Figure 12. Bumetanide prevents upregulation of p75NTR and cell death induction after in vivo ventral root axotomy. a, Schematic representation of the injury. Axotomy of spinal motoneurons was performed at the ventral root between the spinal cord and the junction with the dorsal root (for details, see Materials and Methods). Arrow, Lesion site. b, Representative images from the sections of the spinal cords of unlesioned, lesioned (by ventral root axotomy), and lesioned bumetanide-treated rats at the level of the injury stained immunohistochemically for p75NTR. Scale bar, 30 μm. c, Quantification of p75NTR-positive neurons from the similar sections as in b. Total amount of p75NTR-positive neurons from four sections of the spinal cord and total amount of neurons (NeuN-defined) from four sections consecutive to each p75NTR-stained section was counted by blind stereological counting from ventral horns (for selection criteria, see Materials and Methods) and the p75NTR/NeuN ratio for each animal quantified. Data are presented as percentage of unlesioned control ratio. n = 3 unlesioned, 2 lesioned, 3 lesioned bumetanide-treated rats. ***p < 0.001, one-way ANOVA and Bonferroni's post hoc analyses. d, Representative images of sections as in b stained immunohistochemically for KCC2. Scale bar, 15 μm. KCC2 immunostaining intensity, measured from images similar as in d, is reduced in neurons in the ventral horn of the lesioned animals. n = 18 sections/5 animals; **p = 0.004, one-way ANOVA and Bonferroni's post hoc analyses. e, Quantification of cleaved caspase-3-positive neurons performed as in c from similar sections as in f. NeuN was detected from the same sections as cleaved caspase-3; n = 3 unlesioned, 2 lesioned, 3 lesioned bumetanide-treated rats; **p = 0.012, one-way ANOVA and Bonferroni's post hoc analyses. f, Representative images from the sections of the spinal cords of unlesioned, lesioned, and lesioned bumetanide-treated rats at the level of the injury stained immunohistochemically for cleaved caspase-3 and NeuN. Scale bar, 30 μm. Error bars indicate SEM.