Animals and grouping
Ten-week-old female Sprague–Dawley (SD) rats (200–220 g), specific pathogen free (SPF), available from the animal center of Central South University, were used for experiments in the present study. All animals were housed in cages at the Central South University Animal Department (Changsha, China) under controlled temperature (21 ± 1 °C), humidity (55 ± 5%), and a 12 h light/dark circle. Food and water were available ad libitum. The physical condition of rats was monitored each day during the experiment. The rats with cataracts, or ocular fundus hemorrhage or infection were excluded.
All rats were randomly allocated into seven groups: (i) Control group; (ii) I/R group, rats were treated with anterior chamber compression; (iii) I/R + fluorinated citric acid (FC) group, rats were intravitreally injected with 2 μL FC after the induction of RI/R; (iv) I/R + TSP2 shRNA group, rats were intravitreally injected of 2 μL of TSP2 shRNA adeno-associated virus (AAV) liquid 28 d before RI/R treatment; (v) I/R + scramble RNA group, rats were intravitreally injected of 2 μL of scramble RNA AAV liquid 28 d before RI/R treatment; (vi) I/R + gabapentin (GBP) group, rats were intraperitoneally injected with GBP (100 mg/kg) before and 5 h after RI/R treatment; (vii) I/R + PB group, rats were intravitreally/intraperitoneally injected the same dose of 0.01 mol/L PB.
All animals were treated according to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. All animal experiments were reviewed and approved by the Medical Ethics Committee at Xiangya Hospital of Central South University (Approval ID: 2019030519).
RI/R and administration of FC/GBP
All rats in RI/R groups were treated as described previously [21]. Briefly, a 30-gauge infusion needle connected to the installation instrument with sterile saline was inserted into the anterior chambers of the eyes of the animals after anesthetization (a 1:1 mixture solution (0.4 mL/100 g) of 25% Urethane and 10% chloral hydrate). The intraocular pressure was slowly elevated to 14.63 kPa (110 mmHg), maintained for 60 min, and then gradually lowered to normal pressure. The administration of FC was performed as described previously [22]: After the induction of RI/R, rats were intravitreally injected with 2 μL FC (Sigma Aldrich, USA, 16 nmol/L) or vehicle (0.01 mol/L PB). The needle of the micro-syringe was kept in the vitreous chamber for 2 min and then slowly removed. The administration of GBP was referred to the procedures as a previous description: rats were intraperitoneally injected with GBP (100 mg/kg) or the same volume of vehicle (0.01 mol/L PB) before and 5 h after RI/R.
TSP2 shRNA-AAV transduction
TSP2 shRNA-AAV, Scramble RNA-AAV and AAV-GFP were obtained from HANBIO (Shang Hai, China) as follows: Gene ID: NM 001,169,138.1, the length of CDS: 3519 bp, sources: Rat, interfere sequence: 5′GCAGAUAUCUGCUUCUAA dTdT3′, serotype: II, titer: 1 × 1012. Intravitreal injection was treated as described previously [23], Briefly, the rats were anesthetized with a 1:1 mixture solution (0.4 mL/100 g) of 25% Urethane and 10% chloral hydrate and placed on a heating pad that maintained their body temperature at 35–36 °C throughout the experiments. After anesthesia and pupil dilation with 1% atropine, intravitreal injection of 2 μL of TSP2 shRNA AAV liquid/Scramble RNA AAV liquid/ AAV-GFP liquid was made into one eye of each rat 1 mm behind the limbus, using a 33-gauge needle (Hamilton, Reno, NV, USA) under a surgical microscope. After injection, the rats were maintained normally for 28 days to allow sufficient retinal transduction before the subsequent experiments.
Tissue preparation
Rats were trans-cardiac perfused with saline and then 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) after being deeply anesthetized. For the morphological assay, eyeballs were enucleated, and the cornea, lens and vitreous body were removed. Some remaining eye cups were postfixed in 4% PF overnight, immersed (15% to 30% sucrose solutions) at 4 °C for cryoprotection, then were prepared into 14 μm thick cross-sections successively on slices. Some eye cups were postfixed in 4% PF for 1 h, retinae were dissected, cut into four petals in 24 well plates. For western blotting, retinae were dissected from deeply anesthetized rats and then weighed and quickly frozen on dry ice and stored at − 80 °C for further homogenization.
Immunofluorescence/Double Immunofluorescence
For immunofluorescence of phosphorylated neurofilament (pNF), retinal mounts were pre-incubated for 2 h in 5% donkey serum (Sigma, USA) at room temperature (RT), and then incubated with the anti-pNF antibody (1:200, Mo, BioLegend, USA, Catalog: 801602)/anti-GFAP antibody (1:1000, Mo, Sigma Aldrich, USA) at 4 °C overnight. After several rinses with phosphate Buffered Saline (PBS), mounts were reacted with 488-conjugated donkey anti-mouse secondary antibody (1:400, Jackson ImmunoResearch, USA). The sections were finally covered with a mounting medium containing 40,6-diamidino-2-phenylindole (DAPI, VEC- TOR, CA, USA). For immunofluorescence of GFAP and NeuN, retinal sections were pre-incubated for 1 h in 5% donkey serum (Sigma, USA) at room temperature (RT), and then incubated with anti-GFAP (1:1000, Mo, Sigma Aldrich, USA)/anti-NeuN (1:1000, Mo, Abcam, UK, ab104224) at 4 °C overnight. After several rinses with phosphate Buffered Saline (PBS), mounts were reacted with 488-conjugated donkey anti-mouse secondary antibody (1:400, Jackson ImmunoResearch, USA). The sections were finally covered with a mounting medium containing 40,6-diamidino-2-phenylindole (DAPI, VEC- TOR, CA, USA).
For double immunofluorescence of SYN and PKC-α/GFAP and TSP2, retinal sections were pre-incubated for 1 h in 5% donkey serum (Sigma, USA) at room temperature (RT), and then incubated with anti-SYN (1:500, Rb, Abcam, UK, ab32127) and anti-PKC-α (1:200, Mo, Santa Cruz, USA, sc-8393)/anti-GFAP (1:1000, Mo, Sigma Aldrich, USA) and anti-TSP2 (1:1000, Rb, Abcam, ab84469, Cambridge, UK) at 4 °C overnight. After several rinses with phosphate Buffered Saline (PBS), mounts were reacted with 488-conjugated donkey anti-rabbit secondary antibody (1:400, Jackson ImmunoResearch, USA) and 594-conjugated donkey anti-mouse secondary antibody (1:400, Jackson ImmunoResearch, USA). The sections were finally covered with a mounting medium containing 40,6-diamidino-2-phenylindole (DAPI, VECTOR, CA, USA).
Transmission electron microscopy
Electron microscopy was conducted using retinal electron microscopic sections from controls and post-surgery (three rats per group). Retina tissues were cut into 1 mm3 cube with a vibratome and rinsed with clean saline. Tissues were fixed in 2.5% glutarol solution for 1 h at room temperature or 3 h at 4 °C and then with 1% osmium tetroxide in 0.1 mmol/L cacodylate buffer for 2 h. After rinsing with DDW, sections were treated with 1% aqueous uranyl acetate overnight, dehydrated in ethanol solutions of increasing concentration, up to 100%, followed by dry acetone, and then embedded in durcupan ACM. Ultrathin Sects. (0.1 μm) were cut and mounted on Formvar-coated slot grids, stained with 3% lead citrate, and examined with a HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).
Western Blotting
As previously detailed [21], retinae were homogenized by sonication on ice in a digestion buffer containing a cocktail of protease inhibitors (Sigma, MO, USA). Sonication-digested homogenates were treated with centrifugation, protein concentration determination and degeneration, respectively. Polypeptides were loaded in each lane of 4–20% linear gradient Tris–HCl ready gel (Bio-Rad, CA, USA) and the gel was run in electrophoresis at the voltage of 100 mV. And then, the polypeptides in the gel were electrotransferred to Trans-Blot pure nitrocellulose membrane (Bio-Rad, CA, USA). Following this, they were blocked by 5% non-fat milk for 1 h, the membranes were incubated with anti-GFAP(1:1000, Mo, Sigma Aldrich, USA)/anti-TSP2(1:1000, Rb, Abcam, UK)/anti-α2δ1(1:400, Mo, Abcam, UK)/anti-SYN(1:2000, Rb, Abcam, UK, ab32127)/anti-synapsin 1(1:1000, Rb, Abcam, UK, ab254349)/hormer 1b/1c (1:1000, Mo, Santa Cruz, USA, sc-55463)/ anti-mouse GAPDH (1:2000, Mo, Beyotime, China, AF0006) antibodies overnight and then placed in HRP-conjugated secondary antibodies (1:20,000, Bio-Rad, CA, USA) for 2 h to develop.
qRT–PCR
TSP2 mRNA expression was confirmed using quantitative real-time PCR (qRT–PCR). The total RNA was reverse-transcribed to cDNA using GoScriptTM Reverse Transcription System (A5001, Promega Corporation, CA, USA) according to the manufacturer’s protocol. qRT–PCR was performed using a real-time fluorescence quantitative PCR instrument (ABI 7500, USA). An amplification mixture was prepared using Hieff® qPCR SYBR Green Master Mix (11202ES03, Yeasen Corporation, China) according to the manufacturer's protocol, which contained 10.0 μL SYBR Green Master Mix, 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL cDNA, and 20 μL ddH2O. The results were normalized to GAPDH expression. The primers (synthesized by Tsingke Biological Technology Co., Ltd., Beijing, China) are listed as follows: The primers used were as follows: Tsp2: forward, GTAGGTTTTGACGAGTTTGG, reverse, TCCACATCACCACATAGAAG; GAPDH: forward, CGTCCCGTAGACAAAATGGTGAA, reverse, GCCGTGAGTGGAGTCATACTGGAACA. The relative expression levels of mRNAs were depicted as 2−△△Ct.
f-VEP
The rats were anesthetized with a 1:1 mixture solution (0.4 mL/100 g) of 25% Urethane and 10% chloral hydrate and placed on a heating pad that maintained their body temperature at 35–36 °C throughout the experiments. pupil dilation with 1% atropine. The reference electrode is fixed at the buccal masseter muscle on the same side as the test eyeball, and the recording electrode is fixed at 1–2 mm of the coronal suture of the skull and 2–3 mm of the sagittal suture. An earth clip, serving as a ground electrode, was placed along the tail. The test eye was exposed, while the opposite eyeball was shielded by an opaque eye shield. The visual stimulus was provided by Espion 2 visual electrophysiometer exposure device from Diagnosys (USA), backlight: 0 cd × s/m2 with white light: 600 cd/m2 × 5 ms, 1 Hz. Each f-VEP report was obtained from the average value of 64 visual stimuli, the VEP signal was expanded, and the passband was filtered between 1 and 100 Hz. Then, statistical analysis was performed on the test reports on the amplitude of N2–P2 and the latency of P2 wave.