The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified. Results The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the BuChE-IN-TM-10 nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified. Results The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) Rabbit Polyclonal to CATD (L chain, Cleaved-Gly65) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) BuChE-IN-TM-10 the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes. Conclusions Several quantitative and qualitative indications of microglia activation are recognized both in the contralateral and OHT eyes. Such activation prolonged beyond the GCL, including all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology. access to food and water. Light intensity within the cages ranged from 9 to 24 lux. All surgical procedures were performed under general anesthesia induced with an intraperitoneal (ip) injection of a mixture of ketamine (75 mg/kg, Ketolar?, Parke-Davies, Barcelona, Spain) and xylazine (10 mg/kg, Rompn?, Bayer, Barcelona, Spain). During recovery from anesthesia, the mice were placed in their cages and an ointment comprising tobramycin (Tobrex?; Alcon, Barcelona, Spain) was applied to the cornea to prevent corneal desiccation and illness. Additional actions were taken to minimize distress and pain after surgery. The animals were killed with an ip overdose of pentobarbital (Dolethal Vetoquinol?, Especialidades Veterinarias, Alcobendas, Madrid, Spain). Experimental organizations Two groups of mice were considered for study: an age-matched control (na?ve, n = 12) and a lasered group (n = 12) that was killed two weeks after lasering. Induction of ocular hypertension and IOP measurements To induce OHT, the left eyes of anesthetized mice were treated in one session with a series of diode laser (Viridis Ophthalmic Photocoagulator-532 nm, Quantel Medical, Clermont-Ferrand, France) burns up, following previously explained methods [43,44]. Briefly, the laser beam was directly delivered without any lenses, aimed at the limbal and episcleral veins. The spot size, duration, and power were between 50 and 100 m, 0.5 seconds, and 0.3 W, respectively. Each attention received between 55 and 76 burns up. With the mice under deep anesthesia, the IOP was measured in both eyes having a rebound tonometer (Tono-Lab, Tiolat, Helsinki, Finland) [43,45-47] prior to and 24 hours, 48 hours, and 1 week after laser treatment for the lasered group, and before becoming killed for the na?ve group. At each time point, six consecutive readings were taken for each attention and averaged. To avoid fluctuations of the IOP due to the circadian rhythm in albino Swiss mice [48], or due to the rise of the IOP itself [49], we tested the IOP consistently around the same time, preferentially in the morning and directly after deep anesthesia in all animals (lasered BuChE-IN-TM-10 group and na?ve). Immunohistochemistry The mice were deeply anesthetized, perfused transcardially through the ascending aorta first with saline and then with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.2 to 7.4). The orientation of each attention was carefully managed having a suture placed on the superior pole immediately after deep anesthesia and before perfusion fixation [43]. Moreover, upon dissection of the eye, the insertion of the rectus muscle mass and the nose caruncle were used as additional landmarks [50]. The eyes were post-fixed for two hours in the same fixative and kept in sterile 0.1 M PB. Retinas were then dissected and processed as retinal whole-mounts [51]. For the analysis of the microglia human population in the mice retina and the manifestation of MHC class II.