Rifaximin Improves Spatial Learning and Memory Impairment in Rats with Liver Damage-Associated Neuroinflammation

Abstract

Patients with non-alcoholic fatty liver disease (NAFLD) may show mild cognitive impairment. Neuroinflammation in the hippocampus mediates cognitive impairment in rat models of minimal hepatic encephalopathy (MHE). Treatment with rifaximin reverses cognitive impairment in a large proportion of cirrhotic patients with MHE. However, the underlying mechanisms remain unclear. The aims of this work were to assess if rats with mild liver damage, as a model of NAFLD, show neuroinflammation in the hippocampus and impaired cognitive function, if treatment with rifaximin reverses it, and to study the underlying mechanisms. Mild liver damage was induced with carbon-tetrachloride. Infiltration of immune cells, glial activation, and cytokine expression, as well as glutamate receptors expression in the hippocampus and cognitive function were assessed. We assessed the effects of daily treatment with rifaximin on the alterations showed by these rats. Rats with mild liver damage showed hippocampal neuroinflammation, reduced membrane expression of glutamate N-methyl-D-aspartate (NMDA) receptor subunits, and impaired spatial memory. Increased C-C Motif Chemokine Ligand 2 (CCL2), infiltration of monocytes, microglia activation, and increased tumor necrosis factor α (TNFα) were reversed by rifaximin, that normalized NMDA receptor expression and improved spatial memory. Thus, rifaximin reduces neuroinflammation and improves cognitive function in rats with mild liver damage, being a promising therapy for patients with NAFLD showing mild cognitive impairment.

Keywords: NMDA receptor; hepatic encephalopathy; microglia; mild liver damage; rifaximin; spatial learning and memory.

Figure 1

Astrocyte and microglia activation in the hippocampus. The perimeter of the microglia (stained with Iba1 marker (A)) and the percentage of GFAP stained area (B) was analyzed. Values are the mean ± SEM of 8 rats per group. In (C) the data were analyzed using the non-parametric Kruskal–Wallis test and Dunn’s multiple comparisons test. In (D) the data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons test. The asterisks indicate a significant difference with respect to the control group * p < 0.05, ** p < 0.01, **** p < 0.0001, “aaa” p < 0.001. C: control; C-RIF: Control rats + rifaximin; CCl₄: CCl₄ injected rats and CCl₄-RIF: CCl₄ rats + rifaximin.

Figure 2

IL-1β (A), TNFα (B), and CCL2 (C) content in CA1 region of hippocampus. It was analyzed by immunohistochemistry. The quantification was expressed according to the mean on the grey scale (Mean Grey Value) and as percentage of control rats for IL-1β quantification. Values are the mean ± SEM of 6 rats per group. The data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons test. The asterisks indicate a significant difference compared to the control group * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; “a” compared to the CCl₄ rats “a” p < 0.05, “aa” p < 0.01.

Figure 3

Infiltration of macrophages (A) and CD4+ T lymphocytes (B) into meninges of the hippocampus. It was analyzed by immunohistochemistry with anti-Iba1 or anti-CD4 markers, respectively. Values are the mean ± SEM of 5 rats per group. In (C) data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test and in (D) using a Welch’s ANOVA test and Dunnett’s multiple comparisons test. The asterisks indicate a significant difference compared to the control group * p < 0.05, ** p < 0.01, and “aaa” p < 0.001.

Figure 4

Membrane expression of GluA1 (A) and GluA2 (B) AMPA receptor subunits; of NR1 (C), NR2A (D) and NR2B (E) NMDA receptor subunits in the hippocampus. Samples from slices with (+) or without (-) the crosslinker BS3 were analyzed in the same western blot and the surface expression of receptor subunits was calculated as the difference between the intensity of the bands without BS3 (total protein) and with BS3 (non-membrane protein). Values are the mean ± SEM of 15 rats per group. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparisons test except for GluA2 (B) that Welch’s ANOVA test and Dunnett’s multiple comparisons test was used. The asterisks indicate a significant difference with respect to the control group * p < 0.05. The “a” indicate a significant difference with respect to the CCl₄ rats “a” p < 0.05.

Figure 5

Spatial learning and reference and working memory in the radial maze and novel object location (NOL) and recognition (NOR) memory tests. Reference memory (A,B) spatial learning (C,D), and working memory (E,F) were evaluated in the radial maze. Values are the mean ± SEM of 15 rats per group. In (A,C,E) the data were analyzed using a two-way ANOVA with repeated measures and Tukey’s multiple comparison test. In (A), only for time a significant effect was found (F (2.856, 142.8) = 4.332, p < 0.01); in (C)the effect between groups was significant (F (3, 40) = 2.925, p < 0.05) as well as the time effect (F (2.949, 118.0) = 13.69, p < 0.0001); in (E), only the time was significant (F (3, 72) = 4.24, p < 0.01). In B and F non-parametric Kruskal–Wallis test and Dunn’s multiple comparisons test was used and no significant effects were found. In (D) one-way ANOVA and Tukey’s multiple comparisons test was used and statistic value was F (3, 45) = 3.789, p < 0.05. In multiple comparisons, the asterisks indicate a significant difference with respect to the control group * p < 0.05, ** p < 0.01, “a” with respect to CCl₄ rats “a” p < 0.05 and “b” compared with C-RIF group, “b”, p < 0.05. The symbol # indicates within the same group significant difference with respect to day 1 “#” p < 0.05 and “##” p < 0.01. The $ symbol indicates within the same group a significant difference with respect to day 2 “$” p < 0.05. Rats were evaluated for the ability to identify the change in location of an object through the NOL test (G) and to identify a new object by the NOR test (H). Values are mean ± SEM of 13 rats per group. Data were analyzed using a one-way ANOVA and Tukey’s multiple comparison test (F(3, 34) = 5.389 for NOL test). The asterisks indicate a significant difference with respect to the control group * p < 0.05, and “a” with respect to the CCl₄ rats “a” p < 0.05.

Figure 6

Proposed mechanisms leading to the impaired learning and memory in rats with mild liver damage. These results suggest that the increase in CCL2 in the hippocampus would be an early event in the process leading to cognitive impairment in rats with mild liver damage. The increase in CCl2 would promote both the infiltration of monocytes into the hippocampus to became macrophages and the activation of microglia. Microglia activation in turn would contribute to the increase in TNFα in hippocampal neurons of these rats, leading to the reduced membrane expression of NR1 and NR2A subunits of NMDA receptors which, in turn, would lead to the impairment in spatial learning and memory. All this process is reversed by treatment with rifaximin, that restores cognitive function in rats with mild liver damage. Mild liver damage also triggers infiltration of CD4+ lymphocytes, activation of astrocytes, increased levels of IL-1β and enhanced membrane expression of the GluA2 subunit of AMPA receptors in the hippocampus. These changes are not reversed by treatment with rifaximin. Created with BioRender.com.