Figure 4.

Apoptosis and neurodegeneration in the central nervous system of FVB/Twitcher (Twi) mice. (A) Representative confocal pictures of spinal cord tissue slices from PND30 and PND40 Twi mice showing apoptotic cells (caspase-3, C3; arrows). Nuclei counterstained with To-Pro3 (arrows indicate nuclei of apoptotic cells). (B) Quantification of C3+ cells in spinal cord (SC) tissue slices shows a significant increase of apoptotic cells in Twi mice at both ages (PND30, n=3 mice, 6 slices; PND40, n=6 mice, 14 slices) as compared with wild type (WT) (PND40, n=3 mice, 4 slices. One-way analysis of variance and Dunnett's multiple comparison test, *P⩽0.05, ***P⩽0.001. (C, D) Representative confocal images showing C3+ cells (green, arrows) expressing the oligodendroglial marker APC (C) and the neuronal marker NeuN (D) in the SC of PND40 Twi mice. Lineage markers are stained in red. Nuclei counterstained with To-Pro3 (arrows indicate nuclei of apoptotic cells). (E) Apoptotic oligodendrocytes (C3+O4+) and (F) apoptotic neurons (C3+Map2+) in primary cell cultures prepared from SC tissues of PND40 Twi mice. C3, green; O4 and Map2, red. Scale bars: 60 μm (A); 30 μm (C, F). (G) Age- and region-dependent upregulation of neurodegeneration markers in Twi mice. Cb and SC tissues collected from PND30 and PND40 Twi and WT mice were processed for total RNA extraction. After retrotranscription, cDNAs were analyzed by real-time qPCR for the expression of ATF3 and NCX1. Data are expressed as fold change in the Twi tissues with respect to the content in the WT (set as 1) after normalization by the expression of β-actin as a housekeeping gene. Data represent the mean ±s.e.m. n=4–6 mice/group. Two-way ANOVA and Bonferroni's multiple comparison test. *P⩽0.05, **P⩽0.01, ***P⩽0.001 versus the correspondent region at PND30; ^#P⩽0.05 versus Cb at the correspondent age. ^§P<0.05 versus WT; ns, not statistically significant from WT.