Rounded to 1 cm platinum needle electrodes inserted subcutaneously inside the cheek and tail, respectively. We stored acquired responses on a industrial ERG system (UTAS 3000, LKC Technologies, Gaithersburg, MD), differentially amplified at 1500 Hz having a recording length of 250 ms as well as a digitization rate of 1.92 MHz. Right after testing, yohimbine (2.1 mg/kg) was administered to the rats to reverse effects of xylazine and prevent corneal ulcers (Turner and Albassam, 2005). ERG information have been analyzed offline. Amplitudes have been manually measured for a- and b-waves, PII and oscillatory potentials (OP1-4), as previously described (Mocko et al., 2011). Darkadapted a-waves, which originates in the rod photoreceptors (Hood and Birch, 1990), had been measured from the baseline to the trough with the first adverse wave. B-waves, which originate in the depolarizing bipolar cells (Stockton and Slaughter, 1989), were measured from the trough on the a-wave to the peak from the waveform, or when the a-wave was not present, from baseline towards the peak with the waveform. OPs were digitally filtered making use of the ERG system application (7500 Hz; EM Version 8.1.two, 2008; LKC), and manually measured from trough to peak. Scotopic PII was also filtered and measured, as previously described (Mocko et al., 2011). Baseline ERG testing was carried out prior to commencement of treatment, and after that at 4 weeks, 8 weeks, 12 weeks, and 17 weeks for the duration of treatment.Author Angiopoietin Like 2 Proteins Biological Activity Manuscript Author Manuscript Author Manuscript Author ManuscriptExp Eye Res. Author manuscript; obtainable in PMC 2017 August 01.Hanif et al.Page2.6. Angiopoietin-Like 7 Proteins Recombinant Proteins Retinal structure analysisAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptAfter 20 weeks of stimulation therapy, rats had been euthanized, and eyes had been enucleated and marked superiorly for orientation. Eyes have been immersion-fixed in 4 paraformaldehyde for 30 min, then rinsed in 0.1 M phosphate buffer. Immediately after dissection to get rid of the lens and cornea, the posterior eye cup was dehydrated by way of a graded alcohol series and embedded in plastic resin (Embed 812/DER 736, Electron Microscopy Science, Inc, Hatfield, PA). Posterior hemispheres were sectioned within the superior to inferior plane (0.five m), working with an ultramicrotome (Reichert Ultracut, Leica Inc., Buffalo Grove, IL) using a histo-diamond knife to bisect the optic disc. Retinal sections were then stained with 1 aqueous to-luidine blue (Sigma; St. Louis MO), and imaged utilizing a phase contrast microscope (Leica DMLB, Leica INc., Buffalo Grove, IL). two.7. Measurement of retinal thickness and nuclei Thicknesses of retinal layers (outer segments + inner segments, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, retinal ganglion cell layer) have been measured for treated and non-treated eyes of WES (n = 4) and Sham (n = three) rats from 20magnification photos of retinal cross sections obtained via a phase contrast microscope (Leica DMLB, Leica Inc., Buffalo Grove, IL) applying an image analysis system (Image-Pro Plus five.0; Media Cybernetics, Warrendale, PA). Retinal regions spanning two.five mm superiorly and inferiorly from the optic nerve head were measured. Every 2.5 mm region was subdivided into 5 0.five mm sections and designated ” F” or ” S” 1 for inferior and superior, respectively. Thicknesses for each and every retinal layer had been compared between Sham and WES groups at every location examined. On top of that, thicknesses across all places examined for each and every retinal layer have been averaged inside experimental group.