at the time of hepatectomy) x 100
resected liver weight
For histologic examination, liver specimens routinely processed for embedding in paraffin, sectioned and stained with hematoxylin-eosin for light microscopic examination. Counts of mitotic figures in 20 fields for each group were made under high-power magnification, the results being expressed as mean numbers per 1000 parenchymal cells (mitotic index, MI).
Immunohistochemical staining of incorporated bromodeoxyuridine was performed to generate BrdU-labeling indices (BrdU LIs). The staining procedure was accomplished as previously described (12) and positive cells were counted in 10 randomly selected high-power fields (x200) and the results expressed as the numbers of hepatocytes labeled per 100 cells.
Data are presented as means ‘ή standard errors. Statistical analysis of the results was conducted with the paired Student's t-test.
Histologic changes in the ligated pancreas
Histopathological findings revealed that, at 1 week after the pancreatic duct ligation, the acinar parenchyma was almost completely lost, whereas the islet parenchyma was relatively normal. Ducts were markedly dilated and interstitial tissue demonstrated slight edema, fibrosis and inflammation. Islets progressively proliferated but acinar cells did not essentially reappear during the experimental period.
Liver Wet Weight (LWW)
The liver weight in the PL group was significantly decreased on the postoperation day 2 (8.17±2.16, 11.05±0.96, P < 0.05) of stage two, this continuing on the postoperation days 3 and 4 compared with the sham group (Fig. 1).
Liver regeneration rate (LRR)
The liver regeneration rate in the PL+Hx group was significantly lower on postoperation day 1 (6.66±4.32%, 14.63±4.59%, P < 0.05), and markedly higher on postoperation day 3 (49.93±11.62%, 38.14±7.11%, P < 0.05) than the Hx group (Fig. 2).
BrdU labeling index (BrdU LI)
BrdU positive nuclei were almost absent in the sham and PL groups. In the Hx and PL+Hx groups, peaks were found on postoperation day 1, that for the PL+Hx group (23.46±14.94%) being significantly lower than the Hx group value (54.92±13.28%, P <0.01) (Fig. 3).
Mitotic index (MI)
Mitotic cells were essentially absent in sham and PL groups. In the PL+Hx group, the mitotic cell peak (28.10±12.79 / 1000 cells) after hepatectomy was delayed 24 hours and significantly decreased compared with the Hx group(43.93±7.73 / 1000 cells, P < 0.05) (Fig. 4).
The serum insulin level in the sham group was higher than in PL group and there were significant differences on postoperation day 2 (10.60±4.26, 3.40±2.49, P < 0.05). In the PL+Hx group, on postoperation day 1 insulin levels was significantly lowered, then showed a tendency for increase on postoperation day 2 after hepatectomy compared to the Hx group (Table 1).
The HGF concentration in was increased in the Hx group on the first day after hepatectomy compared with the PL+Hx group; whereas it was delayed until the third day (Table 2).
Hepatic and pancreatic functions
Data for serum Alb, TB, GOT and amylase concentrations are summarized in Table 1. In the sham, PL, and Hx groups the Alb concentrations showed relative increase on the postoperation day 1 in stage two compared with postoperation day 2. The Alb levels progressively increased in the sham group, but fluctuated in the PL group. Both PL and PL+Hx groups later showed low Alb concentrations. TB and GOT concentrations in serum did not exhibit any significant intergroup differences. The amylase concentration in the PL group had a tendency for decrease on postoperation days 1, 3, and 7 in stage two compared with the sham group; similar changes were observed in the PL+Hx group on postoperation days 1 and 2 compared with the Hx group (Table 3).
Liver regeneration is controlled by various growth factors and cytokines such as HGF, EGF, insulin, transforming growth factor-a(TGF-a), interleukin-6, tumor necrosis factor and norepinephrine (1, 4). Among these, HGF, EGF and TGF-a are complete mitogens for hepatocytes in culture (1). HGF displays a 5-10 fold higher potency than epidermal growth factor (EGF) and tumor growth factor-a (TGF-a) on a molar basis (13, 14), and its concentration in plasma rapidly increases 15-17 fold following hepatectomy , above the mitogenic range for hepatocytes (15). EGF and TGF-a plasma levels show only a small increase after partial hepatectomy (16, 17, 18). The kinetics of hepatocytic DNA synthesis and cell division are similar to the HGF levels in plasma (1, 7). Thus it is considered that HGF is a definitive factor for liver regeneration.
Previous studies have indicated that HGF rapid rise in plasma after hepatectomy occurs within 1 to 2 hours (15), whereas its mRNA expression in hepatic Ito cells increases 3-6 hours after partial hepatectomy (19, 20). The elimination rate in the liver, as the main organ for clearing circulating HGF (21), does not appreciably affect the magnitude of the HGF rise in plasma after partial hepatectomy (22), suggesting the rapid elevation is mainly due to the release of preexisting stores from different tissues.
Recent studies demonstrated that matrix break down in the regenerating liver itself is one source of HGF (23, 24). Immunohistochemical studies have shown in fact that HGF is widely distributed in most surface epithelia, lung, kidney, brain, and at high levels in the exocrine pancreas of rats (8). The pancreas plays important supporting roles not only for normal liver but also for liver regeneration (5, 9). We previously showed that 50% pancreatectomy, that has minimal affects on pancreas endocrine function, induced a delay in hepatocytic DNA synthesis and a decrease in the liver regeneration rate (9). Form these results we speculated that the exocrine pancreas may support liver regeneration due to HGF release.
In the present study, almost complete loss of pancreatic acinar cells, with maintenance of islets 1 week after pancreatic duct ligation was associated with a decrease in the HGF concentration in plasma in the PL+Hx on the first day, but increase on the third and seventh days after hepatectomy. This indicats that the exocrine pancreas contributes to the early rapid rise in plasma HGF after hepatectomy. The increase after the second day may be due to endocrine regulatory responses.
Hepatectomy was here found to result in low blood insulin levels in normal pancreas animals compared with sham group after hepatectomy. This may be due to endocrine regulation for maintenance of euglycemia. In the PL+Hx group, the significant decrease in insulin levels on postoperation day 1, despite maintenance of islet structure and normal glucose tolerance tests (25, 26), is in line with the literature (27, 28).
The present study showed that the regenerating liver DNA synthesis peak to be significantly decreased and hepatocytic mitosis markedly reduced and delayed 24 hours in PL+Hx compared with Hx group. Depression of early rapid rise in HGF is the most likely main cause, with low serum insulin levels of secondary importance. HGF is a distinct and potent mitogen for hepatoctes (13, 14, 29) whereas insulin only enhances hepatocyte proliferation and has no mitogenic effect itself (1, 30).
The LRR for the PL+Hx group was markedly depressed on the postoperation day 1 after hepatectomy followed by significant increase on the third day compared with that in the Hx group. These data conform with changes in HGF and insulin concentrations in the plasma. The reduction of HGF and insulin here would be responsible for the LRR reduction on first day; and the elevation of HGF might induce the increase of LRR on the third day. HGF not only stimulates hepatocyte proliferation but also is an essential factor for hepatocytic maturity and develpoment (31). It enhances hepatocytic protein and lipid syntheses to cause hepatocytic enlargement (32, 33, 34). Insulin is the most important trophic factor for liver.
Laparotomy one week after subtotal pancreatic duct ligation was associated with a reduction in the normal liver weight compared with the sham group, in agreement with the insulin and Alb levels. These may be related to changes in endocrine and exocrine pancreas induced by the subtotal pancreatic duct ligation.
In this study, the hepatocyte mitosis peak was early at 24 hours in both Hx and PL+Hx groups compared with previous studies. This may have been due to primary effects of the first operation but this remains to be confirmed.
Our present results suggest an important role of exocrine pancreas in liver regeneration. In the clinical field, the major hepatectomy is attempted for cases of hepatocellular cancinoma with liver cirrhosis, and advanced biliary malignancy (35, 36), and postoperative liver decompensation due to insufficient liver mass is a major complication. Sakai T reported that liver cirrhosis is usually linked with reduced pancreas exocrine function (37) and our results are very interesting in this context suggesting that the early administration of HGF and insulin might be useful for increasing liver volume after major hepatectomy.
In conclusion, subtotal pancreatic duct ligation model partial hepatectomy is associated with decrease in the peak of DNA synthesis, reduction and delay in hepatocyte mitosis, and depression in early liver weight. Change in HGF blood concentrations related to pancreatic duct ligation and depression of early insulin blood levels appear responsible, suggesting that exocrine pancreas plays a supporting role in early liver regeneration.
The authors are grateful to Dr. Tian X. Tang for assistance with the operative procedures for the experimental animals
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FIG. 1. Changes in liver wet weight at various times after second sham laparotomy and subtotal pancreatic duct ligation. Values are means ± SEM. The asterisk represents statistically significa- nt differences from subtotal pancreatic duct ligation (P < 0.05).
FIG. 2. Changes in liver regneration rate at various times after 68% hepatectomy and subtotal pancreatic duct ligation + 68% hepatectomy. Values are mans±SEM. The asterisk represents statistically significant differences from subtotal pancreatic duct ligation + 68% hepatectomy (P < 0.05).
FIG. 3. Bromodeoxyuridine (BrdU) labeling index in liver at various times after second sham laparotomy, subtotal pancreatic duct ligation, 68% hepaterctomy, and subtotal pancreatic duct ligation + 68% hepatectomy. Values are mans ± SEM. *Significantly different from subtotal pancreatic duct ligation + 68% hepatectomy (P < 0.01).
FIG. 4. Mitotic index (MI) in normal liver and in liver regeneration at various times after second laparotomy, subtotal pancreatic duct ligation, 68% hepatectomy, and subtotal pancreatic duct ligation + 68% hepatectomy. Values are means ± SEM. *Signicantly different from subtotal pancreatic duct ligation + 68% hepatectomy. (P < 0.05).
Table 1. Note. Values are means ± SEM. Lapa, sham laparotomy; PL, pancreatic duct subtotal ligation; Hx, 68% hepatectomy; PL+Hx, subtotal pancreatic duct ligation + 68% hepatectomy. The asterisk represents statistically significant differences from the control values. *P < 0.05, **P < 0.05.
Table 2. Note. Values are means ± SEM. Lapa, sham laparotomy; PL, subtotal pancreatic duct ligation; Hx, 68% hepatectomy; PL+ Hx, subtotal pancreatic duct ligation + 68% hepatectomy. No statistically significant differences were found.
Table 3. Note. Values are means ± SEM. Lapa, sham laparotomy; PL, subtotal pancreatic duct ligation; Hx, 68% hepatectomy; PL+Hx, subtotal pancreatic duct ligation + 68% hepatectomy. No statistically significant differences were found.