Figure 4.
Aggravated pathological changes in the brains of HgsnatP304Lmice. (A) Astromicrogliosis in brain hippocampal and cortex regions of MPS IIIC mice is indicative of neuroimmune response. Panels show representative confocal microscopy images of brain tissues of 4-mo-old Hgsnat-Geo and HgsnatP304L mice and their age-matched WT controls stained with antibodies against CD68 (red) and GFAP (green) markers for activated microglia and astrocytes, respectively. DAPI (blue) was used as a nuclear counterstain. Graphs show quantification of fluorescence with ImageJ software. Individual data, means, and SD obtained for five mice per genotypes (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (B) Total brain tissues of HgsnatP304L mice show increased expression of inflammation markers, MIP1α, and TNFα compared with Hgsnat-Geo mice. The cytokine mRNA levels are normalized for the RLP32 mRNA content. Data show individual data, means, and SD. Five to eight mice were analyzed for each genotype. P values were calculated using one-way ANOVA with Tukey post hoc test. (C–F) Somatosensory cortices (layers 4–5) of HgsnatP304L mice show increased levels of markers of impaired autophagy and proteolysis compared with Hgsnat-Geo and/or age-matched WT mice: cytoplasmic LC3-positive puncta (C), granular autofluorescent ceroid materials (D), amyloid-β protein (AP; E), and misfolded SCMAS (F). Panels show representative confocal microscopy images of brain tissues of 4-mo-old (A, B, and D) or 6-mo-old (C, E, and F) Hgsnat-Geo, HgsnatP304L, and WT mice. Bars represent 20 µm in A and C, 100 and 25 µm in D, and 25 µm in E and F. Fluorescence was quantified with ImageJ software. Graphs show individual data, means, and SD obtained for five mice per genotype (three areas/mouse). P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (G) Alteration of sphingolipid levels in the brains of Hgsnat-Geo and HgsnatP304L mice. Levels of glycans produced by enzymatic cleavage of total sphingolipid extracts of brain tissues from WT, Hgsnat-Geo, and HgsnatP304L 2-, 4-, and 6-mo-old mice were measured by normal HPLC. The values show percentage of the specific lipid. Pooled samples of three mice per age per genotype were analyzed. (H) Increased levels of GM2 ganglioside in the brains of Hgsnat-Geo and HgsnatP304L mice. Confocal microscopy images of brain cortex and hippocampus tissues of individual Hgsnat-Geo, HgsnatP304L, and WT 4-mo-old mice stained with antibodies against GM2 (green) and NeuN (red). DAPI (blue) was used as the nuclear counterstain. Scale bar equals 15 µm. Graphs show results of quantification performed using ImageJ software. Individual data, means, and SD obtained for five mice per genotypes (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. MO, mo.

Aggravated pathological changes in the brains of Hgsnat P304L mice. (A) Astromicrogliosis in brain hippocampal and cortex regions of MPS IIIC mice is indicative of neuroimmune response. Panels show representative confocal microscopy images of brain tissues of 4-mo-old Hgsnat-Geo and HgsnatP304L mice and their age-matched WT controls stained with antibodies against CD68 (red) and GFAP (green) markers for activated microglia and astrocytes, respectively. DAPI (blue) was used as a nuclear counterstain. Graphs show quantification of fluorescence with ImageJ software. Individual data, means, and SD obtained for five mice per genotypes (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (B) Total brain tissues of HgsnatP304L mice show increased expression of inflammation markers, MIP1α, and TNFα compared with Hgsnat-Geo mice. The cytokine mRNA levels are normalized for the RLP32 mRNA content. Data show individual data, means, and SD. Five to eight mice were analyzed for each genotype. P values were calculated using one-way ANOVA with Tukey post hoc test. (C–F) Somatosensory cortices (layers 4–5) of HgsnatP304L mice show increased levels of markers of impaired autophagy and proteolysis compared with Hgsnat-Geo and/or age-matched WT mice: cytoplasmic LC3-positive puncta (C), granular autofluorescent ceroid materials (D), amyloid-β protein (AP; E), and misfolded SCMAS (F). Panels show representative confocal microscopy images of brain tissues of 4-mo-old (A, B, and D) or 6-mo-old (C, E, and F) Hgsnat-Geo, HgsnatP304L, and WT mice. Bars represent 20 µm in A and C, 100 and 25 µm in D, and 25 µm in E and F. Fluorescence was quantified with ImageJ software. Graphs show individual data, means, and SD obtained for five mice per genotype (three areas/mouse). P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (G) Alteration of sphingolipid levels in the brains of Hgsnat-Geo and HgsnatP304L mice. Levels of glycans produced by enzymatic cleavage of total sphingolipid extracts of brain tissues from WT, Hgsnat-Geo, and HgsnatP304L 2-, 4-, and 6-mo-old mice were measured by normal HPLC. The values show percentage of the specific lipid. Pooled samples of three mice per age per genotype were analyzed. (H) Increased levels of GM2 ganglioside in the brains of Hgsnat-Geo and HgsnatP304L mice. Confocal microscopy images of brain cortex and hippocampus tissues of individual Hgsnat-Geo, HgsnatP304L, and WT 4-mo-old mice stained with antibodies against GM2 (green) and NeuN (red). DAPI (blue) was used as the nuclear counterstain. Scale bar equals 15 µm. Graphs show results of quantification performed using ImageJ software. Individual data, means, and SD obtained for five mice per genotypes (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. MO, mo.

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