Figure 6.

Progressive astrogliosis and increased 3-NT labeling in ventral horn of VCP^R155H/+ mice. (a) Representative images of GFAP labeled slices. Lumbar sections were immunostained for GFAP and the ventral horns photographed under fluorescence (200 × ). Note the marked increase in reactive astrocytes in VCP^R155H/+ mice at 26 months (iv). Low magnification images of the hemi-spinal cord show the greater degree of astrogliosis in the ventral horn (VH) compared with the dorsal horn (DH) of the VCP^R155H/+ mice (vi). Bar=50 μm (high power); 400 μm (low power). (b) Representative images of 3-NT labeled slices. Lumber slices from 22-month-old WT and VCP^R155H/+ mice were immunostained for 3-NT, and photographed under immunofluorescence. Inserts show pseudocolor (8 bit) representation of fluorescence, in order to highlight the gradient in labeling intensity. Note the moderate labeling of WT MNs, but the markedly increased intensity of labeling not only in MNs of the VCP^R155H/+ mice, but also in the neuropil surrounding and between MNs. (Dotted lines show representative regions marked for one MN, as was used for fluorescence quantification, below). Note that the region of increased 3-NT labeling between ventral horn MNs in the VCP^R155H/+ mice may correspond generally to the region of increased ventral horn astrogliosis (see a, panels iv and vi above) in these animals. Bar=50 μm. (c): Quantification of GFAP and 3-NT labeling. (i) GFAP immunoreactive astrocytes were counted from lumbar ventral horns of WT and VCP^R155H/+ mice of the indicated ages. Each data point reflects counts from 3–5 animals; for each animal, all GFAP labeled astrocytes were counted from 200 × ventral horn microscope fields from five sections. Note the marked age-dependent increase in numbers of reactive astrocytes in the VCP^R155H/+ animals. * indicates difference from WT animals at the same age by two-tailed t-test (P<0.025). (ii) 3-NT labeling intensity was assessed in MNs, and in 10 μm wide zones surrounding MNs, in WT and VCP^R155H/+ mice as indicated. Each data point represents all MNs (>75) present in 5–10 ventral horn micrographs from each of 2–5 animals. Note the age-dependent increase in 3-NT labeling in WT mice, and the greater increase in VCP^R155H/+ mice, both within the MNs (MN) and in the neuropil surrounding them (surrounding). * indicates difference from WT animals at the same age by two-tailed t-test (P<0.001); ^#indicates difference from 18-month-old VCP^R155H/+ mice (P<0.001)