Hyperhomocysteinemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression

Abstract

Hyperhomocysteinemia has been shown to be associated with neurodegenerative diseases; however, lesions or histological changes and mechanisms underlying homocysteine-induced injury in olfactory bulb neurons remain unclear. In this study, hyperhomocysteinemia was induced in apolipoprotein E-deficient mice with 1.7% methionine. Pathological changes in the olfactory bulb were observed through hematoxylin-eosin and Pischingert staining. Cell apoptosis in the olfactory bulb was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Transmission electron microscopy revealed an abnormal ultrastructure of neurons. Furthermore, immunoreactivity and expression of the hairy enhancer of the split 1 (Hes1) and Hes5 were measured using immunohistochemistry, immunofluorescence, and western blot assay. Our results revealed no significant structural abnormality in the olfactory bulb of hyperhomocysteinemic mice. However, the number of TUNEL-positive cells increased in the olfactory bulb, lipofuscin and vacuolization were visible in mitochondria, and the expression of Hes1 and Hes5 decreased. These findings confirm that hyperhomocysteinemia induces injury in olfactory bulb neurons by downregulating Hes1 and Hes5 expression.

Keywords: Nissl body; apoptosis; hairy enhancer of split 1; hairy enhancer of split 5; homocysteine; nerve regeneration; neural regeneration; neurons; olfactory bulb.

Figure 1

Plasma tHcy levels in HHcy mice. Data are expressed as the mean ± SD (n = 12), analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. The unpaired t-test was used between two groups. *P < 0.05, vs. Wt and ApoE^–/– groups, respectively; #P < 0.05, vs. HHcy group. tHcy: Total homocysteine; HHcy: hyperhomocysteinemia; ApoE^–/–: apolipoprotein E-deficient.

Figure 2

Histological changes in the olfactory bulb of HHcy mice. (A) Representative histographs in hematoxylin-eosin staining: Normal histology with mitral cells (thick arrows) and granule cells (arrowheads) in Wt, ApoE^–/–, HHcy, and HFB groups. Scale bar: 50 μm. (B) Representative images of Pischingert staining: Deep blue in mitral cells (thick arrows) and pale blue in granule cells (arrowheads). Scale bar: 10 μm. (C) Quantitative results of the numbers of mitral and granule cells, and the mean optical density of Pischingert staining in the olfactory bulb. Data are expressed as the mean ± SD (n = 12) and analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. The unpaired t-test was used between two groups. Wt: Wild-type; ApoE^–/–: apolipoprotein E-deficient; HHcy: hyperhomocysteinemia; HFB: hyperhomocysteinemia treated with folic acid and vitamin B12.

Figure 3

Ultrastructural change in the olfactory bulb of HHcy mice. (A–D) Transmission electron microscopy images of the olfactory bulb of HHcy mice. (A) Wt group: Mitochondria, rough endoplasmic reticulum, rich ribosome, and nucleus; (B) ApoE^–/– group: mitochondria, rough endoplasmic reticulum, and nucleus; (C) HHcy group: mitochondrial vacuolization and nucleus; (D) HFB group: lipofuscin and nucleus. Scale bar: 1 μm. (E) Quantitative results of the cells with mitochondrial vacuolization and lipofuscin. Data are expressed as the mean ± SD (n = 4) and analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. The unpaired t-test was used between two groups. *P < 0.05, vs. Wt and ApoE^–/–- groups, respectively; #P < 0.05, vs. HHcy group. Wt: Wild-type; ApoE^–/–: apolipoprotein E-deficient; HHcy: hyperhomocysteinemia; HFB: hyperhomocysteinemia treated with folic acid and vitamin B12; M: mitochondria; N: nucleus; VM: mitochondrial vacuolization; L: lipofuscin.

Figure 4

TUNEL-positive granule cells in the olfactory bulb of HHcy mice. (A–D) TUNEL staining of the olfactory bulb of HHcy mice. (A) Wt group: Granule cells (thick arrows); (B) ApoE^–/– group: few scattered TUNEL-positive granule cells (arrowheads); (C) HHcy group: obviously increased TUNEL-positive granule cells (arrowheads); (D) HFB group: few TUNEL-positive granule cells (arrowheads). Scale bar: 30 μm. (E) Quantitative results of TUNEL-positive granule cells. Data are expressed as the mean ± SD (n = 12) and analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. The unpaired t-test was used between two groups. *P < 0.05, vs. Wt and ApoE^–/– groups, respectively; #P < 0.05, vs. HHcy group. Wt: Wild-type; ApoE^–/–: apolipoprotein E-deficient; HHcy: hyperhomocysteinemia; HFB: hyperhomocysteinemia treated with folic acid and vitamin B12; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 5

Expression of Hes1 and Hes5 in the olfactory bulb of HHcy mice. (A) Representative immunohistochemistry of Hes1: In Wt and Apo E^–/– groups, most Hes1-immunostained granule cells (thick arrows); in HHcy group, part of Hes1-immunostained granule cells (thick arrows); in HFB group, a few of Hes1-immunostained granule cells (thick arrows). (B) Representative immunofluorescent of Hes5: In Wt, ApoE^–/– and HFB groups, most Hes5-labelled granule cells (arrowheads); in HHcy group, decreased Hes5-labelled granule cells (arrowheads). Scale bars in A, B: 20 μm. (C) Numbers of Hes1 and Hes5 immunoreactivated granule cells with (n = 12). (D) Western blot assay of Hes1 and Hes5: The representative western blots and bargraph of relative Hes1 and Hes5 value (n = 8). Data are expressed as the mean ± SD and analyzed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. The unpaired t-test was used between two groups. *P < 0.05, vs. Wt and ApoE^–/– groups, respectively; #P < 0.05, vs. HHcy group. Wt: Wild-type; ApoE^–/–: apolipoprotein E-deficient; HHcy: hyperhomocysteinemia; HFB: hyperhomocysteinemia treated with folic acid and vitamin B12; Hes: hairy enhancer of the split.