Exploratory Investigation of Intestinal Structure and Function after Stroke in Mice

Abstract

Stroke is the second leading cause of death worldwide. Patients who have a stroke are susceptible to many gastrointestinal (GI) complications, such as dysphagia, GI bleeding, and fecal incontinence. However, there are few studies focusing on the GI tract after stroke. The current study is to investigate the changes of intestinal structure and function in mice after ischemic stroke. Ischemic stroke was made as a disease model in mice, in which brain and ileal tissues were collected for experiments on the 1^st and 7^th day after stroke. Intestinal motility of mice was inhibited, and intestinal permeability was increased after stroke. Hematoxylin-eosin (HE) staining showed the accumulation of leucocytes in the intestinal mucosa. Myeloperoxidase (MPO) activity and inflammatory proteins (nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS)) in the small intestine were significantly increased in mice after stroke. The expression of tight junction (TJ) proteins (zonula occludens-1 (ZO-1), occludin, and claudin-1) was downregulated, and transmission electron microscopy (TEM) showed broken TJ of the intestinal mucosa after stroke. Glial fibrillary acidic protein (GFAP) and the apoptosis-associated proteins (tumor necrosis factor (TNF-α), caspase-3, and cleaved caspase-3) were notably upregulated as well. Ischemic stroke led to negative changes on intestinal structure and function. Inflammatory mediators and TNF-α-induced death receptor signaling pathways may be involved and disrupt the small intestinal barrier function. These results suggest that stroke patients should pay attention to GI protection.

Figure 1

Animal grouping.

Figure 2

Ischemia model was successfully established. (a) Representative images of the mouse brain and TTC staining: the white part of the brain is the tissue of ischemic infarction. (b) Neurological Deficit Score (NDS) at different time points (n = 10) (Kruskal-Wallis ANOVA on ranks). (c) Mouse body weight at different time points (n = 10) (paired-sample t-test). (d) The impelling ratio (IR) at different times (n = 5) (two-way ANOVA with Tukey's multiple comparison test). Data were expressed as mean ± standard deviation. ^∗∗∗p < 0.001. ns: not significant.

Figure 3

Stroke caused intestinal inflammation. (a) Representative images of hematoxylin-eosin (HE) staining of the ileum (magnification ×200, red arrow points to the leukocyte infiltration). (b) Chiu's score for the intestinal mucosa (n = 7) (Kruskal-Wallis ANOVA on ranks). (c) Myeloperoxidase (MPO) activity was measured (n = 7) (two-way ANOVA with Tukey's multiple comparison test). (d, e) Representative western blots and quantification data of iNOS, NF-κB, and GAPDH for each group (n = 7) (two-way ANOVA with Tukey's multiple comparison test). Data were expressed as mean ± standard deviation. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. ns: not significant. Scale bar: 100 μm.

Figure 4

Ischemic stroke leads to increased intestine permeability. (a) Quantitative analysis of serum FITC-D as a measure of intestinal barrier functions (n = 5). (b) mRNA levels of ZO-1, occludin, and claudin-1 were measured with RT-qPCR and normalized to the expression levels of β-actin (n = 5). The results were presented as fold changes relative to the sham group. (c, d) Representative western blots and quantification data of ZO-1, occludin, claudin-1, and GAPDH for each group (n = 5). Two-way ANOVA with Tukey's multiple comparison test. Data were expressed as mean ± standard deviation. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. ns: not significant. (e) Representative images of transmission electron microscopy (TEM) (magnification ×23000). The ultrastructure of the normal small intestinal mucosa looked like that of sham 1d and sham 7d, including perfect tight junctions (TJ) and normal mitochondria (yellow arrow points to enlarged tight junction, and green arrow points to degenerate mitochondria). Scale bar: 2 μm.

Figure 5

Ischemic stroke activated enteric glia. (a, b) Representative western blots and quantification data of GFAP and GAPDH for each group (n = 5). (c) Representative fluorescence microscopic images of GFAP (green) and DAPI (blue) (magnification ×100). Both images are merged at the top panel. (d) Quantification of GFAP punctae (n = 5). Data were expressed as mean ± standard deviation. ^∗∗p < 0.01, ^∗∗∗p < 0.001 (two-way ANOVA, Tukey's posttest). ns: not significant. Scale bar: 200 μm.

Figure 6

Ischemic stroke promotes apoptosis of the small intestine. (a, b) Representative western blots and quantification data of TNF-α, caspase-3, cleaved caspase-3, and GAPDH for each group (n = 7). Data were expressed as mean ± standard deviation. ^∗∗p < 0.01, ^∗∗∗p < 0.001 (two-way ANOVA with Tukey's multiple comparison test). ns: not significant; CC-3: cleaved caspase-3.