, 2008). Images of GAP-43 immunohistochemistry were also obtained from the injured part of the spinal cord. After standardized background corrections, a mask of each spinal cord section Ixazomib price image was created using an auto-threshold tool from Image J, hence avoiding vacuolization and interrupted tissue integrity. Thereafter, optical densities (OD) of the images were measured from whole injury regions within the area of interest, i.e., the mask itself. OD was calculated using the following formula: OD=−log[(INT(x,y)–BL)]/(INC–BL)]OD=−logINTx,y–BL/INC–BL] Where “OD” is the optical density; “INT (x,y)”
or intensity is the intensity at pixel (x,y), “BL” or black is the intensity generated when no light goes through the material and “INC” is the intensity of the incidental light. Around 6–16 images were analyzed from each rat and 6 animals were analyzed per group. 5-HT and CGRP fiber populations were also identified using a Nikon Microscope Optiphot-2 (Japan) with a green Selleckchem 5 FU excitation filter for the Alexa 555 signal (G-2A, Excitation—510/560). Double-labeling with GFAP antibody was used to delineate the fibrous scar borders and the signal for Alexa 488 was detected using a blue excitation filter (B-2A, Excitation—450/490).
Pictures with resolution of 254 × 254 DPI, were taken at magnification of 200× using a CMOS camera (518 CU, Micrometrics) and analyzed with Image J Software 1.42q. The total area occupied by 5-HT or CGRP axons was determined separately in the rostral, lesion and caudal regions, throughout the width of the tissue sections. To assess 5-HT fibers, pictures were taken of the rostral stump (in the region with abundant visible astrocytes), the central part of the lesion (approximately) and near the scar selleckchem border of the caudal stump. Analogously, images of CGRP fibers were taken of the caudal stump (in the region with abundant
visible astrocytes), in the central part of the lesion (approximately) and near the scar border of the rostral stump. All images (on average, 19 pictures per spinal cord region in each animal, 6 animals per group) were turned into binary (black and white) and a constant threshold value was used to measure the total percentage area (%) occupied by axon fibers. Data were expressed as means ± SEM. Open field locomotor scores were analyzed between groups using analysis of variance (ANOVA) with time as the repeated measure. When there were statistically significant F values (p ≤ 0.05), Bonferroni’s post hoc tests were conducted by comparing OLP transplantation with the corresponding RLP group. Regarding assessment of spinal tissue sparing and regional optical densitometry, all groups were analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. The Kruskal–Wallis test was used for axon profile data (5-HT or CGRP). Values were run on SPSS 11.5 (Statistical Package for the Social Sciences, Inc., USA).