Alteration in the number of neuronal and non-neuronal cells in mouse models of obesity

Abstract

Obesity is defined as abnormal or excessive fat accumulation that may impair health and is a risk factor for developing other diseases, such as type 2 diabetes and cardiovascular disorder. Obesity is also associated with structural and functional alterations in the brain, and this condition has been shown to increase the risk of Alzheimer's disease. However, while obesity has been associated with neurodegenerative processes, its impact on brain cell composition remains to be determined. In the current study, we used the isotropic fractionator method to determine the absolute composition of neuronal and non-neuronal cells in different brain regions of the genetic mouse models of obesity Lep^ob/ob and LepR^Null/Null . Our results show that 10- to 12-month-old female Lep^ob/ob and LepR^Null/Null mice have reduced neuronal number and density in the hippocampus compared to C57BL/6 wild-type mice. Furthermore, LepR^Null/Null mice have increased density of non-neuronal cells, mainly glial cells, in the hippocampus, frontal cortex and hypothalamus compared to wild-type or Lep^ob/ob mice, indicating enhanced inflammatory responses in different brain regions of the LepR^Null/Null model. Collectively, our findings suggest that obesity might cause changes in brain cell composition that are associated with neurodegenerative and inflammatory processes in different brain regions of female mice.

Keywords: isotropic fractionator; neurodegeneration; neuroinflammation; neuronal loss; obesity.

Graphical Abstract

Figure 1

Isotropic fractionator (IF) technique workflow. (A) Representative image of a monogenic mouse model of obesity (10–12 months) and a WT mice (10 months) showing differences in body size. (B) Brain weight of Lep^ob/ob (n = 7; P = 0.058) and LepR^Null/Null (n = 3; P = 0.0465) obese mice when compared to WT mice (n = 7). (C) Workflow diagram illustrating the IF steps that involve the transformation of highly anisotropic brain structures into isotropic suspensions of cell nuclei, allowing for rapid quantitative analysis of cell composition of different brain regions. The isotropic suspension volume (V_F) obtained for each brain region analysed was for hypothalamus—V_F = 2.5 mL; hippocampus—V_F = 5.0 mL; and frontal cortex—V_F = 5.0 mL. (D–F) Representative images of the hippocampus nuclei suspension after fractionation, with staining and immunocytochemistry to DAPI (D), NeuN (E) and merge between DAPI and NeuN (F) and the white arrowheads indicate nuclei doubly stained with DAPI and NeuN. Scale bars: 100 µm. The dot and bar graph were expressed as mean ± SEM; one-way ANOVA with Tukey’s post hoc test. Significant differences were indicated by *P < 0.05.

Figure 2

Changes in total cell number and density, especially in LepR^Null/Null. (A and B) Absolute total cell number (A) and cell density (B) in the hippocampus of WT (n = 4). Lep^ob/ob (n = 6) and LepR^Null/Null mice (n = 3). (C and D) Absolute total cell number (C) and cell density (D) in the frontal cortex of WT (n = 4), Lep^ob/ob (n = 7) and LepR^Null/Null mice (n = 4). (E and F) Absolute total cell number (E) and cell density (F) in the hypothalamus of WT (n = 4), Lep^ob/ob (n = 7) and LepR^Null/Null mice (n = 4). Data are shown as mean ± SEM; symbols represent individual animals; P-values were calculated from one-way ANOVA followed by Tukey’s post hoc test. Significant differences were indicated by *P < 0.05, ***P < 0.001 and ****P < 0.0001.

Figure 3

Neuronal changes in the brain of obese mouse models. (A–C) Neuronal population in the hippocampus including percentage (A), absolute number (B) and density (C) of cells in WT (n = 4), Lep^ob/ob (n = 6) and LepR^Null/Null mice (n = 3). (D–I) Neuronal population in the frontal cortex and hypothalamus, including percentage (D and G, respectively), number (E and H, respectively) and density (F and G, respectively) of cells in WT (n = 4), Lep^ob/ob (n = 7) and LepR^Null/Null mice (n = 4). Data are shown as mean ± SEM; symbols represented individual animals; P-values were calculated from one-way ANOVA followed by Tukey’s post hoc test. Significant differences were indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 4

Increased non-neuronal cell number and density in different brain regions of LepR^Null/Null mice. (A and B) Absolute number (A) and density (B) of non-neuronal cells in the hippocampus of WT (n = 3), Lep^ob/ob (n = 6) and LepR^Null/Null mice (n = 3). (C and D) Absolute number (C) and density (D) of non-neuronal cells in the frontal cortex of WT (n = 4), Lep^ob/ob (n = 7) and LepR^Null/Null mice (n = 4). (E and F) Absolute number (E) and density (F) of non-neuronal cells in the hypothalamus of WT (n = 4), Lep^ob/ob (n = 7) and LepR^Null/Null mice (n = 4). Data are shown as mean ± SEM; symbols represented individual animals; P-values were calculated from one-way ANOVA followed by Tukey’s post hoc test. Significant differences were indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 5

Correlations between total cell number and regional brain weight. (A–C) Pearson correlations between the number of total cells and weight in the hippocampus (A), the frontal cortex (B) and the hypothalamus (C) among WT, Lep^ob/ob and LepR^Null/Null mice. (D–F) Pearson correlations between neuronal number and weight in the hippocampus (D), frontal cortex (E) and hypothalamus (F) among WT, Lep^ob/ob and LepR^Null/Null mice. (G–I). Pearson correlations between the number of non-neuronal cells and weight in the hippocampus (G), frontal cortex (H) and hypothalamus (I) among WT, Lep^ob/ob and LepR^Null/Null mice. Correlation coefficients (r) and P-values are shown in the graphs.

Figure 6

Changes in non-neuron/neuron ratio in different brain regions of LepR^Null/Null mice. (A–C) Non-neuron/neuron ratio in the hippocampus (A), frontal cortex (B) and hypothalamus (C) of WT (n = 4), Lep^ob/ob (n = 6) and LepR^Null/Null (n = 3) mice. (D–F) Pearson correlations between non-neuron/neuron ratio and neuronal density in the hippocampus (D), frontal cortex (E) and hypothalamus (F) of WT, Lep^ob/ob and LepR^Null/Null mice. (G–I) Pearson correlations between non-neuron/neuron ratio and weight in the hippocampus (G), frontal cortex (H) and hypothalamus (I). Data are shown as mean ± SEM; P-values were calculated from one-way ANOVA followed by Tukey’s post hoc test. Significant differences are illustrated by *P < 0.05, **P < 0.01 and ****P < 0.0001. Correlation coefficients (r) are shown in the graphs.