Figure 1.
The effect of GAL over-expression on body weight.
Body weight was measured at the age of 8–9 weeks old as the starting point 0 w. The raw body weight growth in normal control groups (A) and the CPZ-treated groups (B) is shown. Both the cuprozone groups and the cuprizone-challenge plus 3-week recovery groups were measured at the same time. Data are expressed as the mean ± SEM. (n = 12–17 per group). * p<0.05, ** p<0.01, *** p<0.001.
Figure 2.
Over-expression of GAL blocks CPZ-induced myelin breakdown.
(A)–(F) are photographs of the cerebral cortex and the CC, while (a)–(d) are the photographs of whole brain. Both Tg and WT mice were given 0.3% CPZ for six weeks (6wCPZ), and then the two groups of mice were allowed to recover for three weeks on a normal food diet (6wCPZ+3wR). Mice brains were processed for IHC staining using an antibody against MBP. Consistent with previous studies, we found extreme demyelination in WT mice after six weeks of CPZ challenge: compare (B) and (b) with the control groups (A) and (a). However, MBP staining of Tg mice brains, (E) and (d), indicated that myelin breakdown was not significantly different compared with controls, (D) and (c). After three weeks on a normal diet, the WT mice recovered well (as expected) (C). To verify the IHC staining results, we also used luxol fast blue staining on WT CLT (I), WT 6wCPZ (II), Tg CLT (III) and Tg 6wCPZ (IV) samples (6 µm paraffin sections). The bar graphs represent the measurements of optical density of MBP IHC staining in the cerebral cortex and the CC areas. Arrows show the aca area that was used for color-intensity standardization. Data are expressed as mean ± s.e.m values. (n = 3–5 per group). ** p<0.01, *** p<0.001.
Figure 3.
Increased levels of galanin attenuated CPZ-induced oligodendrocyte loss.
Mature oligodendrocytes were detected with IHC using a GST-π antibody. Photographs (A–F) were taken from the knee region of the CC, and (G) and (H) are examples of the full-size pictures taken of WT and Tg brains from the 6wCPZ group. The three CC images in the upper panels (A–C) show the WT mice from the CTL, 6wCPZ and 6wCPZ+3wR groups, and the three images in the middle panels (D–F) show the Tg mice from the same groups. In (A–F), high-magnification micrographs were also taken of the CC area using an oil-immersion lens, as shown in the inserts. (I) A bar chart displaying the numbers of GST-π positive cells, which were counted manually. **p<0.01, ***p<0.001, n = 3 in each group. The longer scale bar represents a length of 200 µm and the shorte scale bar represents 500 µm.
Figure 4.
Increased number of PDGFR-α positive cells in WT mice that underwent the CPZ-induced demyelination challenge.
WT and Tg mice were given CPZ challenge for six weeks (6wCPZ, B and E) while the control mice (CLT, A and D) received normal CPZ-free rodent chow. After six weeks of challenge, two groups of animals (6wCPZ+3wR, C and F) were allowed to recover for three weeks on a normal CPZ-free diet. The bar graph shows the results of the optical-density measurements of the PDGFR-α-positive cells; a significant difference was observed between the 6wCPZ+3wR WT and Tg groups. The scale bar represents 200 µm in the low-magnification. Data are expressed as mean ± SEM values (n = 3 per group). *p<0.05, ** p<0.01.
Figure 5.
The differential expression of GalR1 and GalR2 in the CC area.
RNA samples were extracted from CC areas as demonstrated in Figure 6. The gene expression levels among the groups were normalized to the WT CLT levels (set equal to 1). (A) The expression of GalR1 among the groups. (B) The expression of GalR2 among the groups. Data are expressed as the mean ± SEM values. (n = 3–6 per group). * p<0.05, ** p<0.01.
Table 1.
Primary antibodies used for immunohistochemistrical staining.
Figure 6.
The illustration on the left represents the mouse brain. The top dashed line represents the first coronal cut, 5 mm away from the edge of olfactory bulb. The bottom dashed line represents the second coronal cut, 2.5 mm away from the first cut. The illustration on the right represents the brain section isolated on the left. The tissue within the dashed rectangle, containing mainly corpus callosum and part of the cortex, was used for RNA extraction. Abbreviations: CC – corpus callosum; LV – lateral ventricle; aca – anterior commissure.
Table 2.
Primer pairs provided by SABiosciences™.