Effect of cyclosporine on liver regeneration in partial hepatectomized rats 1

PURPOSE: To evaluate the influence of the cyclosporine in liver regeneration in rats submitted to an experimental model of 70% hepatectomy. METHODS: Forty male rats were randomly divided in four subgroups (C.24h, C.7d, E.24h, E.7d), according to the drug used and the day of sacrifice (24 hours and 7 days). Cyclosporine (10mg/Kg/day) was given to the study subgroup and 1 ml of 0.9% sodium chloride was to the control subgroup. Resection of left lateral lobe and median lobe performing 70% of liver mass. During the animals’ death, KWON formula was applied. Counting of mitotic figures and percentage of positive nucleus with PCNA and Ki-67 were evaluated. RESULTS: In the 2nd, 4th PO and death days, E.7d lose more weight than C.7d. Regarding to the KWON formula, the C.7d regenerated more than the C.24h and the same with the E.7d. Comparing between the groups, only E7d subgroup was statistically significant compared with C.7d, showing the stimulating effect of cyclosporine in liver regeneration. Immunohistochemestry had significant results between the study subgroups. The mitotic index revealed statistical differences in the control subgroups. CONCLUSION: Cyclosporine, in spite of being an immunosuppressive drug, has a positive effect in liver regeneration, although reduce the animal’s body weight.


Introduction
A notable capacity of the liver is the ability to regenerate itself after an injury like hepatectomy.The first report of liver regeneration tells about the punishment of Prometheus in the Greek mythology.He was punished by being chained to a rock after he has revealed to the mortals the secret of fire protect by the gods of Olympus.Every day, an eagle fed of his liver, which regenerated itself during the night 1 .
Liver doesn't regenerate in a true sense, a global tissue hyperplasia occurs until that the original liver mass has been restored 2 .Higgins and Anderson published one of the first studies about liver regeneration in 1931 3 .They removed the left lateral and median lobes of rat liver which amounts 70% of liver rat mass.
At any given time, in rats, about one of 20000 hepatocytes is dividing, and each hepatocyte can divide once or twice at most.After large liver resection, all the liver cells simultaneously go into the G1 phase due to the influence of IL-6 and TNF-α.These cytokines stimulate the hepatocytes to answer the growing factor as HGF (hepatocyte growing factor), TGF-α (transforming growth factor alpha) and HB-EGF (heparin binding -epidermal growth factor).12 to 15 hours after the hepatectomy, the hepatocytes enter in the S phase, and six to eight hours after the DNA synthesis, the liver cells go to the G2 and M phase.The differences between the beginning of proliferation and the peaks of mitosis after partial hepatectomy (24 hours in rats and 42 hours in mice) show the variability of G1 phase in the species 4 .
The degree of hepatocyte proliferation is proportional to the extension of injury, with local mitotic action in small resections; but in injuries greater than 10%, all the liver cells can proliferate.In resections over than 50%, a second peak of mitosis occurs after three to five days in rats 1 .The regenerative process finishes within seven to 14 days 5 .Regulatory growth factors has been studied in the last years, and some of them are well-known and can be divided in three categories: (1) mitogenic agents or growth stimulating factors (HGF, EGF, TGF-α).They are capable in inducing DNA synthesis and mitosis in hepatocytes at rest (G0 phase); (2) comitogenic agents.They stimulate proliferation indirectly, potentiating the effect of mitogenic agents and reducing the effect of inhibiting agents; (3) inhibiting factors.Capable of inhibiting induced mitogenesis in primary hepatocyte cultures 6,7 .Some studies have shown the influence of pretherapy with cyclosporine in liver regeneration after a 70% hepatectomy [8][9][10][11] , although some papers don't confirm the same results 12,13 .
Due to the immunosuppressive properties of cyclosporine and its positive effects on liver regeneration demonstrated in some reports, the possibility of augmentation in hepatocyte proliferation after liver resection, living donor liver transplantation and split liver procedure, appears as a hypothesis of being studied.The purpose of this study is to evaluate the effect of pretherapy with cyclosporine on liver regeneration using a well established animal model of 70% hepatectomy.

Methods
This study was carried out in compliance with the guidelines of the Brazilian Society for Animal Experimentation (COBEA) and Federal Law 6638.Forty male adult Wistar rats (Rattus norvegicus albinus, Rodentia mammalia) divided into two groups of twenty were used.Each one was divided into subgroups of 10 rats.The control group had two subgroups of 10 rats each (C.24h and C.7d) and the study group as well (E.24h and E.7d) according to the sacrifice day.All animals were 180 days old and had an average weight of 508 ± 37.86 g.They were kept in plastic cages, within a controlled light-dark, temperature and humidity environment and had water and commercial feed ad libitum.
Cyclosporine 10mg/Kg/day was administered by gavage for three days before the surgical procedure to the study subgroups and the control subgroups received 1 ml of 0.9% sodium chloride solution by gavage as well.After inhalatory anesthesia with isoflurane, the animals from all subgroups were submitted to hair removal at the ventral abdominal wall and antisepsis with polyvinyl pyrrolidone-iodine.A 4 cm longitudinal incision was performed in the abdomen wall and the peritoneal cavity explored.The left lateral and median hepatic lobes were resected and the pedicle stitched with 3-0 monocryl.This kind of hepatectomy add up to 67% to 70% of the parenchyma of the liver rat.After checking hemostasis, abdominal closure was carried out with 3-0 prolene.
The resected segments was weighted and the value recorded for posterior analysis of the Kwon's formula 14  The body weight of all animals was evaluated in the first day of pretherapy, before the hepatectomy, during the death procedure in all subgroups and on second and fourth post-operative days in the seven days subgroups.

Results
When evaluated initial body weight (PCI) and death body weight (PCM), all subgroups lose weight and the differences were statistically significant (Figure 1).
We found no statistical differences among all the subgroups when compared total liver weight, weight of resected segment during hepatectomy and estimated weight of remaining liver.When evaluated using Kwon' formula, liver regeneration rates in experimental and control groups were found to be different.At 24 hours, the control group had an average liver regeneration rate of 60.72% compared with 62.93% of experimental group (p = 0.49) (Table 1).After one week, the liver regeneration rate in the control group was 83.61% versus 93.05% in the experimental group (p = 0.05) (    When regeneration was assessed according to the average number of mitotic figures in a total count of 100 hepatocytes, group C.24h was found to contain 0.3 ± 0.7 mitotic figures and group E.24h 0.5 ± 0.5 mitotic figures in 100 hepatocytes (p = 0.26).After seven days, group C.7d contained 1.9 ± 1.5 mitotic figures and group E.7d 1.6 ± 1.5 mitotic figures in 100 hepatocytes (p = 0.66).
The PCNA-positive nuclei count in ten fields was assessed in all groups and significant statistical difference was found only when compared the experimental groups (p = 0.0001) (Table 3).
for studying liver regeneration, most of researchers chooses the stimulus like liver resection, where the remaining liver mass is normal, using for this reason the animal model of 70% hepatectomy described by Higgins and Anderson 3 .This is a simple and reproducible model with low mortality, in resistant and low cost animals.The authors used adult Wistar rats, completely developed, weight over 350g, in order to analyze the phenomena of liver regeneration with no humoral, general or specifics mechanisms.
Kim et al. 11 and Daoudaki et al. 8 demonstrated augmentation in hepatocyte proliferation in hepatectomized rats that were submitted to pretherapy with cyclosporine.However, other authors didn't achieve the same results 12,13 .In this study, the authors used oral cyclosporine in a 10mg/Kg/day with rigid plastic orogastric tube as described by other researchers [9][10][11][15][16][17][18] . All grs lost body weight in the moment of death with statistical significance.When compared 7 days experimental and control groups, the first one lost more weight than the last one on the second, fourth and death day (p = 0.03; p = 0.01; p = 0.02 respectively), proving the influence of the drug on animals weight during the research.Tannuri et al. 19 had the same results when resected 70% of newborn liver rats and treated one group with cyclosporine pretherapy (p = 0.01).
Biondo-Simões et al. 20 compared 90 days young rats and 560 days adult rats submitted to 70% hepatectomy.These animals were killed 24 hours and seven days after the procedure and liver regeneration was analyzed with Kwon's formula, mitotic index and immunohistochemistry with PCNA.The young animals regenerated more than the older ones in 24 hours and seven days, but in seven days the adults reached the same hepatic mass that the young rats reached in 24 hours.In this present study, using Kwon's formula, seven days control group regenerated more than the 24 hours group (p = 0.0001) and the same occurred in the experimental group (p = 0.0001).That's because the cell cycle of the hepatocytes has a peak in 24 hours 4 and in non-parenchymal cells that peak occurs 24 hours later 21 .In resections over than 50%, like in this research, a second peak of mitosis in rats occurs three days after the first one 1 .When compared 24 hours control/ experimental groups and seven days control/experimental groups, only in the last one there was statistical significance (p = 0.05), with higher media in the rats treated with cyclosporine.
Dahmen et al. 22  As occurred with PCNA, the Ki67-positive nuclei count in ten fields was assessed in all groups and significant statistical difference was found only when compared experimental groups (p = 0.04) (Table 4).

Discussion
The influence of cyclosporine in liver regeneration has been a controversial issue.In an experimental point of view, . The gavage with cyclosporine or sodium chloride solution was maintained daily until animals' death.The control subgroup (C.24h) and the study subgroup (E.24h) were killed 24 hours after the hepatectomy.The other two subgroups were killed 7 days after the liver resection (C.7d and E.7d).Sacrifice was by inhaled lethal dose of ethylic ether.The regenerated liver was resected and weighted to calculate the percentage of regeneration.A portion of the superior right lobe was sampled, fixed in 10% buffered formalin and sent for histological examination.Slides were prepared with 4 micrometer thick cuts.These were stained with hematoxylin-eosin to evaluate the mitotic figure count while immunohistochemistry was used to evaluate PCNA and Ki67 positive nuclei count.Liver regeneration was assessed using four methods: Kwon's formula, percentage of mitotic figures in 100 hepatic cells, by the average of PCNA and Ki67 positive nuclei in ten fields.Kwon's formula gives the regeneration rate based on weight.% = D / E. 100 where G = R / 0.7 Where: D = liver weight per 100 g body weight on the day that the animal was killed.E = the estimated liver weight per 100 g of body weight before the hepatectomy and is calculated using the weight of the resected liver (R).G = estimated liver weight at the time of the hepatectomy.
Note: min-max -minimum and maximum values, sd -standard deviation, p -statistical significance value.

FIGURE 1 -
FIGURE 1 -Initial and death body weight media ± standard deviation.(A) C.24H: 24 hours control group; (B) C.7d: 7 days control group; (C) E.24H: 24 hours experimental group; (D): E.7d: 7 days experimental group; (E) PCI: initial body weight; (F) PCM: death body weight.Statistical comparations among groups (p).When seven days experimental and control groups were compared during the research, the first one lost weight statistically significant on second and fourth postoperative days and on death's moment too (Figure2).

FIGURE 2 -TABLE 2 -
FIGURE 2 -Seven days control and experimental subgroups during the research.Statistical comparations among groups (p).

TABLE 1 -
Statistical analysis of liver regeneration by kwon' formula in 24 hours and seven days in the control and experimental groups.

TABLE 3 -
Statistical analysis of percentage of PCNApositive nuclei among groups.
Note: sd -standard deviation, p -statistical significance value.

TABLE 4 -
Statistical analysis of percentage of KI67positive nuclei among groups.Note: sd -standard deviation, p -statistical significance value.