Marcos, R. and Correia-Gomes, C. (2016) Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells

24 It has been known that both male versus female differences in liver enzyme activity and 25 gene expression in the liver are attenuated with ageing. Nevertheless, the effect of 26 ageing on liver structure and quantitative cell morphology remains unknown. Male and 27 female Wistar rats of two, six, 12 and 18 months were examined. Stereological 28 techniques and immunohistochemical tagging of Hepatocytes (HEP), liver sinusoidal 29 endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) were 30 applied to assess the total number and number per gram of these cells throughout 31 ageing. The mean cell volume of HEP and HSC, lobular position and liver collagen 32 content were also evaluated with stereological techniques. The number per gram of 33 HSC was similar for both genders and maintained throughout ageing. The mean volume 34 of HSC was also conserved, but differences in the cell body and lobular location were 35 observed. As to HEP, statistically significant gender differences were noted in young 36 rats (females had smaller and more binucleated HEP) but were attenuated with ageing. 37 The same occurred for KC and LSEC, since the higher number per gram in young 38 females disappeared in older animals. As to liver collagen, it increased with ageing, but 39 only in males. Herein, we highlighted that the numbers of these four cell types were 40 related throughout ageing, with well-defined cell ratios. The shape and lobular position 41 of HSC changed with ageing in both males and females. As to HEP, KC and LSEC, the 42 gender dimorphism of the young rat liver disappeared with ageing. 43

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