the significance of differences between the groups in E and F. *P < 0.05 compared with the normal retina, #P < 0.05 compared with the light-only retina, and $P < 0.05 compared with the light + sham retina. Scale bars denote 50 μm.
Figure 2
 
Histologic investigations of the retina after different treatments. Retinal sections of the naïve retina (A), light-only retina (B), light + sHSP27 retina (C), and light + sham control retina (D) were observed 2 weeks after light exposure. Cells in the ONL significantly decreased in the light-only retinas (B) compared with the naïve retinas (A). The ONLs were thicker in the light + sHSP27 retinas (C) than in the light + sham retinas (D), in which the ONLs were considerably decreased. The mean ONL thicknesses of the different groups are also presented (E). The ONL thickness was markedly decreased in all three groups that underwent light exposure; however, the retinas that received an sHSP27 injection after light exposure exhibited significantly better ONL thickness than the light-only and sham control retinas. The ONL thicknesses in different areas of the retina are also presented (F), suggesting that this preservation effect was only observed in some areas of the retina. The values are presented as the mean ± SEM, and the sample size was nine from each different eye preparation in E and F. One-way ANOVA with LSD multiple comparisons was performed for determining the significance of differences between the groups in E and F. *P < 0.05 compared with the normal retina, #P < 0.05 compared with the light-only retina, and $P < 0.05 compared with the light + sham retina. Scale bars denote 50 μm.
Figure 3
 
Apoptotic cells in retinas were observed and counted for all groups. The apoptotic cells were labeled with a fluorescence TUNEL kit (green), and all cell nuclei were labeled with DAPI (blue). Microscopic images of the naïve (normal) retina (A), light-only retina immediately after 7-day light exposure (B, light only 1 W), light-only retina 2 weeks after light exposure (C, light only), light + sHSP27 retina 2 weeks after light exposure (D), and light + sham control retina 2 weeks after light exposure (E) are shown. The apoptotic cells were counted for the entire retinal slide section of each group (F). The values are presented as the mean ± SEM. One-way ANOVA with LSD multiple comparisons was used for statistical analysis; n = 9 in each group. *P = 0.008 for the light-only group and P = 0.004 for the light + sham group compared with the naïve group. #P = 0.003 for the light + sHSP27 group compared with the light + sham (3 W) group. Scale bars denote 50 μm.
Figure 3
 
Apoptotic cells in retinas were observed and counted for all groups. The apoptotic cells were labeled with a fluorescence TUNEL kit (green), and all cell nuclei were labeled with DAPI (blue). Microscopic images of the naïve (normal) retina (A), light-only retina immediately after 7-day light exposure (B, light only 1 W), light-only retina 2 weeks after light exposure (C, light only), light + sHSP27 retina 2 weeks after light exposure (D), and light + sham control retina 2 weeks after light exposure (E) are shown. The apoptotic cells were counted for the entire retinal slide section of each group (F). The values are presented as the mean ± SEM. One-way ANOVA with LSD multiple comparisons was used for statistical analysis; n = 9 in each group. *P = 0.008 for the light-only group and P = 0.004 for the light + sham group compared with the naïve group. #P = 0.003 for the light + sHSP27 group compared with the light + sham (3 W) group. Scale bars denote 50 μm.
Figure 4
 
Examples and quantification of ERG signals. Representative ERG signals from a naïve (normal) adult rat eye (A), an adult rat eye exposed to light for 7 days (B), a light + sHSP27 eye 2 weeks after light exposure (C), and the contralateral light + sham eye 2 weeks after light exposure (D). These images suggest that suppressing HSP27 expression reversed the loss of the physiologic function of the retina after light exposure. The results of the quantification of ERG a-wave and b-wave recordings are presented in E and F, and the values are shown as the mean ± SEM. One-way ANOVA with LSD comparisons was used for statistical analysis, with *P = 0.019.
Figure 4
 
Examples and quantification of ERG signals. Representative ERG signals from a naïve (normal) adult rat eye (A), an adult rat eye exposed to light for 7 days (B), a light + sHSP27 eye 2 weeks after light exposure (C), and the contralateral light + sham eye 2 weeks after light exposure (D). These images suggest that suppressing HSP27 expression reversed the loss of the physiologic function of the retina after light exposure. The results of the quantification of ERG a-wave and b-wave recordings are presented in E and F, and the values are shown as the mean ± SEM. One-way ANOVA with LSD comparisons was used for statistical analysis, with *P = 0.019.
Figure 5
 
Quantitative analysis of the gene expression of different retinal cell markers using real-time PCR. The gene expression levels of rhodopsin (Rho, A), arrestin (Arrestin, B), Thy1 (C), Hsp27 (D), and syntaxin 1a (Stx1a, E) are presented using box and whisker plot charts. The differences in P values between the light-only and relative sHSP27 groups determined using the Mann-Whitney U test are shown at the bottom right. The boxes indicate the interval between the 25th and 75th percentiles, the whiskers denote the interval between the 10th and 90th percentiles, the horizontal lines in the rectangles denote the medians, and the circle denotes an outlier. According to the results, Rho expression significantly increased after HSP27 suppression.
Figure 5
 
Quantitative analysis of the gene expression of different retinal cell markers using real-time PCR. The gene expression levels of rhodopsin (Rho, A), arrestin (Arrestin, B), Thy1 (C), Hsp27 (D), and syntaxin 1a (Stx1a, E) are presented using box and whisker plot charts. The differences in P values between the light-only and relative sHSP27 groups determined using the Mann-Whitney U test are shown at the bottom right. The boxes indicate the interval between the 25th and 75th percentiles, the whiskers denote the interval between the 10th and 90th percentiles, the horizontal lines in the rectangles denote the medians, and the circle denotes an outlier. According to the results, Rho expression significantly increased after HSP27 suppression.
Figure 6
 
Immunofluorescence staining of HSP27 and retinal cell markers. Histologic sections of naive retinas (AD), light-only retinas (EH), light+sHSP27 retinas (I–L), and light + sham retinas (M–P) are presented. The cell nuclei were stained with DAPI (blue), and the slides were double-stained with anti-HSP27 (red), anti-rhodopsin (green, in A, E, I, and M), anti-arrestin (green, in B, F, J, and N), anti-Thy1 (green, in C, G, K, and O), or anti-syntaxin (green, in D, H, L, and P). Moreover, the slides of the light-only and light + sham groups exhibited upregulated HSP27 in both the outer and inner retinal layers and the outer segment of photoreceptors (E–H and M–P); however, rhodopsin and arrestin expression decreased in the outer segment area (E, F and M, N) in these two groups. In the light + sHSP27 retinas, HSP27 expression was not detected (hollow arrows in I–L), and rhodopsin and arrestin were still highly expressed 2 weeks after light exposure (I, J). Scale bars denote 50 μm.
Figure 6
 
Immunofluorescence staining of HSP27 and retinal cell markers. Histologic sections of naive retinas (AD), light-only retinas (EH), light+sHSP27 retinas (I–L), and light + sham retinas (M–P) are presented. The cell nuclei were stained with DAPI (blue), and the slides were double-stained with anti-HSP27 (red), anti-rhodopsin (green, in A, E, I, and M), anti-arrestin (green, in B, F, J, and N), anti-Thy1 (green, in C, G, K, and O), or anti-syntaxin (green, in D, H, L, and P). Moreover, the slides of the light-only and light + sham groups exhibited upregulated HSP27 in both the outer and inner retinal layers and the outer segment of photoreceptors (E–H and M–P); however, rhodopsin and arrestin expression decreased in the outer segment area (E, F and M, N) in these two groups. In the light + sHSP27 retinas, HSP27 expression was not detected (hollow arrows in I–L), and rhodopsin and arrestin were still highly expressed 2 weeks after light exposure (I, J). Scale bars denote 50 μm.
Figure 7
 
Western blot analysis for rhodopsin protein expression in different preparations. Examples of Western blot images of β-actin and rhodopsin are shown in A, and the statistical analysis of the protein expressions of rhodopsin is shown in B. One-way ANOVA with LSD multiple comparison was used for statistical analysis; n = 9 in each group. *P = 0.000, 0.030, and 0.001 in the light-only, light + sHSP27, and light + sham groups, respectively, compared with the naïve retina; #P = 0.016 compared with the light-only group.
Figure 7
 
Western blot analysis for rhodopsin protein expression in different preparations. Examples of Western blot images of β-actin and rhodopsin are shown in A, and the statistical analysis of the protein expressions of rhodopsin is shown in B. One-way ANOVA with LSD multiple comparison was used for statistical analysis; n = 9 in each group. *P = 0.000, 0.030, and 0.001 in the light-only, light + sHSP27, and light + sham groups, respectively, compared with the naïve retina; #P = 0.016 compared with the light-only group.
Table
 
Primer Sequences and Universal Probe Numbers for Quantitative PCR Analysis
Table
 
Primer Sequences and Universal Probe Numbers for Quantitative PCR Analysis
Supplement 1
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