Animal Handling and Groups

Adult female albino Sprague–Dawley rats (n = 224) weighing approximately 180 to 220 g were used. The rats were bred and maintained in the Experimental Animal Facility of the University of Murcia under controlled light (12-hour light-dark cycles with light intensity within the cages ranging from 5 to 30 lux) and temperature conditions (23°C–24°C), and with access to food and water ad libitum.

The animals were treated according to the current European and national regulations and, specifically, according to Directive 86/609/EEC, 2010/63/EU on the protection of animals used for scientific purposes, the R.D.1201/2005 on the protection of animals used for experimental and other scientific purposes, the Law 32/2007 for the care of animals, in their exploitation, transport, experimentation, and slaughter, and the ARVO guidelines for the use of animals in ophthalmic and visual system experimentation.

The IVIs (see IVIs) were performed under general anesthesia, for which a mixture of ketamine (70 mg/kg, Ketolar; Parke-Davies, SL, Barcelona, Spain) and xylazine (10 mg/kg, Rompun; Bayer) was injected intraperitoneally. After the IVIs, the rats were placed in their cages, and tobramycin ointment (Tobrex, Alcon S.A., Barcelona, Spain) was applied over the cornea to prevent corneal desiccation.

The animals were divided into different experimental groups. There were twelve animals in the naïve group that did not receive any treatment, twenty-four animals in the PBS group and forty animals in each of the groups that received IVI of the anti-VEGF factors. Half of the animals in each group were processed 7 days or 1 month after the IVI (Fig. 1; see IVIs). At each survival period, one-half of the animals were immunolabeled with antibodies directed to glial cells and half with antibodies directed to ganglion cells (see below, Immunohistofluorescence).

Experimental design. Diagram depicting the methods used: groups, substances injected, concentrations, processing times after the intravitreal injection (IVI) (days), and number of animals analyzed at each survival time (n).