FIGURE 2.

Axonal degeneration and neuronal loss in Ei24^flox/flox; nestin-Cre mice. A, Nissl staining of cortex and hippocampus of Ei24^flox/+; nestin-Cre and Ei24^flox/flox; nestin-Cre mice at 4 months of age. The arrow indicates an enlarged lateral ventricle. Bar, 200 μm. B, Nissl staining of the fifth layer of the cortex. The graph shows the number of large pyramidal cells per mm² in the indicated areas. Mean ± S.E. of three mice is shown. Bar, 50 μm. C, H&E staining of the hippocampal pyramidal cell layer. The number of pyramidal cells was quantified and divided by the length of the layer. Mean ± S.E. of three mice is shown. Bar, 50 μm. D, compared with control mice, Nissl staining of the cerebrum shows large numbers of vacuolated cells (arrows) in the cingulate cortex and internal capsule of Ei24^flox/flox; nestin-Cre mice. Bar, 50 μm. E, anti-CNPase staining show that vacuolated cells are CNPase-positive oligodendroglial cells in the cingulate cortex and internal capsule of Ei24^flox/flox; nestin-Cre mice. Bar, 10 μm. F, myelin stainning by anti-CNPase exhibits irregular arrangement in the cingulate cortex in Ei24^flox/flox; nestin-Cre mice at 4 months of age. Bar, 100 μm. G, H&E staining shows that the cerebellum is less foliated (arrow) and fissured in Ei24^flox/flox; nestin-Cre mice at 4 months. Bar, 500 μm. H, thickness of the molecular layer, calculated by dividing the distance between lobules III and IV, or lobules V and VI, by 2. Mean ± S.E. of five mice is shown. I, H&E staining of Purkinje cells in control and mutant mice. The number of Purkinje cells in lobules III, IV, and V was quantified and divided by the total length of the lobules. Mean ± S.E. of five mice is shown. Bar, 50 μm. J, anti-calbindin (green) and anti-MBP (red) costaining reveals that Ei24^flox/flox; nestin-Cre mice exhibit dilated calbindin-positive bulbs (arrows) in the DCN region and some of the bulbs are enwrapped by MBP-labeled myelin. Bar, 10 μm. K–M, EM pictures of DCN in Ei24^flox/+; nestin-Cre and Ei24^flox/flox; nestin-Cre mice at 4 months. K, myelinated axons of regular shape and size in the DCN region of control animals. (L–M), Degenerated axons in mutant mice. L, arrowheads indicate electron-dense amorphous structures. The arrow shows a thinned myelin sheath. M, arrowhead indicates undulated and split myelin lamellae. Note that the axons in the mutant sample are conspicuously less abundant than in the control. Bar, 1 μm (K–M). N, number of interneurons per mm² in the lumbar spinal cord. Mean ± S.E. of five mice is shown. O, accumulation of vacuolated cells in the anterior horn of the lumbar spinal cord in mutant mice at 4 months. Bar, 100 μm. P, GFAP signal (red), in sections of cerebral cortex immunostained with anti-GFAP antibody, is stronger in mutant mice. Bar, 20 μm.