Representative confocal microscopy images of IB4 and GAD65 immunoreactivity in the dorsal horn of sham (A-D) and neuropathic (E-H) rats ipsilateral to injury at 5, 7, 10 and 14 days after surgery. Capital letters and lowercase letters denote images taken with 20X and 63X objectives, respectively. Lowercase italic letters show representative images of quantified regions in both lamina I and lamina II, taken with a 63X objective and a zoom factor of 2. IB4-labeling is shown in green and GAD65 in red. Note that for each time point, the image with the largest loss of IB4 staining was chosen for illustration and this IB4 lesion is shown by a yellow rectangle. The smaller regions used for quantification were taken from within these yellow rectangles. In sham animals, IB4 labelling was primarily restricted to a thin band in lamina II, whereas GAD65 labelling was observed throughout the dorsal horn. GAD65-IR boutons were readily observed among the IB4-labelled terminals. Starting at 5 days after surgery (E) but more clearly visible from day 7 (F) there was a region of depletion of IB4 staining that was not observed in sham animals at any time point (A-D). High magnification images of neuropathic rats show a clear lack of IB4 staining in lamina II from days 7 (f and f) to 14 (h and h).GAD65-IR boutons are reduced in cuff animals ( e-h ) compared to sham animals ( a-d ).