Volume 39, Issue 6, Pages 960-966 (December 2003) Apoptosis of hepatic stellate cells in carbon tetrachloride induced acute liver injury of the rat: analysis of isolated hepatic stellate cells  Jung Il Lee, Kwan Sik Lee, Yong-Han Paik, Young Nyun Park, Kwang Hyub Han, Chae Yoon Chon, Young Myoung Moon  Journal of Hepatology  Volume 39, Issue 6, Pages 960-966 (December 2003) DOI: 10.1016/S0168-8278(03)00411-2

Fig. 1 Change of ALT concentrations according to time sequences in acute CCl4 liver injury of Sprague–Dawely rats. Serum concentration of ALT (IU/l) was measured at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point). All results refer to mean±S.D. *P<0.01 compared with that of the control. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 2 Change of HSC proliferation according to time sequence in acute CCl4 liver injury of Sprague–Dawely rats. HSC proliferation was assessed by [3H]thymidine uptake (cpm), measured from isolated HSC at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4, and without treatment, as control (n=10 at each time point); 30 μCi of [3H]thymidine was intraperitoneally injected 4 h before the killing and a β counter was used to count [3H]thymidine incorporation into 10 μg of DNA. All results refer to mean±S.D. *P<0.01 compared with that of the control. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 3 Change of HSC apoptosis, measured from isolated HSC in acute CCl4 liver injury of Sprague–Dawley rats, according to time sequence. HSC apoptosis was assessed using flow-cytometric analysis, measuring percent of annexin-V-FITC bound HSCs at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point). PI was used as dye exclusion test and only PI negative and annexin V immunoreactive cells were regarded as apoptotic. All results refer to mean±S.D. *P<0.01 compared with that of the control. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 4 Change of HSC apoptosis, measured from liver tissue in acute CCl4 liver injury of Sprague–Dawley rats, according to time sequence. Liver sections at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point), were stained with TUNEL and desmin. Number of both TUNEL and desmin immunoreactive cells were regarded as apoptotic HSCs. Immunoreactive cells were counted per 10 random high power fields. All results refer to mean±S.D. *P<0.01 compared with that of the control. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 5 An example of apoptotic HSCs by dual TUNEL and desmin stain. Liver sections at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point) were dual stained with TUNEL/desmin. An example of TUNEL positive HSC nucleus within a fibrotic septum that is desmin positive (A, arrow) indicating an apoptotic HSC at 64 h after CCl4 injection. An example of TUNEL positive but desmin negative apoptotic cell, which is not HSC (B, arrow), and TUNEL negative and desmin positive non-apoptotic HSC (B, arrow head) (TUNEL/desmin dual stain, 400×). Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 6 Correlation between apoptosis of HSC measured from isolated HSCs and from tissue sections in acute CCl4 liver injury of Sprague–Dawely rats. HSC apoptosis was serially estimated in isolated HSCs by measuring the percent of annexin-V-FITC bound, PI negative cells at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point). HSC apoptosis was serially estimated in tissue sections, using TUNEL/desmin dual immunohistochemical study. Both TUNEL and desmin immunoreactive cells were counted in 10 random high power fields at each time points. There was a statistically significant correlation between apoptosis of isolated HSCs and that of the tissue section with r=0.606 and P<0.01. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 7 Change of total number of HSCs according to time sequence in acute CCl4 liver injury of Sprague–Dawely rats. Liver sections at 16, 32, 48, 64, 80 h and 4, 7 days after the single intraperitoneal injection of CCl4 and without treatment, as control (n=10 at each time point), were stained with desmin. The number of immunoreactive cells was estimated in 10 random high power fields. All results refer to mean±S.D. *P<0.01 compared with that of the control. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 8 Change in the expression of Bcl-2, Bax, Fas, and p21/p53 in acute CCl4 liver injury of Sprague–Dawely rats. Western blot analysis of HSCs, isolated from both normal and injured rat livers at the indicated time points, was undertaken using monoclonal antibodies to Bcl-2, Bax, Fas and p21/p53 to detect protein expression. The protein Bcl-2 started to be expressed at 16 h after CCl4 injection and lasted until 80 h after the insult. The protein Bax started to be detected at 64 h after the CCl4 administration and until 4 days after the insult. Expression of p53 was noticed at 32 h after the CCl4 injection and it was not detectable afterwards. Expression of p21 was visible at 32 h after the injection and lasted until 4 days after the insult. Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)

Fig. 9 Relative changes in HSC proliferation, apoptosis and total number HSCs in acute CCl4 liver injury of Sprague–Dawley rats. Each value at particular time points is a relative value compared with that of the peak level. HSC proliferation was measured by [3H]thymidine incorporated to HSC (thymidine). HSC apoptosis was estimated by two means; one was by flow-cytometric quantification of percent of annexin-V-FITC bound cells in isolated HSCs (Annexin-V) and the other was by counting TUNEL and desmin immunoreactive cells in 10 random high power fields in tissue sections (TUNEL). Total number of HSCs was counted from liver sections, stained with desmin, in 10 random high power fields (Desmin). Journal of Hepatology 2003 39, 960-966DOI: (10.1016/S0168-8278(03)00411-2)