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Acta Cirúrgica Brasileira

versão On-line ISSN 1678-2674

Acta Cir. Bras. vol.27 no.1 São Paulo jan. 2012 



The role of N-acetyl-cysteine in the lung remote injury after hepatic ischemia and reperfusion in rabbits1


O papel da N-acetil-cisteína nas lesões pulmonares provocadas à distância pela isquemia e reperfusão hepática em coelhos



Angela Potter de CastroI; Miguel Angelo Martins de Castro JuniorII; Susi LauzIII; Emilio FacinIV; Manuel de Jesus SimõesV; Djalma José FagundesVI

IMSc, Medical Veterinary, Surgery and Veterinary Pathology/Pharmacology, FURG. Fellow PhD degree, Postgraduate Program in Experimental Surgery, UNIFESP, Sao Paulo, Brazil. Main author. Responsible for conception, design, intellectual and scientific content of the study
IIMSc, Associate Professor, FURG, Rio Grande, Brazil. Surgical procedures, manuscript writing
IIIPhD, Associate Professor of Surgery, FURG, Rio Grande, Brazil. Acquisition and interpretation of data, critical revision
IVGraduate student, Scientific Initiation Program, FURG, Rio Grande, Brazil. Surgical procedures, manuscript writing
VPhD, Associate Professor, Histology and Structural Biology Division, Department of Morphology, UNIFESP, Sao Paulo, Brazil. Interpretation of data, histophatological analysis
VIPhD, Associate Professor, Operative Technique and Experimental Surgery Division, Department of Surgery, UNIFESP, Sao Paulo, Brazil. Tutor. Responsible for conception and critical revision of the study





PURPOSE: To study the lesions in the lung of rabbits caused by ischemia/reperfusion hepatic (I/R) after the use of N-acetyl-cysteine (NAC).
METHODS: Twenty-four rabbits distributed in two groups: control group GI (n = 12) 5% glucose solution and experiment group GII (n = 12) NAC. The animals were pre-anesthetized with 1% acepromazine maleate and anesthetized with ketamine 10% and 2% xylazine intramuscularly. The GI and GII were given glucose solution intravenously or NAC 15min before occlusion of the hepatic pedicle (30 min). After the period of reperfusion of 24h (n = 6) or 48h (n = 6), liver and lung samples were collected for histology and immunohistochemistry to assess the impairment of cell.
RESULTS: The animals of GII and GII-24h-48h showed parenchyma liver close to normal, when using NAC. The GII and GII-24h-48h showed lower thickness of alveolar cells that GI and GI-24h-48h. The expression of caspase 3 in lung cells GII presented smaller value compared to the GI group.
CONCLUSION: N-acetyl-cysteine administered 15min prior to the injury ischemia/reperfusion had a significant protective role by minimizing lung injury and apoptotic morphology in the period observed.

Key words: Acetylcysteine. Lung. Reperfusion. Ischemia. Liver. Rabbits.


OBJETIVO: Estudar as lesões no fígado e no pulmão de coelhos, provocadas pela isquemia/reperfusão hepática (I/R) moduladas pelo uso da N-acetil-cisteína (NAC).
MÉTODOS: Vinte e quatro coelhos distribuídos em dois grupos: Grupo controle GI (n=12) solução de glicose 5% e Grupo experimento GII (n=12) NAC. Os animais foram pré-anestesiados com maleato de acepromazina 1% e anestesiados com cloridrato de quetamina 10% e xilazina 2% via intramuscular. Os grupos GI e GII receberam solução glicosada ou NAC respectivamente via endovenosa 15min antes da oclusão do pedículo hepático (30 min). Após iniciou-se o período de reperfusão por 24h (n=6) ou 48h (n=6), terminada a reperfusão, amostras do fígado e pulmão foram coletadas para a histologia e imunoistoquímica para avaliar o comprometimento celular.
RESULTADOS: Os animais do grupo GII-24h e GII-48h apresentaram arquitetura do parênquima hepático próximo do normal, quando se utilizou NAC. Os grupos GII-24h e GII-48h apresentaram menor espessura das células alveolares que os grupos GI-24h e GI-48h. A expressão da caspase 3 nas células pulmonares do grupo GII, apresentou valor menor comparada ao grupo GI.
CONCLUSÃO: A N-acetil-cisteína administrada 15min prévios a lesão da isquemia/reperfusão teve um papel protetor significativo minimizando as lesões morfológicas e apoptóticas pulmonares nos períodos observados.

Descritores: Acetilcisteína. Pulmão. Reperfusão. Isquemia. Fígado. Coelhos.




Ischemia is the interruption of blood flow, consequently the supply of oxygen and nutrients to a tissue or organ. This condition may be reversible or not, depending on the metabolic characteristics of the tissue involved and the environment1.

During the interruption of blood supply to a tissue cell hypoxia occurs, initiating a sequence of biochemical events such as the interruption of the respiratory chain, the consumption, degradation of nucleotide triphosphate, mobilization of calcium from intracellular stores, loss of function of membrane and activation-dependent enzymes calico2-4.

There is evidence that tissue injury is not limited to ischemia, but extends to the process of reperfusion, and this may be defined as the damage that occurs in specific tissue by restoring blood flow after a period of ischemia5. Among the highlights are the injuries necrosis and apoptosis, and the determining factor is the intensity of damage5,6.

Thus, reperfusion damage may extend to organs located at a distance, due to a systemic inflammatory response in which inflammatory mediators are produced and released into circulation4,7.

Ischemia/reperfusion (I/R) play an important role in hepatic resections, liver transplants and in hemorrhagic shock8,9.

The lung is most affected organ by the distance I/R liver injury. Evidenced by significant morphological changes such as perivascular edema, intravascular platelet aggregation and increased tumor necrosis factor alpha (TNF) in bronchoalveolar lavage, and appears to systemic inflammation also known as hepatopulmonary syndrome10.

Because of the systemic inflammatory response, the I/R injury liver induces the production of reactive oxygen species (ROS) whose distance deleterious effects may be more deleterious than local effects11,12.

The ROS play a key role in lung injury resulted from reperfused tissues. These relate to the injury of the alveolar-capillary membrane and consequent transudation that contribute to acute respiratory distress syndrome (ARDS)3.

The main cause of cell injury is oxidizing substances produced by the cells previously ischemic or activated leukocytes11. Many injuries are developed during the tissue reperfusion resulted from reoxygenation. Researches of mechanisms to protect the damage of I/R considers the use of substances antioxidantes13,14. Among these, glutathione (GSH) is considered effective in ischemia stage15,16. GSH is synthesized by the liver in two ways, catalyzed by γ-glutamyl cysteine synthetase and glutathione synthetase, distributed through the bloodstream to all tissues. An important exogenous source of glutathione is represented by NAC17.

NAC is a molecule that shows a link thiol (sulfur) bound to the amino acid L-cysteine. However, is not present in the diet and should be obtained through dietary supplements. This substance has been used in the treatment of pulmonary diseases, as an antidote for poisoning by paracetamol in heavy metal poisoning, diseases of the immune system, and studies with apoptose18.

The antioxidant activity of NAC occurs directly by convert hydrogen peroxide into less reactive compounds and/or indirectly, when induce the biosynthesis of GSH (reduced glutathione)18.

Was observed at literature that the effects of NAC range from 15 to 6 minutes to ischemia and periods of 2 to 4 hours of reperfusion. At our literature review was not observed periods over than 4 hours to reperfusion. Thus, it was pertinent to study the NAC as a protective agent on the I/R liver injury and on the remote damage to the lung parenchyma by morphological analysis in after longer times of reperfusion (24 and 48 hours) in a rabbit experimental model.



The research project was approved by the Ethics Commitee of Federal University of Sao Paulo, ratified by the Federal University of Rio Grande This study follows the standards of animal research, recommended by the Brazilian College of Animal Experimentation, an institution affiliated of International Council for Laboratory Animal Science.

We used 24 rabbits (N = 24), albino, New Zealand (Oryctolagus cuniculus), males, four months, average weight 3Kg from Federal University of Pelotas.

The animals were evaluated randomly into two groups: the control group GI (n = 12) received 5% glucose solution and the experimental group GII (n = 12) received NAC, administered intravenously (IV) 15 min before ischemia. The animals were pre-anesthetized with acepromazine maleate 1% dose of 2mg.Kg-1 and then anesthetized with a combination of ketamine at a dose of 10% 35mg.Kg-1 and 2% xylazine at a dose of intramuscularly (IM). We performed a transverse incision of 4 cm in the right upper quadrant and identified the portal triad (hepatic artery, portal vein and biliary ducts) and the vena cava. Fifteen minutes before clamping the hepatic pedicle was injected intravenously 1mL.Kg-1 glucose solution 5% in the control group. In the Experimental group was injected in the same period the NAC at a dose of 150 mg.Kg-1, diluted in 5% glucose solution. Than was proceed the hepatic ischemia for 30 minutes. After, the clamp was removed and the synthesis of the abdominal wall was performed with sutures. After it was started in the reperfusion time of 24 hours (n = 6) or 48 (n = 6). The animals received analgesia with Dipyrone Sodium 500mg dose of 0.3 mL.Kg-1 at the end of operative procedure intravenously and after 6 hours of procedure. Antimicrobial prophylaxis was performed in all animals at a dose of 1g cephalothin 20mg.Kg-1. After reperfusion, animals were anesthetized and submitted to thoracolaparotomy to remove a fragment of liver and lung.

Samples of liver and lung were processed for hematoxylin eosin staining and immunohistochemistry. Twenty random fields were chosen in each section of each animal and set relevant points of each group. There has been a qualitative and / or quantitative sign of liver or lung lesions.

The liver was analyzed for the presence or absence of congestion of central lobular vein, the portal space and vasodilation, while the lung was evaluated: thickening of alveolar septa, leukocyte infiltration, hemorrhage, edema and the presence of macrophages in the septa.

For lesions absent or present in up to two fields, given the value zero (0), for the presence of mild lesions from 3 to 8 fields set itself the value of a (+), presence of moderate injuries from 9 to 14 fields assigned the value two (++) and for the accentuated presence of injuries from 15 to 20 fields assigned the value three (+++), counting procedure was performed by a software (Image Lab), where they obtained images of 10 fields in each slide. For this we used high-resolution camera AxionCam RLX ™ (Carl Zeiss) light microscope adapted Axiolab™ (Carl Zeiss) and Pentium 4™ with Axiovision™ program REL 4.2 (Carl Zeiss).

Lungs fragments were processed for immunohistochemical labeling of caspase 3 and its expression as a measure of lung cell apoptosis using the avidin-biotin-peroxidase (ABC). The immunohistochemical expression of caspase 3 was identified and quantified by computer program (program AxioVision Rel® 4.2 - Carl Zeiss) and adopted as positivity the appearance of brown color in the core, at region of the boundary cell or the cell cytoplasm. The rate of apoptosis and cell proliferation was calculated using the percentage of labeled cells per 1000 cells counted in twenty random fields and presentation of such measures as mean and standard deviation.

Statistical analysis was prepared by PGS Medical Statistics (Sao Paulo, Brazil). To assess the apoptosis and cell proliferation between groups was used the test of analysis of variance (ANOVA) test and multiple comparison Student-Newman Keuls test for comparing two columns, we adopted the significance level of 0.05 (α < 5%) and levels (p) below this value were considered significant.













Hepatic ischemia is a frequent event today either in trauma surgery or liver transplantation. The evaluation of the phenomenon of I/R has been reported in animal models, but there are restrictions when transposing the results to humans.

The rabbit a rodent to be larger and easier handling was considered appropriate for this research9-21. To these factors is associated with ease of anesthesia protocol, which provided adequate hypnosis and analgesia, an anesthetic for up to 60 minutes.

Among the different experimental models of injury I/R liver stands out the Pringle maneuver, this allows the study of ischemic injury in the affected organ or organs at a distance20.

The changes of ischemia may be evolving into molecular or biochemical histological changes, which in turn, can range from discrete morphological lesions to necrosis. Thus, the choice of the ischemia duration is crucial for the experiment is that depending on time can lead to changes irreversible4-5.

In order to make the proposed objective in this study we chose the ischemic time of thirty minutes in order to mimic a condition that is encountered in clinical practice and to serve as a reliable and reproducible model.

For the period of reperfusion was chosen by a time of 48 hours long considered when comparing to previous studies, because in short periods is well-known how tecidual injury occurs4,22,23.

Substances have been used in order to minimize injuries from I/R including the NAC to be a mucolytic drug which is used in lung diseases, paracetamol poisoning, and application in organs and tissues subjected to ischemia and reperfusion. Its pharmacological effects include suppression of the production of tumor necrosis factor alpha, inhibit platelet aggregation, modulation of cell activation inflammatory18.

Thus, the hypothesis that NAC acts directly protecting the cell damage may be considered, because the properties attributed to the antioxidant NAC and inhibition of inflammatory processes of lung cells are described in studies at different times of reperfusion and with application prior or during ischemia hepátic22,24-26.

The use of NAC 15 minutes before starting the ischemia is based on the biodistribution of drug in plasma and tissues27. Thus its offer directly into the bloodstream are loosely based on reports suggesting that this dose and time prior to application, are suitable for tissue concentrations sufficient to minimize or eliminate the effects of biochemical and structural repercussions on the parenchyma pulmonar27-29.

Lung function depends directly on the cell membrane integrity of the alveolar-capillary network of the lung, whose structure is particularly sensitive to factors related to I/R, as the decrease in cellular energy levels and the action of EROs30.

As described in Table 2, the groups subjected to I/R liver without prior administration of NAC, and a period of 24 hours of reperfusion, showed pathological changes in lung parenchyma (alveolar septal thickening, leukocyte infiltration, hemorrhage and pulmonary edema) in quantity higher than the group that received NAC prior to ischemia (Figure 3, A and C). The same finding was found in animals subjected to reperfusion for 48 hours without prior administration of NAC (Figure 3, B and D). Than concluded that NAC decreased lung lesions observed in microscopic HE staining.

In view of theoretical knowledge about the mechanism of action of ischemia and reperfusion can be inferred that there was a stronger action of ROS in the control group, since it is known that they cause direct cellular injury and stimulate the production of inflammatory mediators, thus may have contributed to the increased capillary permeability and the appearance of pulmonary edema. The swelling may have been the result of dysfunction of vascular endothelium mediated primarily by the release of ROS and an imbalance between vasodilators and vasoconstrictors. Literature reports that rats subjected to liver ischemia for a period of 30min and previous administration of NAC was an attenuation of the injuries I/R in the assessment of optical microscopy31.

The evaluation of the thickness of the pulmonary alveoli is a morphological parameter that is involved in reactive processes to a lung injury. This increased thickness may be due to fluid extravasation into the interstitial space or an increase of cellular elements. In the control group, according to table 3, the thickness of the alveoli was significantly higher than the rates displayed by animals in the NAC group, they did so both in the evaluation of 24h as 48h.

At table 4, was observed lower lesions at lung parenchyma in the groups received NAC, this result may be related to antioxidant effect.

Concluded that the lesions were produced at a distance in response to the period from I/R and probably by oxidizing substances derived from reactive oxygen species, since they were inhibited by NAC.

Considering the theoretical and experimental findings and to provide more substantial support that oxygen free radicals may be involved in the mechanism of cellular injury after lung distance hepatic ischemia and reperfusion, we assessed cell apoptosis by immunohistochemical expression of caspase 3. Since the expression of caspase 3 is related to the implementation phase of the process and therefore irreversible, appeared to be a good indicator of their assessment of the damage26.

NAC is one of the antioxidants that have shown promise in preventing and decreasing the deleterious effects of reactive oxygen species. Thus, NAC demonstrated to be an interesting substance, in studying the development of new solutions for organ preservation with a higher antioxidant potential and effectiveness (Figure 4).

New experimental works, with new techniques of application of NAC, are necessary to prove its actual effectiveness in different organs during reperfusion injury especially in distant lesions in other organs and systems.



The previous application of N-acetyl-cysteine protects the lung morphological changes caused by hepatic ischemia reperfusion after periods of 24 and 48 hours. The NAC promotes a decreased expression of caspase 3 in lung tissue resulting from injury caused by the distance hepatic ischemia for prolonged periods of 24 and 48 hours after reperfusion.



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Angela Pötter de Castro
Rua Moron, 469
96200-450 Rio Grande - RS Brasil

Received: August 09, 2011
Review: October 10, 2011
Accepted: November 14, 2011
Conflict of interest: none
Financial source: none



1 Research performed at Department of Morphology, Laboratory of Histology and Structural Biology, Laboratory of Experimental Surgery and Operative Technique, Federal University of Sao Paulo (UNIFESP) and Laboratory Surgical Technique, Federal University of Rio Grande (FURG). Part of PhD thesis. Tutor: Djalma José Fagundes.

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