Effect of N-acetylcysteine in liver ischemia-reperfusion injury after 30% hepatectomy in mice1

PURPOSE: Evaluate the effect of N-acetylcysteine in liver remnant after hepatectomy associated to ischemia-reperfusion injury in mice. METHODS: Male adult BALB/c mice, weighing 20-22g were used. Animals were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg); received N-acetylcysteine (150 mg/kg, H-IR-NAC group) or vehicle (H-IR group). Surgical procedures were performed under 10X magnification. Partial hepatectomy (30%) was followed by ischemia-reperfusion injury (30 minutes of ischemia and 60 minutes of reperfusion). Blood sample and liver tissue were removed before animal was euthanized. AST and ALT were evaluated in blood samples and histomorphological analyses were performed in remnant liver. Groups were compared by Mann-Whitney test, and it was considered significant when p<0.05. RESULTS: Biochemical evaluations showed reduced levels of ALT in NAC group (H-IR-NAC=376±127U/l vs H-IR=636±39U/l, p=0.023). AST was similar (p=0.456). H-IR group showed hepatic tissue with preserved architecture, large area of steatosis, vascular congestion and rare mitogenic activity. NAC group showed hepatic tissue with small area of steatosis, vascular congestion and elevated mitogenic activity, evidenced by increased binuclear cells (H-IR-NAC=15.88±0.52 vs H-IR=7.4±0.37, p<0.001). CONCLUSION: N-acetylcysteine promotes enzymatic and morphological protection against hepatectomy and ischemia-reperfusion injury.


Introduction
The mortality in the transplant waiting list is still very high, due to scarcity of compatible donors for pediatric recipient 1 .
Some solutions have been made to increase the number of disposable graft: adult liver reduction, division of cadaver liver providing two possible grafts, or use of grafts from live donors 1-3 .
All of these techniques contributed to improve the number of liver grafts. In the other hand, the existing disproportion between graft and recipient is still a relevant problem: the inappropriate size brings hemodynamic and functional implications, occurring primary non-function or dysfunction 3 .
The ischemia-reperfusion lesion related to liver surgery and transplantation, in association to volume of parenchyma reduction, can synergically compromise the organ, stimulating laboratorial research to overcome such problems [2][3][4] .
Antioxidative vitamins, enzymes or other free radical scavengers have been shown diminish or neutralize oxygen and nitrogen free radicals 5 , 6 . A low weight thiol, the N-acetylcysteine (NAC), has its use established in the acetaminophen intoxication, protecting the liver by increasing hepatic glutathione 7 .
NAC has been used in clinical practices in situations as hemodynamic instabilities, protecting hepatic, pulmonary, cardiac and renal tissues, compromised by ischemia and reperfusion. This applicability is supported by experimental animal studies, some done by our research team that showed local and systemic protection after ischemia-reperfusion injury [8][9][10] . NAC acts like an antioxidant beyond improving blood flow in microcirculation 11 .
Since N-acetylcysteine shows protective action against ischemia and reperfusion lesion and that manipulation of liver promotes microcirculation changes, modifying the organ perfusion, it was decided to evaluate if N-acetylcysteine promotes protection to remnant liver after hepatectomy of 30% associated to liver ischemia-reperfusion injury in mice.

Methods
The experimental protocol was submitted and approved by UNIFESP Ethics Committee on Animal Research (CEP#1945/07). It was used twelve adult male BALB/c mice supplied by the Experimental Animal Center of UNIFESP, ranging in age from six to eight weeks and weighing 20-22g. The animals were randomly allocated into two groups: group H-IR (n = 6) subjected to hepatectomy + ischemia/reperfusion; group H-IR-NAC (n = 6) pretreated with N-acetylcysteine (NAC) (150 mg/kg) and then hepatectomy+ischemia/reperfusion, as for the preceding group.
Animal preparation consisted of ketamine and xylazine anesthesia (70 and 10 mg.kg −1 , respectively) intramuscularly, followed by a midline laparotomy and hydration with warm saline solution (3 mL. kg −1 . h −1 ). Animals were kept over a heating pad during all experimental procedures.
The surgical procedures were performed as follows:

Ischemia/Reperfusion
Fifteen minutes after laparotomy, the hepatic hilum was occluded for 30 minutes with an atraumatic clip placed across it interrupting the blood supply, followed by 60 minutes of reperfusion.

Hepatectomy
During the occlusion, 30% of the liver (right lobes, caudate lobe and quadrate process) was excised, as previously

Biochemical dosage
The sampled blood was centrifuged and the sera were used for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) determinations, using Cobas Mira.

Statistical analysis
Nonparametric statistical tests were used to compare differences between experimental groups. Values are considered significantly different for p≤0.05.

Results
The hepatectomy + ischemia/reperfusion group (H-IR) presented a preserved hepatic architecture. However, it was observed presence of vascular congestion, hepatocytes with cytoplasmatic light areas, indicating lipidic infiltration ( Figure   1A). In hepatectomy + ischemia/reperfusion group treated with

Discussion
There are numerous potential mechanisms of action by which NAC may exert protective effects 13 . But the interesting about this experiment was to see the enormous increase in binuclear hepatocytes, after use of NAC. Due to the liver fast metabolism, it is very common to appear polyploidy nucleus (having higher genetic load than normal), so more transcriptions happen. In our study, part of the liver was excised, leading to over function of the remaining hepatocytes. The increase in this short period of time (about two hours) is exciting, because N-acetylcysteine could not only protect but help in recovery.
James et al. 14 observed that IL-6 was increased because of N-acetylcysteine in the liver tissue, denoting a mitogenic stimulus, as well. Glantzounis et al. 18 showed that microcirculation and alanine aminotransferase activity were better when N-acetylcysteine was used, corroborating our findings related to vascular congestion in the parenchyma and ALT levels.
It has been proposed that nitric oxide has an important role in the vascular congestion improvement induced by N-acetylcysteine 15 .
Hogg et al. 15 proposed a protective mechanism by low weight thiol, in which nitric oxide reacts with glutathione, improving the microcirculation, attenuating vascular congestion and tissue injury. Also ALT level was lower after NAC use. AST and ALT are useful screening tests for detecting liver injury. They are found in hepatocytes and cannot diffuse out of the cells in the physiological condition. When the hepatocyte is injured, plasma membrane can be disrupted and the leakage through extracellular fluid of the enzyme occurs where they can be detected at abnormal levels in the serum. So the ALT reduction could be the protection given by N-acetylcysteine as suggested previously 14 .
The ischemia-reperfusion lesion associated to liver transplantation can synergically compromise the organ after transplant. It is in this context that N-acetylcysteine, as protective

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strategy, could minimize the lesion, promoting best functionality of the graft. Further experiments are currently under way to address this hypothesis.

Conclusion
The N-acetylcysteine promotes protection to the remnant liver after hepatectomy and ischemia-reperfusion in mice, based on biochemical and morphological analysis.