Exp Toxic Pathol 2003; 54: 281–286 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath
Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
Impaired liver regeneration after partial hepatectomy in db/db mice HIROFUMI YAMAUCHI, KOJI UETSUKA, TARO OKADA, HIROYUKI NAKAYAMA, and KUNIO DOI With 7 figures Received: October 24, 2002; Revised: November 18, 2002; Accepted: November 29, 2002 Address for correspondence: HIROFUMI YAMAUCHI, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan; Tel. & Fax: +81-3-58418185, e-mail: [email protected]
Key words: Fatty liver; db/db mice; liver regeneration; partial hepatectomy.
Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.
currence of graft dysfunction after liver transplantation and of mortality and morbidity after liver resection (BEHRNS et al. 1998; SELZNER and CLAVIEN 2001; KONERU and DIKDAN 2002). Impaired liver regeneration is supposed to be a cause of poor prognosis after liver transplantation and hepatic resection. To clarify this point, many studies using animal models of fatty liver have been carried out. Animal models of fatty liver are divided into two categories; diet-induced models such as that induced by choline-deficient diet feeding and genetically occurring models such as leptin or leptin receptor deficient animals. Recently, it is said that the true impairment of liver regeneration is observed only in the latter models (ZHANG et al. 1999; RAO et al. 2001; PICARD et al. 2002). Of genetic fatty liver models, ob/ob mice lack leptin and obese Zucker rats have dysfunction of leptin receptor. db/db mice, like obese Zucker rats, have abnormal leptin receptor (COLEMAN 1982), and they also suffer from impaired angiogenesis because of dysregulated balance of vessel remodeling factors (KAMPFER et al. 2001). These animals are also used as a diabetic model (KOTEISH and DIEHL 2001). The purpose of this study was to analyze the process of liver regeneration until 10 days after 70% partial hepatectomy (PH) in db/db mice.
Materials and methods
Fatty liver is the most common hepatic disorder in humans. It can be caused by a variety of aetiologies such as obesity, diabetes mellitus and alcohol consumption (SELZNER et al. 2000). While often considered benign conditions, steatosis is associated with more frequent oc-
Thirty-five C57BL/KsJ db/db female mice (40–45 g) and thirty-four normal lean (+m/+m) littermates (17–21 g) were obtained from Saitama Experimental Animals Co. (Saitama, Japan). The animals were subjected to the experiments at 10 weeks old. Animals were kept in an animal
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Fig. 1. Restitution of liver weight after PH in +m/+m mice (m) and db/db mice (h). Data are shown as the mean ± SE (5 to 7 mice). * P < 0.05 compared with +m/+m mice.
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Fig. 3. Mitosis index of hepatocytes after PH in +m/+m mice (m) and db/db mice (h). Data are shown as the mean ± SE (4 mice).
room under controlled conditions (temperature: 23 ± 2 °C; relative humidity: 55 ± 5%; 14 h-light and 10 h-dark cycle) and fed standard diet (MF, Oriental Yeast Co. Ltd., Tokyo, Japan) and water ad libitum. A 70% partial hepatectomy (PH) was performed under light ether anesthesia as described by HIGGINS and ANDERSON (1931). Five to seven animals were killed at 1, 2, 3, 5, 7 and 10 days after PH, respectively. In addition, 4 +m/+m and 4 db/db mice were killed before PH and served as untreated controls. Two hours before sacrifice, animals were injected intraperitoneally with 100 mg/kg of bromodeoxyuridine (BrdU, Sigma, St Louis, MO). At autopsy, the liver was weighed and then fixed in 10% neutral buffered formalin for histological and immunohistochemical examinations. The protocol of this study has been approved by the Animal Care and Use Committee of Graduate School of Agricultural and Life Sciences, The University of Tokyo. The restitution of liver weight was calculated using the following equation: 100 × (C – (A – B))/A; B being the excised liver weight at the time of PH, A being the estimated total liver weight (= B × 100/70), and C being the weight of the regenerated liver at the time of sacrifice (SELZNER and CLAVIEN 2000). The mitotic cells were counted on paraffine sections stained with hematoxylin and eosin (HE) under light microscopy (×400). The mitosis index was expressed as the percentage of mitotic hepatocytes per total numbers of hepatocytes counted in 8 fields for each animal. For immunohistochemical studies on BrdU and proliferating cell nuclear antigen (PCNA), paraffine sections were stained by the avidin-biotin-peroxidase complex (ABC) method using Vector ABC kits (Vector Lab. Inc. USA). BrdU is a thymidine analogue, which is incorporated into the DNA during S phase. PCNA is a cell-cycle nuclear protein that is expressed in the late G1 and throughout the S phase of the mitotic cycle. Anti-BrdU (Dako, Carpinteria, CA) and anti-PCNA mouse monoclonal antibodies (Novocastra Labo. Ltd., UK) were used as the primary antibodies, and counterstaining was done with methylgreen. BrdU and PCNA labelling indices of hepatocytes were determined on immunostained sections under light microscopy (×50), and expressed as the percentage of positive hepatocytes per total numbers of hepatocytes counted in 6 fields for BrdU or in 4 fields for PCNA in each animal. Cells with fragmented DNA were detected by the TUNEL method using an apoptosis detection kit (Apop Tag; Intergen, Purchase, NY). The TUNEL method was first proposed by GAVRIELI et al. (1992) and is now widely used for the detection of apoptotic cells. The restitution of liver weight, mitosis index and BrdU and PCNA labeling indices were expressed as the mean ± standard error (SE) (n = 5 to 7 for liver weight restitution and n = 4 for other parameters). Statistical analysis was done by Student’s t-test between +m/+m mice and db/db mice. When individual differences were large, Welch’s ttest was employed. A value of P < 0.05 was judged to be significant.
Results The restitution of liver weight was significantly inhibited in db/db mice compared with +m/+m mice after PH (fig. 1). In +m/+m mice, the liver mass increased rapidly from 1 (18%) to 5 days (57%), and restored the original mass at 10 days after PH (67%) (fig. 1). On the other hand, the liver weight of db/db mice hardly changed until 3 days (8.5%) and started to increase gradually from 5 days (17%) (fig. 1). However, the restituted liver mass was still half of that of +m/+m mice at 10 days after PH (28%, fig. 1). The liver of control +m/+m mice showed slight vacuolization of hepatocytes (fig. 2a) while that of control db/db mice showed prominent swelling of hepatocytes filled with fine vacuoles mainly in the centrilobular area (fig. 2b). At 2 days after PH, hepatocytes with small-sized vacuoles were observed almost all over the hepatic lobules in +m/+m mice (fig. 2c). In db/db mice, similar hepatocyte changes to those in control mice expanded to midzonal area of hepatic lobules (fig. 2d). At 5 days after PH, histological features of the liver almost returned to those before PH in both +m/+m and db/db mice (fig. 2e and f). The mitosis index in +m/+m mice peaked at 2 days after PH, decreased rapidly towards 5 days, and showed a slight increase again thereafter (fig. 3). In db/db mice, the mitosis index gradually increased from 2 to 5 days, and then decreased thereafter (fig. 3). The BrdU labelling index didn’t change at 1 day after PH in both +m/+m and db/db mice (figs. 4, 5a and b), and it peaked at 2 days in both +m/+m mice (29%) and db/db mice (17%) (figs. 4, 5c and d). The index returned to the baseline at 5 days after PH in +m/+m mice while the recovery was delayed in db/db mice (figs. 4, 5e and f). In +m/+m mice, the PCNA labelling index peaked at 2 to 3 days after PH (62–63%) (figs. 6, 7a and b). Then the index rapidly decreased at 5 days (12%), and maintained the similar value until 10 days after PH (14%) (figs. 6, 7c and d). In db/db mice, the PCNA labelling index showed no increase at 1 day. It reached a plateau (around 40%) at 2 to 5 days after PH, although the maximal value was lower than that in +m/+m mice (figs. 6, 7e and f). Thereafter the index decreased slowly and returned to the similar level to that in +m/+m mice at 10 days after PH (fig. 6). There were few TUNEL-positive hepatocytes in both db/db and +m/+m mice at any time points examined.
Discussion In the present study, the regenerative process after PH was examined in db/db mice and +m/+m mice. Among ge-
Fig. 2. Liver histology in +m/+m mice (a, c and e) and db/db mice (b, d, and f). Slight vacuolization of hepatocytes (a) and prominent swelling of hepatocytes mainly in the centrilobular area (b) before PH. Hepatocytes with small-sized vacuoles all over the hepatic lobules (c) and swollen hepatocytes in centrilobular to midzonal area of hepatic lobules (d) at 2 days after PH. Histological features of the liver similar to those before PH (e and f) at 5 days after PH. HE, ×200. Exp Toxic Pathol 54 (2003) 4
Fig. 4. BrdU labelling index of hepatocytes after PH in +m/+m mice (m) and db/db mice (h). See the foot note of figure 3.
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Fig. 6. PCNA labelling index of hepatocytes after PH in +m/+m mice (m) and db/db mice (h). See the foot note of figure 3. * P < 0.05 compared with +m/+m mice.
Fig. 7. Immunohistochemistry for PCNA in the liver of +m/+m mice (a, c and e) and db/db mice (d, b and f). Only a few positive hepatocytes (a, b) before PH. The number of positive hepatocytes is larger in (c) than in (d) at 2 days after PH, and in (f) than (e) at 5 days after PH. Immunostaining, ×200.
netic animal models of fatty liver, ob/ob mice, which lack leptin secretion, are said to show impaired restitution of liver mass as well as significantly suppressed BrdU labelling index of hepatocytes (YANG et al. 2001; TORBENSON et al. 2002). On the other hand, obese Zucker rats, which have dysfunction of leptin receptor, are also said to show inhibited liver mass restitution after PH. However, the data on the changes in BrdU labelling index are controversial (SELZNER and CLAVIEN 2000; PICARD et al. 2002).
In this study, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m
Fig. 5. Immunohistochemistry for BrdU in the liver of +m/+m mice (a, c and e) and db/db mice (b, d and f). Only a few positive hepatocytes (a, b) before PH. The number of positive cells is larger in (c) than in (d) at 2 days after PH. Compared with (e), several positive cells are seen in (f) at 5 days after PH. Immunostaining, ×200. Exp Toxic Pathol 54 (2003) 4
mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as another type of animal models. In db/db mice, many metabolic abnormalities may have an indirect negative effect on hepatocyte proliferation (KAPLAN 1998). For example, impaired glucose transport to hepatocytes might deprive the liver of part of the energy necessary for the anabolic process of cell proliferation, and up-regulation of gluconeogenesis could reduce the availability of amino acids necessary for the massive protein synthesis required after PH. Leptin may also play a direct role in the regenerative process after PH. Leptin is a member of the cytokine super family mainly synthesized by adipocytes. In recent years, leptin has been shown to be a potent mitogen for numerous cell lines in peripheral tissues (HARRIS 2000). Moreover, leptin is reported to enhance wound re-epithelialization (FRANK et al. 2000) and to promote angiogenic process in vitro and in vivo (BOULOUMIE et al. 1998). Impaired liver regeneration in db/db mice seemed to be brought about by abnormal metabolism, dysfunction of leptin receptor and inhibited angiogenesis. To evaluate the effects of steatosis on the liver regeneration after PH and to elucidate their mechanisms, comparative studies using various steatotic models are needed. db/db mice are considered to be useful as one of these animal models.
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