Animals and Induction of Retinal Injury by Light or Lipopolysaccharide (LPS)
The animal protocols used in the study were approved by the Animal Ethics Committee of the Eye and Ear Nose Throat Hospital of Fudan University, Shanghai, China and the experiments complied with the Association for Research in Vision and Ophthalmology’s Statement on the Use of Animals in Research. Sprague–Dawley rats (male, 6–8 weeks old, approximately 200 g) were kept in a colony room on a 12-h light/12-h dark cycle at 22–24 °C. Normal food and water were available ad libitum. All experiments were performed on the animals after an intraperitoneal injection of 10% chloral hydrate (0.4 ml/100 g). At the end of the experiments, the animals were anesthetized with an overdose of 10% chloral hydrate and killed by cervical dislocation. All operations were performed in such a way as to minimize animal suffering. Before light exposure, the animals’ pupils were dilated with 1% atropine eye drops (Santen Pharmaceuticals Co., Ltd, Osaka, Japan). In the light-injury model, the rats were separated into individual transparent boxes with wire tops and exposed continuously to bright light (5000 lux) in a light box to induce retinal degeneration. After exposure for 24 h, the rats were returned to the normal light/dark cycle and room conditions. In the intravitreal LPS injection model of retinal damage, phosphate-buffered saline (PBS; HyClone, Logan, UT, USA) was used to dissolve and dilute LPS (Sigma-Aldrich, St. Louis, MO, USA) solution to a concentration of 125 ng/μl. Rats were anesthetized and the pupils were dilated with atropine sulfate (Santen Pharmaceuticals Co., Ltd). Then a Hamilton microinjector (Hamilton, Reno, NV, USA) was used to perform the intravitreal injection with LPS (2 μl, 125 ng/μl) at the 1 mm posterior to the limbus. The right retinal samples were collected before exposure or 1, 3, 5, and 7 days after light- or LPS-induced injury for further analysis.
Isolation of Cytosolic and Mitochondrial Fractions and Detection of Cytosolic mtDNA
The anterior segment of each eye was removed and the retina was isolated after the eye had been enucleated after light- or LPS-induced injury, according to the experimental schedule. The retinas were weighed and cut up. Each fresh retina (usually ≤ 1 h after the animal was killed) was processed with the Mitochondria Isolation Kit for Tissue (C3606, Beyotime, Shanghai, China). The tissue was then mixed with 10 times the volume of mitochondrial separating reagent A and homogenized with a Dounce tissue grinder (10 passes). The homogenates were centrifuged at 600 × g for 10 min at 4 °C. The supernatants were pipetted, transferred to fresh 1.5 ml tubes, and centrifuged at 11,000 × g for 10 min at 4 °C. The mitochondrial DNA was isolated from the cytosolic supernatant. All experimental procedures were performed on ice. The PCR assay was used to detect the cytosolic mtDNA, as described in our previous work. In brief, the above-mentioned mtDNA was mixed with Buffer FG1 and Buffer FG2 of the FlexiGene DNA Kit (no. 51206, Qiagen) in a 2 ml tube. After inverting the tube three times, the mixture was incubated at 65 °C for 10 min. Isopropanol (100%) was used to induce DNA precipitation. The tube was centrifuged for 3 min at 10,000 × g. Then Buffer FG3 was used to dissolve the DNA precipitation at 65 °C for 30 min. Quantitative PCR (qPCR) was used to amplify the sequence encoding mitochondrial cytochrome c oxidase 1 (mt-Co1) to detect the mtDNA and to amplify the 18S rDNA sequence to detect the nuclear DNA. The levels of mtDNA were normalized to the nuclear DNA and compared between groups. Primer sequences are listed in Supplementary Table S1.
Preparation and Retinal Transfection of mtDNA
The mtDNA was isolated from normal rat retinal cells as previously described. The rat retinal microvascular endothelial cells were collected, washed with ice-cold PBS, and resuspended in 1 × Cytosol Extraction Buffer. After incubation on ice for 10 min, the cells were homogenized with an ice-cold Dounce tissue grinder. The homogenized mixture was centrifuged at 700 × g for 10 min at 4 °C. The supernatant was decanted into a new 1.5 ml microcentrifuge tube and centrifuged at 10,000 × g for 30 min at 4 °C. The pellet was resuspended in 1 × Cytosol Extraction Buffer and centrifuged again at 10,000 × g for 30 min at 4 °C, after which the supernatant was discarded. The pellet was resuspended in Enzyme Mix (5 μl) and incubated in a 50 °C for at least 60 min. The mixture was then centrifuged at12500 × g for 5 min. The supernatant was discarded and the pellet (mtDNA) was resuspended in Tris–EDTA (TE) buffer. The mtDNA was then diluted to a concentration of 1 μg/μl for the subsequent experiments. A solution (0.02 μg/μl mtDNA) containing 1 μl of mtDNA (1 μg/μl), 47.5 μl of PBS, and 1.5 μl of Attractene Transfection Reagent (Qiagen, 301,005) was prepared and incubated at room temperature for 15 min before intravitreal injection. The control solution contained 48.5 μl of PBS and 1.5 μl of Attractene Transfection Reagent (Qiagen, 301,005). According to our previous study in retinal microvascular endothelial cells stimulated with mtDNA, the concentration of mtDNA solution of intravitreal injection was 0.02 μg/μl. The rats were anesthetized and injected intravitreally with the mtDNA solution (0.02 μg/μl, 2 μl) or control solution (2 μl) 1 mm posterior to the limbus. After stimulation with mtDNA or control solution, the rats were maintained under their original feeding conditions. For electroretinography (ERG), the right eye of each rat was injected intravitreally with mtDNA and the left eye was injected with control solution. Only the right eye of each rat was included in the western blotting, TUNEL staining, and PCR analyses.
At 1, 3, 5, and 7 days after the intravitreal injection of mtDNA or control solution, retinal function was evaluated with ERG, recorded as a scotopic electroretinogram (Espion Electrophysiology System; Diagnosys LLC, Lowell, MA, USA). The rats were dark-adapted for 2 h and anesthetized. After the pupil was dilated with atropine sulfate (Santen Pharmaceuticals Co., Ltd), oxybuprocaine (Santen Pharmaceutical Co., Ltd) and carbomer (Bausch & Lomb, Rochester, NY, USA) were applied topically for corneal anesthesia and hydration, respectively. Under illumination with dim red light, platinum ring electrodes were placed on the corneal surface. A subdermal grounding electrode was placed hypodermically on the tail and an identical electrode inserted into the rat’s nose as the reference electrode. After 10 min dark adaptation, ERG signal recording was commenced as previously described . The amplitude of the a-waves was measured from baseline to the troughs of the a-wave. The b-wave amplitude was measured from the negative peak of the a-wave to the positive peak of the b-wave.
TUNEL staining was performed on the retinal sections using the In Situ Cell Death Detection Kit, TMR red (cat. no. 12156792910, Roche, Germany). The eyes were prepared and and removed the anterior segment of the eye 7 days after the intravitreal injection of mtDNA or control solution. 4% paraformaldehyde was used to fixed the eyecups, and then the eyecups were dehydrated in sucrose solutions (20% for 2 h and 30% for overnight). The tissues were embedded and stored at −80 °C and sagittal sections cut (10 μm). The tissue sections were fixed with 4% paraformaldehyde for 20 min at room temperature. The sections were immersed and washed in PBS for 30 min. The sagittal sections were then incubated with Permeabilization Solution (0.1% Triton X-100) for 30 min. The Enzyme Solution (vial 1) was added to the Label Solution (vial 2) to obtain the TUNEL reaction mixture (vial 1: vial 2 = 1:9). The sagittal sections were immersed in the TUNEL components and equilibrated for 60 min at 37 °C in a humidified atmosphere in the dark. After the sections were washed twice in PBS for 10 min each, 4¢,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) was used to counterstain the samples. The slides were observed and photographed under a fluorescence confocal microscope (Leica TCS SP8 WLL, Wetzlar, Hesse-Darmstadt, Germany). The approximate excitation/emission peaks of the TUNEL stain and DAPI were 364/454 and 540/580 nm, respectively.
The retinas were collected at 1, 3, 5, and 7 days after the intravitreal injection of mtDNA or control solution. The retinas processed with the Mitochondria Isolation Kit for Tissue (C3606, Beyotime, Shanghai, China), as previously described and the proteins collected from the supernatant and sediment were used for the detection of cytochrome c. Western blotting was performed according to methods described in previous study. The following primary antibodies were used: rabbit anti-BAX (#2772, Cell Signaling Technology), rabbit anti-BAK (#12105, Cell Signaling Technology), rabbit anti-caspase 9 (AF6348, Affinity Biosciences Ltd), rabbit anti-cleaved caspase 9 (AF5240, affbiotech), rabbit anti-caspase 3 (ab44976, Abcam), rabbit anti-cleaved caspase 3 (ab49822, Abcam), anti-β-actin (ab8227, Abcam), anti-cGAS (ab179785, Abcam), rabbit anti-STING (D1V5L) (#50494, Cell Signaling Technology), rabbit anti-TBK1/NAK (D1B4) (#3504, Cell Signaling Technology), rabbit anti-phospho-TBK1/NAK (Ser172) (D52C2) XP® (#5483, Cell Signaling Technology), rabbit anti-IRF3 (D83B9) (#4302, Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) (D6O1M) (#29047, Cell Signaling Technology), anti-interferon β (ab140211, Abcam), rabbit anti-cytochrome c (10993-1-AP, Proteintech), rabbit anti-VDAC1 (ab154856; Abcam). Three eyes from each group were tested.
The retinas were isolated 1, 3, 5, and 7 days after the intravitreal injection of mtDNA or control solution. The total RNA was isolated from each retina with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, and quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized with the PrimeScript RT Reagent Kit (Takara, Ohtsu, Shiga, Japan). A LightCycler® 480 II Real-Time PCR instrument (Roche, Basel, Switzerland) was used to perform all real-time PCRs as previously published study. The primer sequences are shown in Table S1. Three eyes from each group were analyzed.
Data were analyzed using the SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The ERG results are the means of six rats were used per experiment. Differences between two groups were compared with the Mann–Whitney U test. The Kruskal–Wallis test was used to compare differences among three or more groups. Values of P < 0.05 were considered statistically significant. At least 3 independent experiments were conducted for the quantitative results.