Effect of Ischemic Postconditioning and Atorvastatin in the Prevention of Remote Lung Reperfusion Injury

Objective The aim of the present study was to evaluate the ability of ischemic postconditioning, atorvastatin and both associated to prevent or minimize reperfusion injury in the lung of rats subjected to ischemia and reperfusion by abdominal aortic clamping. Methods We used 41 Wistar norvegic rats, which were distributed into 5 groups: ischemia and reperfusion (I/R), ischemic postcondictioning (IPC), postconditioning + atorvastatin (IPC+A), atorvastatin (A) and SHAM. It was performed a medium laparotomy, dissection and isolation of the infra-renal abdominal aorta; except for the SHAM group, all the others were submitted to the aortic clamping for 70 minutes (ischemia) and posterior clamp removal (reperfusion, 70 minutes). In the IPC and IPC+A groups, postconditioning was performed between the ischemia and reperfusion phases by four cycles of reperfusion and ischemia lasting 30 seconds each. In the IPC+A and A groups, preceding the surgical procedure, administration of 3.4 mg/day of atorvastatin was performed for seven days by gavage. After the surgical procedure, the right caudal lobe was removed from the lung for histological study, using tissue injury score ranging from grade 1 (normal tissue) to grade 4 (intense lesion). Results The mean lung injury was 3.6 in the I/R group, 1.6 in the IPC group, 1.2 in the IPC+A group, 1.2 in the A group, and 1 in the SHAM group (P<0.01). Conclusion Ischemic postconditioning and atorvastatin were able to minimize lung reperfusion injury, alone or in combination.


INTRODUCTION
Reperfusion is a fundamental step in the treatment of ischemia. However, clinical and experimental evidence shows that the main events leading to cell and tissue dysfunction are related to reperfusion [1] . maintained gas exchange, since reactive oxygen species (ROS) act systemically. In surgeries with temporary aortic occlusion, pulmonary edema is a frequent complication, with a multifactorial etiology, including reperfusion injury. Already during ischemia, there is an increase in pulmonary arterial pressure, a factor that may favor the formation of edema in the lungs. This increased resistance in the pulmonary circulation results in part from a greater blood flow due to its redistribution to the territory above the occlusion and from the increase in left ventricular end-diastolic volume, whose emptying is impaired by the increase that the occlusion of the aorta imposes on post-loading [4] .
IR is associated with the production of another inflammatory mediator, tumor necrosis factor (TNF). Lesion of the intestinal mucosa by IR allows the release of endotoxin into the portal circulation, inducing the production of TNF by liver macrophages. Increased TNF in the systemic circulation is capable of leading to inflammatory lung injury, characterized by accumulation of neutrophils. This sequence of events was demonstrated by Caty et al. [5] in an IR model by temporary occlusion of the superior mesenteric artery in rats. After reperfusion, endotoxin levels in portal venous blood and TNF were increased in the systemic circulation. In parallel, there was accumulation of neutrophils in the lungs and increased pulmonary capillary permeability.
Some techniques for protection against reperfusion injury have already been tried and tested, and among them, ischemic postconditioning (IPC), which consists of one or more short cycles of reperfusion, followed by one or more short cycles of ischemia, immediately after the ischemic phase and before permanent reperfusion occurs. Although IPC has already shown a protective effect in many organs submitted to IR as well as in distance protection [6] , its efficacy in the prevention of remote lung injury is still very early [7] .
Much has been studied about the pathophysiology of reperfusion injury and some mechanisms have already been well evidenced, such as the role of free radicals, vascular endothelial dysfunction, and neutrophil-mediated injury [1] . Recently, there has been an increase in interest in statins, drugs known for their antidislipidemic effect, this time due to its pleiotropic effect, which is characterized by anti-inflammatory properties, immunomodulating, antithrombogenic actions and improvement of endothelial function [8] . Recent experimental studies [9] have shown promising results with the use of statins demonstrating their role in the protection against IR injury, a fact that led us to inquire about their benefits facing reperfusion injury.
The aim of the present study was to evaluate the effect of IPC and atorvastatin, alone and in combination, in the prevention of reperfusion injury in the lungs of rats subjected to IR by aortic clamping.

METHODS
The study was approved by the Committee of Ethics in Animal Experimentation of the Universidade Anhanguera-Uniderp. A total of 41 Wistar norvergic male rats weighing 250 g to 300 g were collected from the Hospital Veterinário da Universidade Anhanguera-Uniderp. The animals were kept in cages at ambient temperature of approximately 23°C with 12h light cycles and received water and food ad libitum.
The animals were distributed in the following groups: • and isolation of infra-renal aorta. The animals were anesthetized by intraperitoneal injection of a 2:1 solution of ketamine hydrochloride (Cetamin ® ), 50 mg/mL, and xylasine hydrochloride (Xilazin ® ), 20 mg/mL, respectively, at a dose of 0.1 mL/100 g.
After anesthesia, the rats were submitted to a median longitudinal laparotomy of approximately four centimeters, exteriorization of the small intestine, identification and dissection of infra-renal abdominal aorta artery.
In all groups except SHAM, the abdominal aorta was occluded by atraumatic vascular clamp that remained for 70 minutes (ischemia phase). After clamp placement, the small intestine was repositioned into the abdominal cavity and the surgical wound was closed with continuous suture of the skin with 4-0 monofilament nylon. After the ischemia phase, the abdominal wall was reopened by removal of the suture and in the I/R and A groups, the vascular clamp was removed, initiating the reperfusion phase, lasting 70 minutes. In the IPC and IPC+A groups, preceding the reperfusion phase, the IPC was performed by four cycles of reperfusion (removal of the atraumatic vascular clamping of the abdominal aorta) with duration of 30 seconds each, interspersed by four cycles of ischemia (occlusion of the abdominal aorta artery by atraumatic vascular clamp), also with duration of 30 seconds each.
In all groups after the beginning of the reperfusion phase, the abdomen was closed again by continuous suturing of the skin with 4-0 monofilament nylon thread until the end of the experiment.
In the SHAM group, only a median longitudinal laparotomy of approximately four centimeters was performed, with exteriorization of the small intestine, identification and dissection of the infra-renal abdominal aorta artery, remaining anesthetized for 140 minutes.
After the reperfusion phase, all animals were submitted to median thoracotomy and resection of the right caudal lung lobe, and these specimens were washed with saline solution and especially in the lung. As a result, many evaluated the impact of IPC in subsequent apoptotic and inflammatory responses. In experimental IR models in rats with 30 minutes of ischemia and three hours of reperfusion there was a significant decrease in tissue necrosis with IPC. There is also a decrease in ROS generation and protection of mitochondrial integrity, suggesting that the protective effect of IPC may be the result of a reduction in the inflammatory response. However, few studies have directly assessed the impact of IPC on inflammation. IPC may limit the expression of P-selectin, which is required for neutrophil bearing and recruitment. In addition, it may reduce the accumulation of neutrophils in the affected region, decrease to ischemic vascular endothelial adhesion, and attenuate the endothelial dysfunction of the involved vessel, events that normally occur in IR [11] .
The main histological findings observed in the different groups can be observed in Figures 2 to 6.
In the present study, we observed pulmonary protection with IPC, demonstrating the efficacy of the method against this IR model, which can be justified by the fact that ROS, regardless of placed in 10% formaldehyde solution for histological analysis.
Euthanasia was performed by intraperitoneal administration of a lethal dose of ketamine + xylazine hydrochloride (0.4 mL/100 g).
The slides were prepared with the harvested material, which was stained with hematoxylin-eosin and analyzed by optical microscopy by a single observer, without prior knowledge of it on the group belonging to each rat.
Pulmonary segments were classified according to the degree of tissue injury as stated by Greca et al. [10] .
• • Grade 1 (normal): normal parenchyma under optical microscopy; • • Grade 2 (mild): focal edema in few alveolar septa, mild congestion, neutrophils in alveolar septa, less than 50 per large increase field; • • Grade 3 (moderate): moderate edema in alveolar septa or mild edema in several septa, moderate congestion, neutrophils in alveolar septa between 50 and 100 per large increase field; • • Grade 4 (intense): severe edema in alveolar septa or mild edema in several septa, moderate congestion, neutrophils in alveolar septa, more than 100 per field. Measurements were expressed as mean and standard deviation in the variables whose distribution was normal, and in the median and interquartile range when normality was not observed. For the verification of normality, the Shapiro-Wilk test was used. For the intergroup comparison, Kruskal-Wallis test with Dunn's post hoc test was used. Significance level was considered P<0.05.

RESULTS
The averages of degrees of tissue injury were 3.6 in the I/R group, 1.6 in the IPC group, 1.2 in the IPC+A and A groups, and 1 in the SHAM group (Table 1 and Figure 1).

DISCUSSION
Ischemia followed by reperfusion may induce apoptosis and an inflammatory response that affects tissue repair,  IPC was performed on the lower limbs of rats, with three hours of ischemia and four hours of reperfusion. Santos et al. [6] also performed mesenteric IR in rats, but used 30 minutes of ischemia and 60 minutes of reperfusion, causing intestinal lesions but not pulmonary lesions, thus demonstrating that these animals did not present significant damage to the lungs with these reduced periods of intestinal IR. As mentioned, there are few publications aimed at evaluating remote lung damage against IR, especially using IPC as a protection method. Existing publications point to a promising role in this method, similar to other IR situations in which IPC provides tissue protection [14] . Despite this, there are few studies that have used IPC in clinical practice, which reinforces the importance of finding a pharmacological method that presents the same or greater efficacy, increasing interest in safe drugs such as statins and that at least their pleiotropic effect could be useful in such situations.
In the present study, pulmonary protection was obtained with the use of atorvastatin, in the same intensity as with IPC. As there are no studies with the same design used here, i.e. aortic clamping Santos CHM, et al where they are produced, when reperfusion occurs throughout the body causing the remote reperfusion injury, so much so that in the I/R group a marked lesion in the lungs was observed. By acting as a moderator of ROS production, IPC causes less local and distant injury [4,7] .
The study of the remote reperfusion injury in the lungs is a relatively new subject, therefore, without publications that allow us to compare further with the present results. Dorsa et al. [7] also performed ischemia by aortic clamping and evaluated the protective effect of IPC, confirming the results presented here that this method may decrease the degree of lung injury. Another study that obtained similar results was that of Garbaisz et al. [12] , in which the authors also applied infrarenal aortic clamping as a method of ischemia and obtained pulmonary protection with IPC, although using times superior to those presented here for both ischemia (3h) and for reperfusion (4h). This shows that, for Wistar rats, 70 minutes of IR are sufficient to produce pulmonary lesions, and no further prolonged experiments are needed as the used by those authors.
simulating what is practiced in humans, that is, the absorption by the gastrointestinal tract, aiming its clinical applicability. Statins have been successfully tested for this purpose in several situations. Wu et al. [15] performed renal IR in rats and demonstrated that atorvastatin decreased tissue injury in the and atorvastatin use, the comparison with the literature is also impaired. Moreover, since the use of statins for the prevention of reperfusion injury is relatively new, the best route of administration and ideal dose are items to be better clarified in future research. Administration by gavage was chosen here with the intention of control group. The same results were obtained by Cusumano et al. [16] in renal IR of rats using atorvastatin.
Statins also protect other tissues in the presence of IR, such as heart [17][18][19] , nervous system [20] and liver [21] . In the lung, the efficiency of statins was also demonstrated, as published by Matsuo et al. [22] , however, with a method different from the one used here, since these authors performed IR directly in the pulmonary hilum, thus not being a study of remote protection. In addition, these authors used rosuvastatin as a protective medicine and not atorvastatin as in the present research.
The mechanism of protection of statins to IR situations is due to its pleiotropic effect. By inhibiting a conversion of HMG-CoA to L-mevalonate, statins prevent a synthesis of isoprenoids, which are precursors of cholesterol biosynthesis, which serve as important lipid ligands for post-translational modification of intracellular proteins such as small GTPases, Rho, Rac and Ras. This protein isoprenylation allows a suitable subcellular localization and an intracellular circuit of proteins, which control various cellular functions, and an inhibition and pathways are important components of the pleiotropic effects of statins. The Rho pathway is related to oxidative stress, atherosclerosis  Greca et al. [10] . Focal edema (fe); alveolus (A). HE, 10x and 40x.  [10] . Focal edema (fe); alveolus (A). HE, 10x and 40x.
and elevated blood pressure, signaling the pathway between the two crucial mechanisms, such as cytoskeletal remodeling and the ROS synthesis [16] .
In the development of this project, we did not know that the therapeutic methods applied would present the results shown here, so that an association group (IPC+A) was created aiming at enhancing tissue protection. However, there was no advantage in the association, since, in isolation, these therapeutic methods obtained mean tissue lesion statistically similar to the SHAM group, i.e., it would not be possible to have a lower lesion than was already achieved. Thus, it can be verified that atorvastatin has the capacity to protect the lungs in situations of reperfusion at a distance, at the same intensity as IPC, and it is possible to invest in research that confirms the best method of using these therapies to apply them in the clinical practice.

CONCLUSION
IPC and atorvastatin were able to minimize lung reperfusion injury, alone or in combination.