Mice
C57BL/6J mice were provided by the Institutional Animal Care of Beijing Friendship Hospital Affiliated to Capital Medical University. Four-week-old mice (males and females) were randomly assigned to groups, and maintained under a standard 12-hour light/dark cycle at 24.5°C. All experimental procedures were approved by the Office of Research Ethics Committee at Beijing Friendship Hospital Affiliated to Capital Medical University (ethics approval number: 18-2020).
Intravitreal Injections
Animals were anaesthetized with 1.5%–2% isoflurane. Intravitreal injection of NMDA (100 mM in PBS) was performed using a sharp 32-guage needle (micro-syringe equipped of Hamilton Storage, United States). Two days post NMDA challenge, intravitreal injection of recombinant SCF (Novus Biologicals, United States, 50 ng/ml in PBS) and anti-c-Kit neutralizing antibody (Tocris Bioscience, United Kingdom, 50 ng/ml in PBS) were performed. Mice injected with an equal volume of PBS (2 μl per eye) were served as control.
Tissue Preparation and Immunofluorescence
Immunohistochemistry was performed as described previously [16, 17]. Briefly, mouse eyeballs were prefixed in prefixation buffer (5% acetic acid, 0.4% paraformaldehyde, 0.315% saline, and 37.5% ethanol), then incubated in 4% paraformaldehyde overnight at 4°C, followed by embedded in paraffin. Eyecups were sectioned at 5 μm on a microtome (Leica, Germany). Slides were then deparaffinized, rehydrated, and boiled in 10 mM citrate buffer, followed by incubation in 5% donkey serum for 30 minutes at room temperature. Slides were then incubated with indicated primary antibodies at 4°C overnight, rinsed with PBS, and then incubated in species-matched fluorophore-conjugated secondary antibodies for 1 hour at 37°C. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Images were obtained using confocal microscopy of Fluo View FV1000 (Olympus, Japan). To perform the whole retina quantification, at least 6 sections across the optic disc were analyzed.
The primary antibodies used were as follows: anti-c-Kit at 10 μg/ml (AF1356, R&D Systems, United States), anti-glutamine synthetase (GS) at 1:200 (ab73593, ab64613, Abcam, United Kingdom), anti-SCF at 1:200 (ab64677, Abcam), anti-Connexin 43 (Cx43) at 1:100 (ab78055, Abcam), anti-Iba1 at 1:200 (ab178847, Abcam), anti-NeuN at 1:200 (ab209898, Abcam), and anti-Calretinin at 1:400 (MAB1568, Millipore). The secondary antibodies used were as follows: donkey anti-goat IgG Alexa Fluor 488 at 1:500 (ab150129, Abcam), donkey anti-rabbit Alexa Fluor 555 at 1:500 (ab150074, Abcam), donkey anti-mouse Alexa Fluor 555 at 1:500 (ab150106, Abcam), donkey anti-mouse Alexa Fluor 647 at 1:500 (ab ab150107, Abcam), donkey anti-rabbit IgG Alexa Fluor 488 at 1:500 (ab150073, Abcam), goat anti-chicken IgG Alexa Fluor 555 at 1:500 (ab150170, Abcam).
Analysis of Microglia
The method was performed as described previously [14]. Briefly, five 40× field views were captured from three 15 μm-thick retinal sections per eye using the Olympus confocal imaging system with 1-μm z-steps. By using a grid system, the number of grid-crossing points per individual microglia cell was counted (n > 3 eyes per group). The number of Iba1+ cells was counted in 5 eyes per group.
Electroretinogram Recording
Corneal scotopic flash electroretinogram (fERG) of mice was performed at corresponding time point after intravitreal injection of NMDA (at lease 5 mice in each time point) as described previously [17]. Briefly, after adaption darkness overnight, mice were anesthetized with 1.5%–2% isoflurane. The animal body temperature was maintained at 37°C by using a heating pad. The pupils of mice were dilated with tropicamide and phenylephrine eye drops (Santen Pharmaceutical, Japan). The recording electrodes of gold loops were placed on the cornea. The reference electrodes and grounding electrodes of gold needles were inserted subcutaneously into angulus oculi and tail respectively. We obtained flash recordings at the light intensities of −2.5, −0.5, −0.02, and 0.5 log (cd*s/m2) using Reti-scan system (Roland Consult, Germany). Waves measured at 0.5 log10 (cd*s/m2) were presented. The fERG procedures were performed under the environment of dim red light. The amplitudes of a-wave and b-wave were analyzed among groups.
Scotopic threshold responses (STRs) were elicited using a -4.5 log10 (cd*s/m2) stimulus using Reti-scan system (Roland Consult) as described previously [18]. Thirty flashes with an interstimulus interval of 2s were averaged. Amplitudes of the positive STR (pSTR) and negative STR (nSTR) were measured for about 140 and 220 ms after the stimulus flash, respectively.
For photopic negative response (PhNR) analysis, flash strength was 10 log10 (cd*s/m2), and 50 responses were averaged for each eye as described previously [19]. The PhNR was measured from baseline to the trough immediately following the b-wave.
Light/dark Transition Test
Light/dark transition test was performed as described previously [17]. The light/dark box consists of one light chamber (45 × 30 × 40 cm) and one dark chamber (15 × 30 × 40 cm), and these two compartments were connected with a door (10 × 10 cm). Mice were maintained in dark environment overnight, and adapted in the dark chamber for 2 minutes. The door was then opened, and mice were allowed to freely move into the light chamber for 5 minutes with 300 lux of tungsten filament bulb over the center of the compartment. All of the mice were tested naïve (only one test per mouse). Four paws completely through the door were defined as entering the light chamber. The time of exploratory behavior in the light compartment was analyzed.
Western Blotting
Eye samples were prepared after mice with euthanized. Retinas were then isolated and homogenized in an ice-cold mixture of RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Beyotime). Extracts were separated using 12% sodium dodecyl sulfate poly-acrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were incubated in TBST (12.5 mM Tris–HCl, pH 7.6, 75 mM NaCl, 0.1% Tween 20) containing 5% fat-free milk for 1 hour at room temperature, then transferred into solution containing primary antibodies at 4°C overnight, and probed with indicated secondary antibodies in TBST for 2 hours at room temperature. Membranes were exposed on an Odyssey infrared imaging system with the Odyssey Application software V1.2.15 (LI-COR Biosciences, United States). All blots were analyzed by ImageJ (National Institutes of Health, United States). The relative levels of SCF were determined by normalizing against β-actin. The primary antibodies used were as follows: anti-SCF at 1:1000 (ab64677, Abcam), anti-β-actin at 1:1000 (ab179467, Abcam). The secondary antibody used was peroxidase-conjugated goat anti-rabbit IgG at 1:2000 (Beyotime).
Gene Functional Annotation Analysis
For transcriptome analysis, total retinal cells were incubated in RNAiso Plus (Takara, Japan) at a concentration of 2 × 106 cells/ml and stored at −80 °C. All samples were transported to the Genomics Institute on dry ice for the transcriptome study. The mRNA sample was enriched using oligo (dT) magnetic beads and fragmented into short fragments using fragmentation buffer. The corresponding cDNA libraries were produced and qualified using an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR System. Primary raw reads produced by HiSeq 4000 (Illumina, United States) were qualified and filtered to obtain clean reads. The Pearson correlation coefficients were based on all gene expression levels. A heatmap analysis of gene expression levels were created based on the averaged fragments per kilobase of exon per million fragments mapped (FPKM) values of genes. Genes with fold change ≥2 and adjusted P values ≤ 0.001 were considered as the differentially expressed genes (DEGs). Annotation analysis of Gene Ontology (GO) was performed to determine the on-going biological process. The KEGG database was used to perform pathway analysis of DEGs.
Statistical Analysis
All statistical differences were performed on SPSS 23.0 by one-way ANOVA test among comparisons groups. Data are presented as mean ± standard deviation (SD). Differences were considered as significant at P < 0.05.