To ensure continuous ocular hypertension within the eye IOP was measured using a TonoLab jump tonometer at 5 min before IOP elevation, then every 15 min for the first 120 min of IOP Foretinib GSK1363089 xl880 elevation, and every 60 min for the remaining period of elevation. The elevated IOP was preserved for the suggested length and up to 7 h. Through the procedure, the mean arterial blood pressure was monitored and reported by a Powerlab/8SP data acquisition system. A month after ocular hypertension, the animals were euthanized. The optic nerve of every eye was isolated and fixed immediately in 2 and 2% paraformaldehyde. Five minutes glutaraldehyde in a 0. 1 M cacodylate buffer immediately, placed in 0 and in 10 percent OsO4. 25 percent uranyl acetate for 2 h each, dehydrated with a number of acetones, and then embedded in epoxy resin. Next, 1 um sections were cut, positioned on glass slides, and stained with 1000 toluidine blue. Stained sections were photographed at 10 magnification using a camera and printed therefore the complete nerve was obvious in the field of view. The intensity of ON damage in each part was alone graded by three masked Skin infection investigators having an Optic Nerve Damage Score, as follows, Grade 1 regular, Grade 2 up to 200-mile dead and darkly stained axons with initial gliosis, Grade 3 up to 50% dead axons with gentle gliosis, Grade 4 up to 800-682 dead axons with notable gliosis, and Grade 5 nearly hundreds of dead axons with severe gliosis. The mean ONDS of every ON determined by the three investigators was evaluated and noted using statistical analysis. Eyes of euthanized rat were fixed in four to five paraformaldehyde over night and embedded in paraffin. Next, 4 um thick sections were stained with hematoxylin and eosin and cut throughout the optic papilla. For quantitative analyses, sections perpendicular to the retinal area were examined under a stereomicroscope. Thicknesses of five retinal layers were calculated in a masked fashion at three adjacent regions within map kinase inhibitor 0. 5 mm of the ON in the poor peripapillary region and the mean values were reported. The five retinal layers are, 1) general retinal thickness from the outer limiting membrane to the inner limiting membrane, 2) the outer nuclear layer, 3) the outer plexiform layer, 4) the inner nuclear layer, and 5) inner retinal thickness from the inner plexiform layer to the limiting membrane. Measurements were performed in the same topographic area of the retina to minmise local anatomic variations. Cell counts of the GCLs were performed manually across a length of 300 um in the same topographic area of the retina. One day before euthanasia, rats were anesthetized with a drink of xylazine and ketamine and their ONs were absolutely transected at about 2 mm behind the planet, without injuring the ophthalmic artery. Dextran tetramethylrhodamine crystals were employed at the cut end of the ON stump. Twenty-four hours later, eyes were enucleated and fixed in a 4% paraformaldehyde solution at 4 C for 120 min.