Fluoro gold

Five days before killing, rats were deeply anesthetized. Then, 2 μl of 3% of FluoroGold (Sigma-Aldrich, St. Louis, MO, USA) was injected into the superior colliculus on each side, as previously reported [37 (link)]; notably, FluoroGold is taken up by RGC axon terminals and bilaterally transported in a retrograde manner to the cell somata. At the time of killing, the rats’ eyeballs were enucleated and directly fixed in 4% paraformaldehyde for 1.5 h at room temperature. The retinas were then carefully dissected and prepared as flatmounts. To quantify the densities of labeled RGCs, each retina was divided into four quadrants. Sixteen microscopic fields of each retina were counted: two from the central region (1.5 mm from the optic disc) and two from the peripheral region retina (3 mm from the optic disc) for each quadrant. RGC densities (cells/mm²) were grouped according to retinal eccentricity (central and peripheral) and expressed as means ± standard errors of the mean (means ± SEMs).

Under anesthesia, a 2-cm incision was created in the animal’s scalp and two small holes were drilled into the skull as illustrated [8 (link)]. Next, injections of 10-μl of 5% fluorogold (Sigma-Aldrich) were carried out using a micropipette at 3.8, 4.0, and 4.2 mm below the surface. The fluorogold was injected 3 days before the animals were sacrificed. Retrieval, fixation, dissection and processing of retinal samples were performed as described previously [8 (link)]. The RGC density was calculated as the ratio of the total number of RGCs divided by the total area of the retinal sample [8 (link)].

The anesthetic rat is placed onto the stereotaxic frame, and a 2 cm deep cut is created on the rat's scalp and 2 small holes drilled into its skull [7 (link)]. Afterwards, a micropipette was used to inject 10 μl of 5% fluorogold (Sigma-Aldrich) at 3.8, 4.0, and 4.2 mm below its skull. Of note, this step was carried out 3 days prior euthanasia of rat. Then, the retina was dissected and collected, as described by Chao et al. [7 (link)]. Finally, the RGC count was determined through the equation of total RGC number over total area of retina used [7 (link)].

Following anesthetics administration, the scalp of the rat was incised with a 2 cm cut along with 2 small holes drilled into the skull on both sides behind the bregma and 1.5 mm lateral to the midline [12 (link)]. Through a micropipette, 2 μL of 5% fluorogold (Sigma-Aldrich, St. Louis, MO, USA) was injected at depths of 3.8, 4.0, and 4.2 mm beneath the cranium. Three days following RGCs’ retrograde immunolabeling, pressure-induced retinal ischemia was performed on the animals’ right eyes, with the adjacent eyes being the untreated ones. The twelve o’clock location of the rat’s eye was highlighted using a suture to carry out orientation; then, the eyes were removed from their sockets. Following this, the retina was carefully retrieved, fixated, dissected, and processed [11 (link)]. Then, the retina was placed onto a slide and divided into 4 quadrants. Then, each retinal quadrant was split into 3 zones (i.e., central, middle, and peripheral) at distances of approximately 1, 2, and 3 mm from the optic disc, respectively [20 (link)]. Inside the aforementioned zones, and with the assistance of a microscope, 6 fields of 0.430 × 0.285 mm² each along the medial line were tallied. In this case, a total of 72 fields of the entire retina was tallied [20 (link)]. The average RGC density was evaluated by obtaining the ratio of the total RGC number against the total retinal area [20 (link)].

Rats from the different groups were anesthetized and injected with the fluorescent tracer 3% FluoroGold (2 μL; Sigma) diluted in saline into the bilateral superior colliculi (6.0 mm posterior and 2.0 mm lateral to bregma and 4–4.5 mm deep) via a microsyringe as previously described (Zhou et al., 2017a (link)) 5 days before sacrifice. Five days after FluoroGold injection, the retinas were isolated, dissected, divided into four quadrants, and flat-mounted on glass slides with the GCL facing up. Each quadrant was further divided into central (1.5 mm from the optic disk) and peripheral regions (3 mm from the optic disk). Two fields in each region were randomly selected and counted. Totally 16 microscopic fields in each retina were counted and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems) at a final magnification of 3,200 (scale bar, 50 μm). For each image taken, the number of labeled cells was counted two times by an investigator who was blinded to the grouping, and the average was used as the final result.

Seven days after EVC, anesthetized rats were injected with the fluorescent tracer 3% FluoroGold (2 µL; Sigma) diluted in saline via microinjection into the bilateral superior colliculi (6.0 mm posterior and 2.0 mm lateral to bregma and 4–4.5 mm deep) as previously described^{60 (link)}. Twenty-one days after EVC (14 days after FluoroGold injection), the retinas were dissected, divided into four quadrants, and flat-mounted on glass slides with the GCL facing up. Twenty images per retina (two from the central and two from the peripheral retina for each quadrant) were captured using a laser scanning confocal microscope (TCS SP8; Leica Microsystems) at a magnification of 20×. The cells were counted by an investigator who was blinded to the study treatments.

Cocaine (15 mg/ml saline; Macfarlan Smith Ltd., UK), Fluorogold (20 mg/ml saline; a retrograde tracer; FG; Fluorochrome, USA) and ibotenic acid (5 mg/ml saline; Sigma, USA) were used.