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)].
Free full text: Click here
