Cold Shock Induced Oxidative Stress, Apoptosis and Genome-wide Gene Expression Perturbation in the Eyes of Zebrafish and the Mitigation of Blue Wavelength Light


 As global climate changes, severely temperature variations have significant impacts on survival and development of fish. While the potential effects of light wavelength on cold shock and associated mechanisms remain largely unknown in fish. Here, zebrafish were pre-exposed to white LEDs (an irradiance of 0.9 W/m2) and blue LEDs (LDB, peak at 450 nm, 0.9 W/m2) for 2 weeks, and then exposed to 26℃ or 11℃ for 48 hours, respectively. Cold shock led to low survival rate. Cold shock altered retinal structure, increased the number of apoptotic cells and Caspase-3 activity, inhibited superoxide dismutase (SOD) and catalase (CAT) activities, up-regulated mRNA expression of (NF-E2-related factor 2) nrf2, p53, casp3 and casp9, and down-regulated cat expression in fish eyes. These results demonstrated that acute cold exposure induced oxidative stress and apoptosis in zebrafish eyes, which may lead to mortality. However, cold shock in combination with LDB apparently mitigated these negative effects, which might be involved in the up-regulation of antioxidant response and down-regulation of apoptotic responses at transcriptional and translational levels. Furthermore, cold shock also caused dysregulation of genome-wide gene expression involved in circadian rhythm, phototransduction and il-17 signaling pathway, indicating that cold shock disturbed phototransduction cascade and circadian rhythm signals and caused inflammatory responses. Ten key genes involved in circadian rhythm, phototransduction, cell cycle arrest, RNA processing or inflammatory responses were identified, including muc5d, rnps1, si:dkey-243i1.1, opn1mw1, gadd45ba, cebpd, btg2, si:dkey-242g16.2, nr1d1 and zgc:122979, which may play an important role in the protection of LDB against cold shock. Finally, our study suggested the relationship between spectrum and cold stress and demonstrated LDB could protect fish against the negative effect of cold stress in the eyes.


Introduction
Water temperature is considered as the "abiotic master factor" for sh, which controls nearly all life activities, including growth, development, behavior, and reproduction (Donaldson et al., 2008). Although sh species own the ability to adapt to gradual temperature variations (Wang et al., 2022), sharp changes commonly lead to sublethal disorders and even mortality (Bacchetta et al., 2020). A rapid decline in water temperature, namely cold shock, has negative effects on metabolic processes (Islam et al., 2021;Jiao et al., 2020;Zhang et al., 2021), swimming ability (Szekeres et al., 2014), antioxidant and immune systems (Ale et al., 2021;Reda et al., 2020;Wen et al., 2018;Singh et al., 2021), and ultimately result in cellular apoptosis (Cheng et al., 2017). Furthermore, several documented events, referred to as "winterkills", have occurred in wild and cultured sh populations (Doménech et al., 1997;Hoag, 2003;Conover et al., 2006), which lead to episodic die-off of sh and great economic losses.
In natural sh habitat, penetrability of different colors or wavelengths of light varies greatly, sh vision and spectrum perception match their living ethology (Brüning et al., 2016). This means that each species has an optimal wavelength where sh prefer to aggregate, grow and develop (Villamizar et al., 2011).
Light emitting diodes (LEDs) have a longer life span and lower electrical running costs than traditional metal bulbs, in particular, could provide much more e cient lighting systems by using high energy short wavelength in sh farming (Migaud et al., 2007). Several studies have reported effects of different colors of LEDs on growth and development in juvenile Atlantic cod and turbot (Sierra-Flores et al., 2016), European seabass larvae (Villamizar et al., 2009), and yellowtail damsel sh . Other researches have con rmed the interactive effects between light spectrum and other environmental factors such as starvation, bacteria or virus (Choi et al., 2012;Roh et al., 2018a;Roh et al., 2018b;Ho et al., 2020). However, to the best of knowledge, few researches investigated the relationship between light spectrum composition and cold shock in sh (Kim et al., 2014).
Among the functional organs in sh, the eyes are an extension of the brain , and are important sensory organs which provide approximately 80% of the information from all external stimuli (Carrasco, 2018). No eyelids and xed pupils make the sh retina more vulnerable to the external environment (Park et al., 2013). The damage of eyes and retinal cells results in disorders of brain neuromodulation and hormone secretion, reduces nding/intake and predator avoidance, and ultimately results in a reduction of sh survival (Baumann et al., 2016). Although the morphological damage of eyes has been well known during cold stress and speci c light spectrum for a long time in sh (O'Day and Young, 1979;Pointer et al., 2005), the underlying mechanisms are largely unknown in sh eyes.
Zebra sh are an excellent model for understanding the mechanisms of effects of cold shock or light spectrum on sh (Wang et al., 2014). The molecular mechanisms of cold stress have well been elucidated by RNA-seq in zebra sh larvae (Long et al., 2013;. Our studies have suggested that blue LEDs (LDB) play a positive role in immune and antioxidant systems (Zheng et al., 2016a;Yuan et al., 2017a), and mitigated cadmium-induced toxicity in the liver of zebra sh (Yuan et al., 2017b). To test the hypothesis that LDB can protect sh against cold shock in sh, zebra sh were pre-exposed to white LEDs and blue LEDs for 2 weeks, and then exposed to 26℃ or 11℃, respectively. Body weight, body length, and survival rate were measured. Eyes were sampled to examine histological changes, oxidative stress, apoptosis and transcriptomic responses. Oxidative stress was evaluated by measuring lipid peroxidation and the activity and mRNA levels of antioxidant enzymes. Cellular apoptosis was con rmed by detecting the activity levels of several caspases and mRNA expression of genes involved in apoptosis pathway, as well as TUNEL staining. RNA-seq was used to identify some key genes or pathways which were sensitively responsive to LDB and/or cold shock. The present study investigated the effects of LDB on cold shock and associated mechanisms in sh, which would provide a new insight for improving the overwintering ability of sh in aquaculture.

Experimental design
Three months old zebra sh (AB strain) were initially acclimated to white LEDs (LDW, control) for 2 weeks at 26.2 ± 0.5°C with a photoperiod of 14 h light and 10 h darkness before the experiment. The sh were fed twice daily (10.00 h and 18.00 h) with Artemia nauplii and commercially manufactured feeds. At the beginning of the experiment, healthy and uniform zebra sh were subjected to white LEDs (an irradiance of 0.9 W/m 2 ) and blue LEDs (LDB, peak at 450 nm, 0.9 W/m 2 ) for 2 weeks, and then exposed to 26℃ or 11℃ for 48 hours, respectively. The pre-acclimation to LDB for 2 weeks was selected because it could induce sh resistance to cold shock. The setting of irradiance is determined according to our previous research (Yuan et al., 2017b;Zheng et al., 2016a). The low temperature at 11°C was selected for the cold shock experiment because sh started to die at this temperature after 12 h. The low water temperature was maintained by a constant circulation of cold water by using a thermostat. There were four groups in the experiment including the control, LDB, Cold, and LDB + Cold. Each treatment was assigned to 3 tanks with 40 sh per tank. In the present study. Survival rate was monitored at 6, 12, 24, 36 and 48 h. Body weight and body length were recorded at 24 h to con rm the effect of LDB on growth. Fish displaying no gill movement and no response to touch were considered as dead and removed. Eyes were sampled at 6 h and divided into three parts. The rst part was immediately stored in RNAlater solution (Takara) for RNA extraction. The second part was xed for histological and apoptotic observation. The nal part is immediately placed in dry ice and transferred into refrigerator at -80 ℃ for biochemical parameters.

Histological and apoptotic observation
Eyes were xed in eyeball xed liquid (Servicebio), embedded in para n wax, strained with hematoxylin/eosin (H & E), and prepared for light microscopy according to our previous studies (Zheng et al., 2011). TUNEL (terminal deoxynucleotidyl transferase (TdT) -mediated dUTP nick end labeling) staining was performed using In Situ Cell Death Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany) following the instruction of manufacturer. Generally, cells labeled with green uorescence were considered to be apoptotic.

Biochemical analysis
Malondialdehyde (MDA) and activity levels of superoxide dismutase (SOD) and catalase (CAT) were investigated by commercial kits (Nanjing Jiancheng Biological Engineering Company), referred to our recent study (Zheng et al., 2016b). Caspase activities were examined by cleavage of colourless substrates speci c for caspase-3 (Ac-DEVD-pNA), caspase-8 (Ac-IETD-pNA) and caspase-9 (Ac-LEHD-pNA) and release of the chromophorep nitroaniline (pNA). The examination methods were performed to the methods provided by Zheng et al (2021). Generally, aliquots of homogenate (10µL) were incubated at 37°C with assay buffer including 50 mm HEPES pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA and 10% glycerol. The reaction mixture was incubated at 37°C for 5 h and absorbance of the product was measured at 405 nm in a 96-well plate reader (In nite 200 PRO, TECAN). Enzyme activity and protein content were calculated by normalizing the values to protein levels which were examined using Bradford method (Bradford, 1976). Enzyme activity was expressed as fold change relative to the control.

Real-time PCR
Examination of mRNA expression was carried out according to our recent study , including the following steps: RNA extraction, cDNA synthesis, and real-time PCR. The primer sequences of all genes were referred to Supplementary Material in Table S1. The speci city of primer pair sequences was con rmed by electrophoresis detection and melting curve analysis with a single DNA band or peak. Ampli cation e ciency was stable among all genes and different treatments. Relative mRNA levels of genes were calculated according to the 2 −△△Ct method, normalizing to the geometric mean of the best combination of β-actin and gapdh (Zheng et al., 2015).

Transcriptome analysis
The entire operation process of transcriptome analysis includes the following steps: RNA preparation, sequencing, assembly, annotation, identi cation of DEGs and KEGG enrichment pathway, as suggested by Zhu et al., (2020a). Generally, total RNA was isolated using TRIzol reagent (Takara Bio Inc., Tokyo, Japan). RNA quality analysis was performed using Bioanalyzer 2100 (Agilent, USA) (RIN≥7.0). Twelve cDNA libraries (three replicates per treatment) were constructed using the RNA Library Prep Kit for Illumina and sequenced on an Illumina HiSeq platform by Beijing Genomics Institute (BGI, China). Raw data were ltered to obtain clean reads by removal of adaptors, low-quality reads, and ambiguous reads.
A total of 24932 non-redundant unigenes and their FPKM values (Fragments PerKilobase Million) were provided in Supplementary Material. Genes that met the cut-off criteria of FDR < 0.01 and a |log2 foldchange | of > 1.0 were assigned as DEGs. FPKM values determined by RNAseq analysis signi cantly correlated with expression levels analyzed by qPCR (R 2 = 0.8734, p < 0.05) (Fig. S1), which supported the reliability of transcriptome data. To nd target enrichment pathways, the DEGs were further carried out using online database (http://www.webgestalt.org). Functions with FDR < = 0.05 were considered as signi cant enrichment.

Statistical analysis
Results are showed as mean ± standard error (SEM). Prior to statistical analysis, all data were tested for normality of distribution using the Kolmogornov-Smirnov test. Data were analyzed using a two-way ANOVA to identify the main effects of LDB and cold shock and their interactions. Pairwise comparisons were carried out by Bonferroni t-test if signi cance was found (p < 0.05). Analyses were performed using SPSS 18.0 for Windows (SPSS, Michigan Avenue, Chicago, IL, USA).

Growth and survival
Body weight (Fig. 1A) and body length (Fig. 1B) were not affected by LDB, cold shock, and their interaction (p > 0.05) ( Table 1). On the contrary, survival rate was signi cantly impacted by LDB, cold shock, and their interaction at 12 h, 24 h, 36 h and 48 h (p > 0.05) (Table 1). Generally, cold shock sharply reduced survival rate at 24 h, 36 h and 48 h under LDW and LDB (p < 0.01), however, the LDB + Cold group showed higher survival rate than the Cold group (p < 0.05).

Histological changes
Histological alterations of retina were shown in Fig. 2. The retinal cross-section of zebra sh from inside to outside is inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nucleus layer (ONL), and outer segment (OS), respectively (E) (Fig. 2E). There were high cell densities in the different layers of retina in the control and LDB groups, particularly in the ONL and INL ( Fig. 2A and  2C). The cell densities in the ONL and INL were low in the Cold group (Fig. 2B), but were not apparent in the LDB + Cold group (Fig. 2D). The thickness of different retina layers including OS, ONL, OPL and IPL increased in the Cold group (p < 0.05), and remained relatively unchanged in the LDB group compared with the control (p > 0.05) (Fig. 2F). However, cold shock with LDB reduced the thickness of OPL and IPL compared with cold shock singly (p > 0.05). Two-way ANOVA analysis con rmed that the thickness of OS, ONL, OPL and IPL was strongly associated with cold shock (p < 0.05) (Table 1). Besides, LDB and the combination of LDB and cold exhibited a signi cant difference on the OPL and IPL thickness, respectively (p < 0.05) ( Table 1).

Apoptotic response
Apoptotic cells were found in the OS of retina in the all groups including the control (Fig. 3A), Cold (Fig. 3B), LDB (Fig. 3C), and LDB + Cold (Fig. 3D). However, the Cold group showed more apoptotic cells than the other groups (Fig. 3E).

Oxidative stress and apoptotic responses
The MDA content, the activity levels of SOD, CAT, Caspase-3, Caspase-8 and Caspase-9, and the mRNA levels of nrf2, sod1, sod2, cat, p53, casp3 and casp9 were signi cantly responsive to LDB or cold or their interaction (Table 2). Compared with the control, cold shock reduced cat expression and activity levels of SOD and CAT, and increased Caspase-3 activity and mRNA levels of nrf2, p53, casp3 and casp9 (p < 0.05) (Fig. 4). Compared with the single cold stress, cold shock in combination with LDB decreased MDA content, casp9 expression, and activity levels of Caspase-3, Caspase-8 and Caspase-9, and enhanced mRNA expression of sod1 and cat and activity levels of SOD and CAT (p < 0.05). Compared with the control, LDB up-regulated mRNA expression of nrf2, sod2, cat, casp3 and casp9 (p < 0.05).

Transcriptomic analysis
A total of 1246, 11 and 6 differently expressed genes (DEGs) were observed in the eyes of zebra sh from sample groups Cold/Control, LDB/Control, and LDB + Cold/Cold respectively (Fig. 5A). The number of upregulated DEGs was 838, 3 and 5 while the number of down-regulated DEGs was 408, 8 and 1 for Cold/Control, LDB/Control, and LDB + Cold/Cold respectively (Fig. 5B). A total of 3 and 7 DEGs were shared between LDB + Cold/Cold and Cold/Control and between LDB/Control and Cold/Control, respectively (Fig. 5C). Among these genes, muc5d (mucin 5d) was up-regulated in the Cold group compared with the control (p < 0.001) while LDB with cold shock reduced its expression compared with the single cold exposure (p < 0.01) (Fig. 5D). Expression levels of rnps1 (RNA-binding protein with serinerich domain 1) and si:dkey-243i1.1 declined in the Cold group compared with the control (p < 0.01) while both genes were up-regulated by LDB + Cold compared with cold shock alone (p < 0.01) (Fig. 5E and 5F).

Discussion
Acute decreases in water temperature have led to large losses of production and economy for many sh species in aquaculture and wild environments (Wu et al., 2011), which results in that studying the mechanisms of cold resistance are fascinating. In the present study, we found that cold shock damaged retinal structure and reduced survival rate in zebra sh, and demonstrated that the related mechanisms were involved in oxidative stress, apoptosis, and genome-wide gene expression dysregulation. Furthermore, we found that LDB could mitigate cold stress and improve sh survival, which may provide a cheap and effective method to improve cold tolerance of sh in aquaculture.
The injury of eyes may be due to excessive reactive oxygen species (ROS), as many existing studies have shown that ROS is produced when sh are subjected to cold stress (Wu et al., 2015;Cheng et al., 2017;Ghisaura et al., 2019). To scavenge excess ROS and reduce its toxic effects, organisms have evolved an antioxidant system. SOD promotes the conversion of •O 2− into H 2 O 2 which is subsequently catalyzed into H 2 O by CAT, playing a vital role in the removal of ROS. However, when ROS-scavenging system cannot effectively neutralize or eliminate the excess of ROS, oxidative damage would happen . In the present study, the reduction in SOD and CAT activities and cat expression were observed in the eyes of zebra sh exposed to cold shock, which may be associated with oxidative damage of eyes.
Similarly, several studies have reported that cold shock changed activity and mRNA expression of antioxidant genes and caused oxidative damage in the liver, gill and brain of zebra sh (Tseng et al., 2011;Wu et al., 2015). This concept was further supported by that cold shock induced apoptosis as re ected by increases in Caspase-3 activity and mRNA expression of casp3, casp9 and p53, as well as the increased apoptotic cells in the eyes of zebra sh. Among apoptosis related genes, p53 was the rst tumor suppressor gene linked to apoptosis, and can promotes apoptosis via up-regulating pro-apoptosis genes such as casp3 and casp9 (Perfettini et al., 2004). Low temperature induced apoptosis by up-regulating casp3, casp9 and p53 in the liver of puffer sh and grouper (Cheng et al., 2017;Sun et al., 2019). In the present study, the positive relationships between CAT activity and cat expression and between Caspase-3 activity and casp3 expression were con rmed during cold stress, suggesting these genes were transcriptionally regulated. On the contrary, enzyme activity levels did not always parallel with corresponding gene expression such as Caspase-9 activity and casp9 expression. This may be associated with time-lag effect between transcription and translation, post-translational protein modi cation or RNA stability, as suggested by Zheng et al (2016c). Therefore, our results indicate that cold shock may cause eye damage by inducing oxidative stress and apoptosis at transcriptional and post-transcriptional levels.
Up to now, no published data is available on the relationship between between light spectrum and cold stress and the potential mechanisms that LDB could mitigate cold stress. However, several previous studies have indicated that there are positive effects of LDB on growth, development, immune and antioxidant responses (Villamizar et al., 2011;Shin et al., 2013;Kim et al., 2014;Sierra-Flores et al., 2016;Choi et al., 2017;Ho et al., 2020). The present study observed demonstrated that LDB alleviated oxidative stress and apoptosis induced by cold shock. For example, compared with the This may be another possible mechanism that enabled sh to respond more rapidly to subsequent cold stress. The increased nrf2 expression paralleled with the up-regulated mRNA levels of sod2 and cat, suggesting that both antioxidant genes are transcriptionally regulated by nrf2 (Osburn and Kensler, 2008;Zheng et al., 2019). However, the potential negative effects of LDB on sh should be not neglected. Although low intensity blue light cannot cause acute stress and histological damage of eyes, the retina may be damaged during chronic irradiation for a long time or high intensity blue light (Migaud et al., 2007). In the present study, LDB alone did not signi cant affect growth, lipid peroxidation and caspase activity, but the casp3 and casp9 were signi cantly up-regulated compared with the control. This may be an early warning sign of eye injury. Therefore, light intensity and irradiation time should be considered with precautions when LEDs are used as a technique to improve cold resistance in sh.
To understand the potential mechanisms of cold shock and the protective role of LDB on cold stress, comparative transcriptomic pro les were examined. Studies on transcriptomic responses have focused on the liver and larvae when sh are subjected to cold stress (Long et al., 2012;Ge et al., 2020;Zhu et al., 2020b;Song and McDowell, 2021;Zhuo et al., 2021), with little information on effects of cold stress or light spectrum on transcriptome of eyes. In the present study, DEGs were signi cantly enriched in the pathways of circadian rhythm in the eye of zebra sh exposed to cold shock. It has well been known that eyes and brain are responsive to circadian oscillators (Zhao and Fent, 2016). Circadian rhythm genes in eyes exhibit higher intensity of oscillations than brain in zebra sh (Whitmore et al., 1998). There are a few transcription factors or core circadian genes which control all circadian gene expression, including clock, arntl, per, cry, nr1d, npas2, bhlhe40/41 in mammals (Ko and Takahashi, 2006). CLOCK or NPAS2 and ARNTLs heterodimers activate the transcription of core clock genes and clock-controlled genes and enhance metabolic processes by a positive limb of the feedback loop. The heterodimers also activate nuclear receptors which repress transcription of CLOCK/ARNTL and CLOCK/NPAS2 heterodimers by a negative limb of the feedback loop. Similarly, the core clock genes PER and CRY which are transcriptional repressors interact with the heterodimers by a negative limb of the feedback loop and inhibit its activity and thereby negatively regulate their own expression (Sen and Hoffmann, 2020). BHLHE40 represses the activity of CLOCK/ARNTL heterodimer by competing for the binding to E-box elements in the promoters of its target genes (Sato et al., 2004). Therefore, the up-regulation of per1a, per1b, per2, per3, bhlhe40, bhlhe41, nr1d1 and cry2 and the down-regulation of clocka, clockb, arntl1a, arntl1b, npas2 may indicate a negative regulation of circadian rhythm in the eyes of zebra sh exposed to cold stress. Furthermore, we also found that DEGs in phototransduction were dysregulated and DEGs in il-17 signaling pathways were up-regulated in zebra sh exposed to cold shock, possibly indicating that cold stress disturbed phototransduction cascade signal and caused in ammatory responses.
In the present study, 10 genes may play an key role in the protection of LDB against cold shock by transcriptomic analysis, including muc5d, rnps1, si:dkey-243i1.1, opn1mw1, gadd45ba, cebpd, btg2, si:dkey-242g16.2, nr1d1 and zgc:122979. Zebra sh muc5d is a gel-forming secreted mucin gene, which is highly homologous to other vertebrate mucins in their genomic and protein domain organization (Jevtov et al., 2014). Transcription of muc5 results in mucus hypersecretion, which are closely associated with oxidative stress and many chronic in ammatory airway diseases in mammals (Rada et al., 2011). RNPS1 is a subunit of the postsplicing complex and directly serves as a mark for mRNA surveillance which subjects aberrant mRNAs to nonsense-mediated decay (Lykke-Andersen et al., 2001). si:dkey-243i1.1 participates in centriole replication which is important for cell polarization and division (Howe (Howe et al., 2013;Fırat-Karalar and Stearns, 2014). In the present study, LDB cold shock up-regulated muc5d and down-regulated rnps1 and si:dkey-243i1.1, indicating that cold stress may cause in ammation and inhibit RNA processing and cell division. However, LDB mitigated the negative effects of cold stress on gene expression. The opsin encoded by opn1mw1 is a light-sensitive protein (Lamb, 2013), which is important for maintaining the normal function in the visual cycle in zebra sh (Shi et al., 2019). Although opn1mw1 was dramatically down-regulated by cold stress, LDB signi cantly increased opn1mw1 expression under normal temperature and cold shock conditions compared with cold shock alone, suggesting that LDB could alleviate phototransduction damage induced by cold stress. The other 6 genes such as gadd45ba, cebpd, btg2, si:dkey-242g16.2, nr1d1 and zgc:122979 showed a similar change that cold stress upregualted these genes under LDW and LDB while LDB signi cantly down-regulated their expression under normal temperature condition. CEBPD is a transcription activator regulating the expression of genes involved in in ammatory responses (Kinoshita et al., 1992). GADD45 and BTG2 negatively regulate of mitotic cell cycle (Jin et al., 2000;Gaudet et al., 2011). NR1D1 negatively regulates transcription of CLOCK/ARNTL and CLOCK/NPAS2 heterodimers to inhibit metabolomic processes (Ko and Takahashi, 2006). Therefore, the down-regulation of gadd45ba, cebpd, btg2 and nr1d1 induced by LDB may contribute to reduce in ammation and increase cell division and metabolism, which is possibly involved in the protection of LDB against cold shock.

Conclusion
The use of LED technology has increased considerably in the last few years because LEDs have a longlife span and low electrical running costs, in particularly, can be manufactured to yield speci c wavelengths according to a species' environmental requirements (Sánchez-Vázquez et al., 2019). For zebra sh, blue LEDs are an effective light source, which can be used to mitigate cold stress and increase survival rate by inhibiting oxidative damage and apoptosis and regulating genome-wide gene expression in the eyes. The present study con rmed the importance of light spectrum in resisting cold shock in sh. However, further studies are required to investigate whether other cultured sh species have a preference for a speci c light spectrum and whether the light spectrum can increase the tolerance to cold shock.

Declarations Author Declarations
Funding P LB, operated the experiment, collected samples, extracted data, carried out the data analysis, interpreted results, and prepared the manuscript draft; H T, C X, W D, Z QL and W Z: operated the experiment and collected samples; W P and Z JL: developed the protocol and search strategy, supervised the study, screened all samples for eligibility, reviewed and revised the manuscript, and provided nancial support.

Data availability
The data and materials that support the ndings of this study are available from the corresponding author upon reasonable request.
Code availability Not applicable.
Informed consent Not applicable.

Figure 3
Effects of treatment with blue LEDs (LDB) irradiation on apoptosis in the eyes of zebra sh subjected to cold shock. Transverse sections of retina in zebra sh subjected to Control (A), Cold (B), LDB (C), LDB + Cold (D) groups. Green uorescence by TUNEL staining denotes apoptosis in the outer segment (OS) of eyes of zebra sh. E, number of apoptotic cells relative to the control. *, ** and *** means signi cant differences (p < 0.05), (p < 0.01) and (p < 0.001) between Cold and Control or between LDB and LDB + Cold. #, ## and ### means signi cant differences (p < 0.05), (p <0 .01) and (p < 0.001) between LDB and Control or between Cold and LDB + Cold.