11 Mounting in vitro and in vivo evidence suggests that progressive

loss of telomeres is an important component of aging.12-14 Telomere shortening eventually reaches a critical point that triggers replicative senescence (irreversible growth arrest). There is a direct correlation between telomere length, the proliferative capacity of somatic cells and aging in normal healthy individuals.15, 16 Telomere length is a validated biomarker of aging.17-20 Real-time polymerase chain reaction (PCR) is the gold standard for measuring buy BMS-354825 telomere length, but using this technique for liver homogenates has limitations, because distinct intrahepatic cell lineages cannot be analyzed separately. Quantitative fluorescence in situ hybridization (Q-FISH) is a reliable indirect measure of telomere length.21, 22 Studies in diseased liver have revealed Talazoparib cell line significant reductions in telomere length in small series of patients with cirrhosis or hepatocellular carcinoma, where small numbers of cells were analyzed.23, 24 Only two studies25, 26 examined the relation between age and telomere length in “healthy” liver. These were limited by small sample size, limited age range, and the use of tissue derived from individuals with an increased risk of senescence.

Furthermore, only 64% of cells in liver tissue are hepatocytes,27 hence analysis of telomere length in whole liver homogenates is unlikely to reflect hepatocyte telomere length. The effect of aging on other intrahepatic lineages is unknown. Our study is the first to examine the effect of aging in normal liver, distinguishing between each intrahepatic lineage, using a large volume Q-FISH in situ approach and archival liver. DAPI, 4′,6-diamidino-2-phenylindole; PCR, polymerase chain reaction; for Q-FISH, quantitative fluorescent in situ hybridization; TBS, Tris-buffered saline. The Norfolk and Norwich Research Ethics Committee approved the use of archived liver tissue. Finding normal liver tissue for studies

across a wide age range is problematic. Liver biopsy is not performed in healthy individuals, and it would be unethical to subject healthy controls to liver biopsy for research. In other circumstances, investigators elect to use liver obtained at resection for hepatic metastases, particularly colorectal malignancy, using tissue distant from the tumor that appears normal microscopically. However, colorectal malignancy and hepatocellular carcinoma arise with increasing frequency with increased age and are associated with telomere shortening28-30; malignancy generally arises more often in accelerated aging or senescence. It is improbable that liver tissue could be obtained readily across a wide age range in this context. Based on the premise that liver donors by their nature are “unselected” and often present following trauma or disease unrelated to aging, intraoperative liver biopsies from implanted donor livers taken immediately after reperfusion were studied (time-zero liver biopsies).