Rd10 mice and FA treatment
Rd10 mice (The Jackson Laboratory) were housed in a specific pathogen-free facility in the Animal Laboratories of Yantai Yuhuangding Hospital. All animal studies adhered to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. It has been reported that in rd10 mice, retinal abnormalities begin on postnatal day (P)7[14, 15] and that photoreceptor death peaks around P25 and is nearly complete by P35[16, 17]. For treatment, rd10 animals were intragastrically administered 25 mg/kg, 50 mg/kg or 100 mg/kg FA (1270311, Sigma) every day from P4 to P24. Before intragastric administration, the mice were anaesthetized with isoflurane in animal anaesthesia ventilator system (Matrx, America); the mice experienced no pain and awoke quickly. The animals were sacrificed via sodium pentobarbital injection (200 mg/kg; intraperitoneal) on P25, and their eyeballs were enucleated for further investigation.
Electroretinogram (ERG) recordings
Before ERG recordings, the mice were adapted to the dark overnight. The mice were anaesthetized by intraperitoneal pentobarbital sodium(50 mg/kg) prior to pupil dilation using 1% tropicamide. ERGs were recorded with a Ganzfeld stimulator (Roland Consult, Germany) that generated and controlled light stimuli. Scotopic ERGs were recorded following a 1.3-ms single flash with an intensity of-1.52, -0.52, 0.48 or 1.0 log cd s/m2. A total of 5 responses per intensity were averaged for each flash stimulus. Intraperitoneal sodium pentobarbital (200 mg/kg) was then used to euthanize the mice. The amplitudes of the major ERG components (a- and b-waves) were measured (RETI System software) using automated and manual methods.
Hematoxylin and eosin (HE) staining
Eyeballs were fixed in formalin overnight prior to being embedded in and sectioned into 3-µm slices. Before staining, xylene was used for deparaffinization, and the sections were rehydrated in an ethanol gradient prior to being washed in PBS. Then, the sections were stained with HE. A microscope (Leica DM4000, Germany) was used to analyse retinal histology and count nuclei in the outer nuclear layer (ONL).
Cell culture and FA treatment
BV2 murine microglial cells (purchased from cell bank, Kunming Institute of Zoology) were cultured as previously described. In brief, the cells were maintained in DMEM (high-glucose) containing 10% FBS and penicillin/streptomycin. The microglia were activated by lipopolysaccharide (LPS, 50 ng/ml, L6529, Sigma) for 1 h and then treated with FA (0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/ml, 1 mg/ml, 2 mg/ml or 5mg/ml, 6529, Sigma). After 24 h, the cells were collected for downstream analyses.
For retinal wholemounts, eyes were immersed in 4% paraformaldehyde (PFA) fixative for 30 minutes, and the retinal cups were separated carefully from the eyeballs. Both retinal wholemounts and cell slides were stained using primary and secondary antibodies, washed extensively and flat-mounted. The following primary antibodies were used: anti-iba1 (019-19471, Wako Chemicals) and anti-iNOS (sc-7271, Santa Cruz). The following secondary antibodies were used: Alexa Fluor 488-conjugated donkey anti-rabbit IgG H&L and Alexa Fluor 555-conjugated donkey anti-goat IgG H&L. The retinal wholemounts and cell slides were visualized via confocal microscopy (Carl Zeiss LSM710, Germany).
RNA was isolated from retinae using TRIzol Reagent (Invitrogen), and a Bioanalyzer 2100(Agilent) was used to gauge the quality of the resultant nucleic acid. RNA preparation, library construction, and sequencing were conducted using a BGISEQ-500 instrument at the Beijing Genomics Institute (BGI, Shenzhen, China).
Total RNA was isolated from the retina of rd10 using the RNAiso Plus kit (TAKARA Bio Inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio Inc, Japan) was then used to prepare cDNA following the instructions. Rea-time PCR was performed in a 20-μL reaction system, containing 10 μL of 2×SYBR Premix Ex Taq, 2 μL of cDNA, and 10 μmol/L primer pairs. The thermocycler settings were 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 34 s.
Western blot analysis
RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quantification. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8(sc-365042, SANTA) and β-actin (ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Each experiment, including the immunostaining, qPCR and western blotting experiments, was replicated 3 times. All quantitative data was analysed using 2-tailed Student’s t test or one-way ANOVA by SPSS 21.0. The data are the means ± standard errors of the mean (SEMs). P<0.05 indicated significance.