Light damage induces inflammatory factors in mouse retina and vitreous humor

Experimental animals, reagents, and antibodies

BALB/c mice (5–8 weeks) purchased from Sun Yat-Sen University Laboratory Animal Center were used for all experiments. Mice were housed under a 12:12 light:dark cycle with ad libitum access to standard mouse chow. The study included 50 mice, with an equal distribution of males and females. All procedures conformed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center at the Sun Yat-Sen University, Guangzhou, China (Approval form #2022031). The reagents, antibodies, and other resources used in this study are listed.

Light exposure

The mice were placed in constant darkness 12 h before exposure to light. Non-anesthetized mice were then exposed to typical laboratory lighting (50 lx) or white light (15,000 lx) for 2 h using light-emitting diode (LED) lights placed at the top of a standard mouse cage. To maintain orientation during light exposure, each mouse was placed inside an end-cut 50 ml conical tube, which was secured inside a box used for light exposure. During light exposure, the mouse's front and hind paws were fixed with tape to ensure that the mouse's body did not flip over. The level of illumination was measured using a digital light meter (TASI, T8121, Jiangsu, China).

RNA-seq analysis

Mice were exposed with or without bright light (15,000 lx, 2 h), and retinas were collected 48 h after exposure. Total RNAs were extracted using TRIzol. The purified RNAs were then sent to Chi Biotech for sequencing. Bulk RNA-seq data were accessed from the NCBI Sequence Read Archive (SRA) database under accession number PRJNA916821. Adapters were removed using Trim Galore v1.18. Raw sequencing data were mapped to the GRCm39 genome assembly using HISAT2 v2.2.1 [14]. We calculated fragments per kilobase of exon per million (FPKM) using featureCounts v2.0.1 [15]. Using DESeq2 [16], we identified differentially expressed genes (DEGs) between control and light damage groups (fold change ≥2 and false discovery rate [FDR] ≤0.05). All upregulated genes (fold change ≥2 and FDR ≤0.05; n = 1384) were used for gene set enrichment analysis (GSEA) by R package clusterProfiler [17].

Immunofluorescence (IF) staining and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

After euthanasia, the eyeballs were enucleated into PBS to remove excess connective tissue and fixed in 4% PFA for 10 min. A small cut was made in the cornea of each mouse eye after fixation for 10 min and further fixation for 50 min. The eye was then dehydrated using 10% and 20% sucrose in 0.1 M phosphate-buffered saline for 1 h and dehydrated in 30% sucrose overnight. The dehydrated eyes were placed in a 1:1 mixture of 30% sucrose and optimal cutting temperature for 1 h before being embedded in the optimal cutting temperature medium. Retinal cryosections along the superior and inferior retinal meridians were cut at 8-μm thickness, and the sections were subjected to standard immunofluorescence protocols, as previously described [18]. The antibodies and dilution conditions are as follows: rabbit monoclonal (EPR16588) to IBA1 (Abcam, # ab178846), 1:100; Anti-rabbit IgG (H+L), F (ab') 2 Fragment (Alexa Fluor ® 594 Conjugate; Cell Signaling Technology, #8889), 1:500. TUNEL assay was performed using a TUNEL BrightRed Apoptosis Detection Kit (Vazyme, #A113) according to the manufacturer's procedure. The stained retinal sections were imaged with TissueFAXS microscopy.

Coomassie blue assay

The VH was diluted with radioimmunoprecipitation (RIPA) assay buffer (VH: RIPA = 1:1) containing a proteinase inhibitor cocktail and a protein phosphatase inhibitor. An equal volume of VH was used for electrophoresis. After electrophoresis, the gel was fixed/stained with 0.05% Coomassie brilliant blue R-250 prepared in 50% methanol and 10% acetic acid. Staining was performed for 4 h at room temperature with gentle shaking. The gel was then washed in a washing buffer (50% methanol, 10% acetic acid) for 24 h, and images were taken using a Bio-Rad imaging system.

Optical coherence tomography (OCT)

Before imaging, the mice were anesthetized with 1% pentobarbital sodium (70 μl/10 g, prepared in normal saline solution). Their pupils were dilated with 1–2 drops of tropicamide phenylephrine eye drops, and the corneas were lubricated with normal saline solution. OCT was performed on both eyes with a Heidelberg Spectralis OCT device (Heidelberg Engineering) to investigate structural changes. Thickness measurements were performed with a circular ring scan (circle diameter 1, 3, and 6 ETDRS) centered on the optic nerve head, which represents the average retinal thickness in a certain field. Central retinal thickness was calculated from four fields around the optic nerve head using Heidelberg Eye Explorer software.

Electroretinography (ERG)

Briefly, the mice were dark-adapted overnight and anesthetized with 1% pentobarbital sodium. Their pupils were dilated with 1–2 drops of tropicamide phenylephrine eye drops, and the corneas were lubricated with hypromellose gel. The ERG was recorded using a Diagnosys Celeris rodent ERG device. Electrodes were placed on top of each cornea. Mice were stimulated with flashlight of various intensities, ranging from 0.003 to 10 cd sec/m².

Hematoxylin and eosin (HE) staining of the retina

After euthanasia, the eyeballs were enucleated and fixed in FAS eye fixation solution (Servicebio #G1109) for 24 h. After fixation, the eyes were dehydrated in 60%, 70%, 80%, 90%, and 100% ethanol and processed for paraffin embedding. Paraffin sections of 8-μm thickness were subjected to hematoxylin and eosin (HE) staining, as previously described [18]. The stained retinal sections were imaged with TissueFAXS microscopy.

ELLA analysis

The VH was obtained using a Hamilton needle, with a volume of 3–5 μl obtained from each eye. VH from the control (n = 4 mice) and LIRD mice (n = 9 mice) were diluted 2–10 times and loaded in a total of 50 μl volume, assayed in triplicate for a panel of four inflammatory mediators using the Simple Plex Cartridge (ProteinSimple, SPCKC-MP-004230) on an Ella^TM System (ProteinSimple). Data were acquired on Simple Plex Explorer with an instrument default setting.

Statistics

The results are expressed as the mean ± SD. GraphPad Prism 9 software (GraphPad software, Inc., La Jolla, CA) was used for statistical analysis, as described in the Results section. All tests are two-tailed, unpaired t-tests unless otherwise indicated.

Ethics approval and consent to participate

All procedures conformed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center with the IACUC at the Sun Yat-Sen University, Guangzhou (#2022031), China.

Light damage induces photoreceptor death, retinal degeneration, and function impairment

We determined the LIRD mouse model using retinal morphological and functional analyses. The BALB/c mice were exposed to bright light, and the effects of LD were first investigated by in vivo OCT imaging. Thinning of the outer nuclear layer (ONL) was evident in the mouse retina after LD (Figure 1A). Histological analysis by HE staining confirmed that LD led to disruption of the retinal structure and loss of photoreceptors (Figure 1B). Further, TUNEL assays demonstrated prominent photoreceptor death after LD (Figure 1C). Concomitant with retinal degeneration and photoreceptor death, ERG analysis showed that the a- and b-wave amplitudes were significantly decreased, indicating LD-induced retinal response impairment (Figure 1D). Together, these results demonstrate the successful establishment of the LIRD mouse model.

LD induces inflammatory responses in the retina

We explored retinal transcriptome alteration in response to LD by RNA sequencing analysis (RNA-seq, n = 4 mice per group). Robust upregulation of inflammatory genes was detected in the mouse retina after LD (Figure 2A). The chemokines Ccl2, Ccl4, and Ccl3 ranked top in the profile of upregulated genes (Figure 2A). Our recent study revealed that interferon signaling was increased in sodium iodate-induced retinal degeneration [18]. Interestingly, the interferon-inducible gene Ifi204, and Casp1, an effector of interferon signaling, were induced by LD (Figure 2A), suggesting that activation of the interferon pathway may be a shared mechanism in retinal degeneration. Further, metalloproteases (Mmp8, Adam8), interleukins (Il1b), complement components (C3), and toll-like receptors (Tlr6, Tlr2) were also significantly increased by light exposure (Figure 2A). GSEA further demonstrated a significant enrichment of genes in the positive regulation of the inflammatory response (Figure 2B).

Because activation of macrophage/microglia is a hallmark of retinal inflammation, we performed immunohistochemistry staining and labeled the macrophages/microglia with an anti-IBA1 antibody. In the control retina, macrophages/microglia were mainly detected in the inner and outer plexiform, with typical thin and branched processes representing the resting phenotype (Figure 2C). After LD, a substantial increase in IBA1-positive cells was found in the ONL, exhibiting a swollen, amoeboid-like reactive phenotype (Figure 2C). These findings suggest that LD induces an inflammatory response and infiltration of macrophages/microglia in mouse retinas.

Light damage increases VH protein concentration

We then determined the overall VH proteins using Coomassie blue staining. The VH samples were loaded at the same volume, revealing an overall increased protein concentration based on the degree of Coomassie blue staining (Figure 3A), as evidenced by increased band intensity.

Light damage increases inflammatory factor expression in VH

We then measured the VH inflammatory factor using Ella, a highly sensitive automated immunoassay system. We chose a cartridge coated with four inflammatory mediators that were previously reported to be involved in retinitis pigmentosa [9]. In the control group, the mean concentrations of CCL2, IL6, IL1β and TNFα in VH were 118.4 ± 104.8, 1.7 ± 1.2, 9.6 ± 6.8, and 3.1 ± 2.1 pg/ml, respectively (Figure 4A-D). After LIRD, the concentrations of CLL2 (3000.9 ± 1077.0 pg/ml; Figure 4A) and IL6 (9.5 ± 4.1 pg/ml) were increased ~3000- and 10-fold, respectively (Figure 4B). Notably, this dramatic upregulation of CCL2 in the VH of LRID mice is consistent with the results of the RNA-seq analysis that showed CLL2 ranking top among upregulated inflammatory factors in the LD retina (Figure 2A). Increased levels of TNFα (12.6 ± 11.0 pg/ml; Figure 4C) and IL1β (42.3 ± 60.5 pg/ml; Figure 4D) were also detected in the VH after LIRD, although not significant.