a-d, Representative transverse lumbar spinal cord sections stained with NeuN (green) and B8H10 (magenta) antibody in mice that were (a) 489 ± 6 days old wild-type nontransgenic, (b) 348 ± 2 days old SOD1G37R, (c,d) 404 ± 14 days old, end stage SOD1G37R injected subpially with (c) AAV9-scrambled virus or (d) AAV9-shRNA-SOD1 and then assayed 90 days later at ages between 398 and 465 days. (b) α-motoneurons and early accumulation of aggregates of mutant SOD1 are seen in 348 ± 2 day old SOD1G37R mice, while those neurons and NeuN staining intensity are (c) nearly completely lost by end-stage accompanied by marked accumulation of mutant SOD1 in intermediate zone and in ventral horn. d, Remaining α-motoneurons in AAV9-shRNA-SOD1-treated SOD1G37R mice and potent suppression of aggregates of misfolded SOD1 in the ventral horn. e-h, Quantitative analysis of α-motoneuron survival, NeuN staining intensity, and misfolded SOD1 protein accumulation. Each dot represents an individual animal; at least 4 sections per animal were used. i-t, Representative transverse lumbar spinal cord sections stained with NeuN (green), GFAP (magenta), Iba1 (green) or vimentin (blue) antibodies in (i) 348 ± 2 day old wild-type nontransgenic, (j) 348 ± 2 day old SOD1G37R, and (k,l) SOD1G37R subpially injected at ~348 days of age with (k) AAV9-scrambled virus and harvested at 404 ± 14 days of age (end-stage) or (l) AAV9-shRNA-SOD1 and subsequently aged to 398-465 days (~90 days post-treatment). u-w, Quantitation of GFAP, Iba1, and vimentin in each genotype/experimental group. Each dot represents an individual animal; at least 4 sections per animal were used. Experimental animals used: SOD1G37R (348 ± 2 days of age, n = 3); Sham-operated (or injected with scramble-AAV9) SOD1G37R (n = 4); AAV9-shRNA-SOD1-treated SOD1G37R (n = 4); and wild-type nontransgenic (n = 4) mice. Data are mean ± S.E.M. Statistical significance was determined with (e) two tail unpaired t-test with Welch’s correction (P = 0.0150, t = 4.680, df = 3.288) and with (f-h, u-w) one-way ANOVA followed by Bonferroni post hoc test (f: ANOVA, P = 0.0061, F = 10.29; g: ANOVA, P = 0.0009, F = 11.71; h: ANOVA, P = 0.0394, F = 4.980; u: ANOVA, P = 0.0060, F = 7.223; v: ANOVA, P < 0.0001, F = 20.73; w: ANOVA, P = 0.0004, F = 14.30). P values are shown between the indicated groups. DH, dorsal horn; VH, ventral horn.