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. 2012 Feb;55(2):609-21.
doi: 10.1002/hep.24713.

A mouse model of accelerated liver aging caused by a defect in DNA repair

Siobhán Q Gregg  1 , Verónica Gutiérrez, Andria Rasile Robinson, Tyler Woodell, Atsunori Nakao, Mark A Ross, George K Michalopoulos, Lora Rigatti, Carrie E Rothermel, Irene Kamileri, George A Garinis, Donna Beer Stolz, Laura J Niedernhofer
Affiliations

A mouse model of accelerated liver aging caused by a defect in DNA repair

Siobhán Q Gregg et al. Hepatology. 2012 Feb.

Abstract

The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging-related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1(-/Δ) mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1(-/Δ) mice, we systematically compared liver from 5-month-old progeroid Ercc1(-/Δ) mice to old (24-36-month-old) wild-type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1(-/Δ) mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1(-/Δ) mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1(-/Δ) liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1(-/Δ) and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome-wide transcriptional changes between old WT and 16-week-old, but not 5-week-old, Ercc1(-/Δ) mice, emphasizing that the Ercc1(-/Δ) mice acquire an aging profile in early adulthood.

Conclusion: There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging-related hepatic changes.

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Figures

Figure 1
Figure 1. Premature onset of age-related pathology in Ercc1−/Δ mouse liver
(A) Hematoxylin and eosin (H&E) stained liver sections from 7 wk, 23 wk, and 34 mth-old WT mice, and Ercc1−/Δ littermates (10X). The bottom right panel is a high magnification (40X) image of Ercc1−/Δ liver (23 wk) illustrating inflammatory infiltrate (black arrows), abnormal nuclei with inclusion (green arrows) and dead hepatocytes (blue arrows). (B) Masson’s trichrome stained liver sections. Insets (40X) in the bottom right panel show perisinusoidal fibrosis in Ercc1−/Δ and old WT liver samples. (C) LipidTOX™ stain of liver sections to detect neutral lipids (20X). (D) Immunofluorescence detection of desmin and α-SMA, markers of hepatic stellate cells and their activation, respectively, in frozen liver sections (20X).
Figure 2
Figure 2. Age-related thickening of the sinusoidal endothelium in Ercc1−/Δ mouse liver
(A) Immunofluorescence detection of CD31, a surface marker of endothelial cells in frozen liver sections (20X). (B) Transmission electron micrographs (TEM; 12,000X) of liver illustrating thickening of the basement membrane in sinusoids of mutant and old WT mice compared to young WT. Insets show enlargement of a portion of the original image and arrows indicate deposition of basement membrane and pseudocapillarization of the sinusoidal endothelium. (C) Immunoblot to detect the extracellular matrix protein laminin in liver lysates from 20 wk-old Ercc1−/Δ mice, control littermates and old WT (2–3 year) mice (n=3 per group). Densitometry was used for quantification and averages are listed as a ratio compared to 20 week-old WT mice.
Figure 3
Figure 3. Premature defenestration in Ercc1−/Δ mouse liver
(A) Scanning electron micrograph (10,000X) of liver, illustrating defenestration in sinusoids of mutant and old WT mice. Insets show enlargement of a portion of the original image. (B) Quantification of porosity shown in A. 3 SEM images per mouse (n=3 mice per group) were quantified, and the % open area of the sinusoid is represented ± S.E.M. Asterisks indicate significant differences. (C) Serum cholesterol and (D) triglyceride levels in WT and Ercc1−/Δ mice of various ages. The average ± S.E.M. are plotted (n=6 for 7 wk-old mice, n=9 for 21 wk-old and old WT mice). Asterisks indicate significant differences as calculated by a paired Student’s t test.
Figure 4
Figure 4. Premature loss of liver function in Ercc1−/Δ mice
(A) Representative liver sections from 10 week-old WT and Ercc1−/Δ mice immunostained for the proliferation marker Ki67 48 hr post-partial hepatectomy. The fraction of proliferating hepatocytes is graphed (5 random fields of view analyzed from n=4 mice per genotype; asterisk indicates p<0.05, Student’s two tailed t-test). (B) Serum chemistries reflecting liver function. Plotted are the average ± S.E.M. for 6 mice per age and genotype. WT animals are graphed in reds; Ercc1−/Δ mice in blues. Darker colors indicate increasing age. AST is aspartate transminase; ALT is alanine transaminase. Asterisks indicate significant differences.
Figure 5
Figure 5. Premature senescence of Ercc1−/Δ mouse liver
(A) Senescence-associated (SA) β-galactosidase histochemical stain on flash frozen liver sections. (B) Immunoblot detection of the senescence marker p16INK4a in liver lysates of a 20 week-old WT mouse and an Ercc1−/Δ littermate, and a 26 month-old WT mouse. Tubulin was used as a loading control to calculate the fold-increase in p16 expression relative to adult WT mice. (C) DAPI staining of hepatocyte nuclei illustrating increased nuclear size and heterogeneity in old WT (34 months) and adult Ercc1−/Δ mouse liver (1000X). Quantification of nuclear size using Metamorph imaging software and images of DAPI-stained frozen liver sections. Plotted are the average nuclear areas ± S.E.M. calculated from 10 random fields from 5 mice per group. The asterisks indicate significant differences. (D) Transmission electron micrographs of representative hepatocyte nuclei (12,000X).
Figure 6
Figure 6. Increased oxidative damage in liver of old and progeroid mice
(A) Immunodetection of lipid hydroperoxides, a by-product of lipid peroxidation, in liver lysates from 20 wk-old WT and Ercc1−/Δ littermate mice vs. old WT mice (26–34 months of age). The box plot represents the mean (in colored square), along with the 25th and 75th percentile for each group (n=5 mice). Average lipid hydroperoxide levels for groups were as follows: 20 wk WT (4.8µM/g liver), 20 wk Ercc1−/Δ (7.3µM/g liver; p=0.12, one-tailed Student’s t-test), and old WT (7.2µM/g liver). (B) Immunofluorescence detection of acrolein, a by-product of lipid peroxidation. The staining intensity was quantified using AxioVision imaging software. Plotted are the averages ± S.E.M. determined from analysis of 10 random fields of liver sections from 2 mice per group. Asterisks indicate significant differences. (C) Representative images illustrating lipofuscin accumulation in 20 week-old WT and Ercc1−/Δ mice and 26–34 month-old WT mouse liver. AxioVision software was used for quantification of 10 random fields measured from 5 mice per group. Plotted is the average fluorescence intensity for each group ± S.E.M. Asterisks indicate significant differences.
Figure 7
Figure 7. Age-associated transcriptional reprogramming in liver of Ercc1−/Δ mice
(A) Significant gene expression changes (p<0.05, two tailed t-test) in the liver of 5- and 16-week old Ercc1−/Δ mutant livers as compared to littermate control animals. Note the progressive dampening in the expression of genes involved in oxidative metabolism and the GH/IGF1 axis along with the upregulation in the expression of genes involved in cell cycle arrest, DNA damage responses, oxidative stress and detoxification (red color: up-regulated; green color: down-regulated, FC: fold change, wks: weeks). (B) qPCR measuremtn of mRNA levels of a subset of genes identified to be differentially expressed in the liver of 16-week old Ercc1−/Δ animals compared to wild-type littermates. For each gene, expression levels in the mutant tissue are plotted relative to those of controls (red dotted line). Error bars indicate S.E.M. between replicates (n ≥ 4). (C) Spearman’s r correlation reflecting transcriptome similarities between the 5 or 16 week-old Ercc1−/Δ and the 130 week-old mouse livers where −1.0 is a perfect negative (inverse) correlation, 0.0 is no correlation, and 1.0 is a perfect positive correlation.
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