Abstract
Cellular senescence is a molecular hallmark of ageing that is associated with multiple pathologies, and DNA damage marker γH2AX, together with cell cycle inhibitor p21, have been used as senescence markers in multiple species including dogs. Idiopathic canine chronic hepatitis has recognised breed-related differences in predisposition and prognosis, but reasons behind this are poorly understood. This retrospective study using archived post mortem tissue aimed to provide insight into liver ageing in 51 microscopically normal canine livers across seven breed categories, including those with and without increased risk of chronic hepatitis. Immunohistochemistry was conducted for γH2AX, p21, and cell proliferation marker Ki67, and the mean number of positive hepatocytes per high power field was determined. All three markers were strongly correlated to each other, but no age-dependent expression was seen in the combined study population. Overall expression levels were low in most dogs, with median values representing less than 1.5% of hepatocytes, but this increased to 20–30% in individual dogs at the upper end of the range. Individual breed differences were noted in two breeds that have increased risk of chronic hepatitis, with English Springer Spaniels having lower expression of Ki67 than other dogs, and Labradors having higher expression of Ki67 and γH2AX than other dogs. These results warrant further investigation in these breeds and highlight a need to validate reliable markers of cellular senescence in dogs.
Highlights
The concept of healthy ageing and longevity is an increasing focus in veterinary and human medicine (Kaeberlein et al 2016)
Archived samples of microscopically normal livers from 51 dogs were included in the study (Table 1 and electronic supplementary material), with ages ranging from four months to 18 years, and representing seven different breed categories: 10 small breeds; nine English Springer Spaniels (ESS), six English Cocker Spaniels (ECS), 10 Cavalier King Charles Spaniels (CKCS), five Staffordshire Bull Terriers (SBT), seven Labradors, and four other large breed dogs
These categories were chosen to encompass small, medium, and large breed dogs, as body size influences the absolute rate of ageing, and smaller body size is associated with a longer life expectancy (Kraus et al 2013)
Summary
The concept of healthy ageing and longevity is an increasing focus in veterinary and human medicine (Kaeberlein et al 2016). Among the nine interlinked molecular hallmarks of mammalian ageing is cellular senescence: a state of irreversible cell cycle arrest induced either through replicative exhaustion or through a variety of cellular stresses, but in Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. The histochemical stain for senescence-associated β-galactosidase activity (SA-β-gal) has been a mainstay of in vitro experiments, but cannot be visualised in formalin fixed tissue that constitutes the majority of retrospective samples in veterinary research. In both human fibroblasts and mouse hepatocytes, DNA damage marker γH2AX is the best surrogate marker for SA-β-gal (Passos et al 2007; Wang et al 2009). Expression of γH2AX and cell cycle inhibitor p21 have recently been
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