Aging Deteriorates Blood Brain Barrier Function and Polarizes Adaptive T Cell Expansion Contributing to Neurocognitive Damage in Experimental Cirrhosis

Abstract

Cirrhosis incidence is significantly increased with age and frequently complicated with neurocognitive dysfunction. We have evaluated the contribution of aging to neuroinflammation in the liver-brain axis in advanced chronic liver disease. Young (6-week-old) and old (9-month-old) mice were included in a 12-week protocol of CCl₄-induced cirrhosis. Liver damage, neuromotor and cognitive capacities, blood brain barrier integrity and function, liver and brain T cell subpopulations and ammonia levels were evaluated. Timp1 and Acta2 gene expression was upregulated in old cirrhotic mice. Increased liver damage was confirmed histologically by Sirius red staining, expression of alpha-SMA, collagen 1-alpha1 and vimentin in aged CCl₄-treated mice. Aging further compromised the neuromotor and cognition capabilities in cirrhotic animals. Stress axis components Crh and its receptor Nr3c1 gene expression levels were upregulated in the paraventricular nucleus and hippocampus of old cirrhotic mice. CCl₄-damage significantly increased ammonia levels in the liver, brain and serum of cirrhotic mice. Circulating ammonia was significantly higher in old cirrhotic mice. Significant correlations were established between brain ammonia, neuromotor capabilities and results on the object recognition tests. A decreased integrity of blood brain barrier was accompanied by astrocyte activation and increased apoptosis-linked cleaved Caspase 3 in old cirrhotic mice. Liver resident CD4⁺ T-cell subpopulations were contracted in cirrhosis, although they showed a pro-inflammatory Th17 profile. Liver and brain resident CD8⁺ T-cell subpopulations were expanded in old cirrhotic animals, along with reduced tissue cytolytic activity. CD8⁺ T cell expansion and reduced perforin levels in the brain correlated with neuromotor and cognitive dysfunction. In conclusion, aging aggravates liver fibrosis, worsens neuromotor and cognitive functions and shifts liver and brain adaptive T cell profiles compromising the BBB integrity in experimental advanced chronic liver disease. Results strengthen the impact of aging in the liver-brain axis and neuroinflammation in cirrhosis.

Figure 1.

Liver damage in young and old mice with cirrhosis. (A-D) mRNA relative expression of Timp1, Col1a1, Acta2 and Mmp2 in total liver homogenates. Mean ± standard deviations are represented (4 animals/group). (E) Fibrosis-related stains in the liver. Representative liver images stained with Sirius red, α-SMA, collagen I and Vimentin and their respective quantifications. Signal was blindly measured in user-specified regions of interest (ROIs) as brown or red area percentage in respect to the total area of the ROI using the ImageJ software. Mean ± standard deviations are represented (6 animals/group). Scale bar = 100 µm. (F) Biochemical characterization liver function parameters in blood samples. Mean ± standard deviations are represented (6 animals/group). Abbreviations: Alanine transferase (ALT), Aspartate transferase (AST), albumin (ALB), Tissue inhibitor of metalloproteinases 1 (Timp1), Collagen 1 (Col1a1), Alpha smooth muscle actin (α-SMA and Acta2), Matrix metalloproteinase-2 (Mmp2), Vimentin (Vim), Young (Y) and Old (O). P values are indicated as follows (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; and (****) p < 0.0001.

Figure 2.

Behavior alteration in young and old mice with cirrhosis. (A-D) Panel of behavioral tests designed to evaluate alterations derived from liver disease and aging. The Open Field (A) and the Neurological Severity Score (B) evaluated motor performance and neurological state, respectively. The Elevated Plus Maze (C) evaluated anxiety-like behavior. The Object Recognition test (D) evaluated recognition memory. Mean ± standard deviations are represented (15 animals/group). (E-H) mRNA relative expression of Crh, Nr3c1, Bdnf and Ntrk2 in either the PVN or the HPP of mouse brains. Mean ± standard deviations are represented (6 animals/group). (I-K) Ammonia levels in the liver, serum and brain respectively of the mice subjected to the behavioral tests. Mean ± standard deviations are represented (7 animals/group). (L-N) Correlation between brain ammonia levels and Open Field Test (L), Neurological Severity Score (M) and Object Recognition test (N) respectively. Abbreviations: Elevated Plus Maze (EPM), Corticotropin-releasing factor (Crh), Nuclear Receptor Subfamily 3 Group C Member 1 (Nr3c1), Brain-Derived Neurotrophic Factor (Bdnf), Neurotrophic receptor Tyrosine Kinase 2 (Ntrk2), Paraventricular nucleus (PVN), Hippocampus (HPP), Neurological Severity Score (NSS), Young (Y) and Old (O). P values are indicated as follows (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; and (****) p < 0.0001.

Figure 3.

Blood brain barrier alterations in young and old mice with cirrhosis. (A) Representative Western blot of three biological replicates and protein relative expression of Zo1 and Claudin1 in brain tissue homogenates. Band densitometry is shown by the image as relative to b-actin levels. Mean ± standard deviations are represented (5 animals/group). (B) Representative images of hippocampus stained with GFAP and its respective quantification. Signal was blindly measured in user-specified ROIs as green area percentage in respect to the total area of the ROI using the ImageJ software. Nuclei, stained by DAPI, are represented in blue. Mean ± standard deviations are represented (4 animals/group). Scale bar = 100 µm. (C) Representative Western blot of three biological replicates and protein relative expression of Cleaved Casp3 in brain tissue homogenates. Band densitometry is shown by the image as relative to b-actin levels. Mean ± standard deviations are represented (4 animals/group). (D) Gene and protein expression levels of MMP9 and MMP12 in liver and brain, and serum of all study groups. Mean ± standard deviations are represented (5 animals/group). Abbreviations: Zonula occludens 1 (Zo1), Glial fibrillary acidic protein (GFAP), Immunofluorescence (IF), Caspase 3 (Casp3), matrix metallo-proteinase (MMP), Young (Y) and Old (O). P values are indicated as follows (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; and (****) p < 0.0001.

Figure 4.

Liver immune alterations in young and old mice with cirrhosis. (A) Representative liver images stained with CD3 and CD45 and their respective quantifications. Signal was blindly measured in user-specified ROIs as brown area percentage in respect to the total area of the ROI using the ImageJ software. Mean ± standard deviations are represented (6 animals/group). Scale bar = 100 µm. (B-C) Flow cytometry analysis of T CD4⁺ (B) and CD8⁺ (C) cells from liver tissue. Mean ± standard deviations are represented (5 animals/group). (D) Ratio between CD4⁺ and CD8⁺ cells from liver tissue. (E-F) Representative dot plot images from flow cytometry analysis of T CD4⁺ cells expressing either IFNγ⁺ (E), IL-17⁺ (F) or IL-10⁺ (G). Data is expressed as percentage of positive cells of the previous gate. Mean ± standard deviations are represented (5 animals/group). (H) Representative protein relative expression of Perforin and Granzyme B by Western blot in liver tissue homogenates. Band densitometry is shown by the image as relative to b-actin levels. Mean ± standard deviations are represented (5 animals/group). (I-L) Representative histogram images from flow cytometry analysis of T CD4⁺ expressing PD-1⁺ (I) or CD69⁺ (J) and CD8⁺ expressing PD-1⁺ (K) or CD69⁺ (L). Mean ± standard deviations are represented (5 animals/group). Abbreviations: Cluster of differentiation (CD), Interferon Gamma (IFNγ), Interleukin (IL), Programmed cell death protein 1 (PD-1) Young (Y) and Old (O). P values are indicated as follows (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; and (****) p < 0.0001.

Figure 5.

Brain immune alterations in young and old mice with cirrhosis. (A-C) Flow cytometry analysis of T CD3⁺ (A), CD4⁺ (B) and CD8⁺ (C) cells from brain tissue. Mean ± standard deviations are represented (5 animals/group). (D) Ratio between CD4⁺ and CD8⁺ T cells from brain tissue. (E) Representative protein relative expression of Perforin and Granzyme B by Western blot in brain tissue homogenates. Band densitometry is shown by the image as relative to b-actin levels. Mean ± standard deviations are represented (5 animals/group). (F) Correlation between percentage of CD8+ T cell population in brain and results in the Open Field test for all included animals. (G) Correlation between percentage of CD8+ T cell population in brain and results in the Object Recognition test for all included animals. (H) Correlation between perforin levels in the brain and results in the Object Recognition test for all included animals. Old cirrhotic mice are represented in white dots whereas the rest of groups are represented in colored dots. Abbreviations: Cluster of differentiation (CD), Young (Y) and Old (O). P values are indicated as follows (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; and (****) p < 0.0001.