The ischemic process is involved in the pathophysiology of various diseases, including coronary artery disease, acute limb ischemia (ALI), and cerebrovascular disease, all of which carry a high morbidity and mortality [1]. During ischemic conditions, reduced blood flow leads to decreased delivery of oxygen, glucose, and other substances required for cell metabolism, leading to tissue injury [2,3].

Although reperfusion to ischemic tissue is needed to prevent permanent cellular damage, there is a systemic side effect known as ischemia-reperfusion injury (IRI). In various medical and surgical procedures, including thrombolytic, organ transplantation, or cardiopulmonary bypass machines, IRI remains a significant problem due to its systemic effect on distant organs. The injury can affect the lungs, kidneys, and heart, leading to systemic inflammatory response syndrome and multiple organ dysfunction syndrome, with high mortality rates ranging from 30% to 40% [3-5].

ALI is an emergency condition caused by a sudden loss of perfusion to the lower limbs that threatens limb viability and has an onset of fewer than two weeks. Its incidence is 1.5 cases per 10,000 individuals per year, with a mortality rate of 18% [6,7]. Revascularization is the primary treatment for ALI; however, the associated IRI can cause local and distant organ damage, thereby increasing morbidity and mortality rates [8,9].

Lung injury induced by IRI is mediated by reactive oxygen species (ROS), inflammatory mediators, and neutrophil activation, resulting in increased microvascular permeability and lung edema [10]. Several therapies proposed to reduce lower limb IRI-induced injury include ischemic preconditioning, allopurinol, N-acetylcysteine, statin, mannitol, angiotensin-converting enzyme inhibitors, and antioxidants like superoxide dismutase, catalase, selenium, and vitamin E.

Selenium is an essential component of the antioxidant enzyme glutathione peroxidase, which can bind to ROS, thereby protecting against ROS-mediated cellular injury [11]. Several studies have reported the protective effect of selenium administration in reducing IRI injury in various organs, including the brain, intestines, kidney, spinal cord, heart, and ovaries [12]. This study aimed to compare the degree of lung injury resulting from lower limb IRI in Sprague-Dawley (SD) rats administered selenium versus those not receiving selenium.

An experimental study on rats was conducted to analyze the impact of selenium administration on lung injury resulting from IRI. The experimental part of this study was conducted at the Veterinary Medical Teaching Hospital, Bogor Agricultural University, while sample analysis was conducted at the Department of Anatomical Pathology, Cipto Mangunkusumo Hospital, in September 2019. The study population consisted of experimental SD rats obtained from the Laboratory Animal Management Unit, Faculty of Veterinary Medicine, Bogor Agricultural University. The inclusion criteria for the samples were male rats aged 2.5-3 months, weighing 250-350 grams, and in healthy conditions with thick, slick, shiny, and clean fur. The exclusion criteria included discharge of mucus, pus, or blood from the eyes or ears, excessive saliva, diarrhea, and rapid breathing. The chosen SD rats were acclimatized for 1 week and provided with standard feed and adequate water. Three treatment groups were determined. Using the Federer formula for animal studies, a sample size of nine rats was allocated for each treatment group, with an additional ten rats reserved for potential study-related mortality. However, after careful consideration from the Ethics Committee, the sample size was reduced to five rats per group. Ethical clearance of this study was granted by the Animal Ethics Committee, Faculty of Veterinary Medicine, Bogor Agricultural University (Clearance number 148/KEH/SKE/VIII/2019). All procedures were conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Fifteen sample SD rats were divided into three groups: Group A or the control group (lower limb IRI without selenium administration), Group B (selenium administration before reperfusion), and Group C (selenium administration after reperfusion). Before the study, each SD rat was anesthetized intramuscularly with Ketamine (15-20 mg/kg body weight) and Xylazine (5-13 mg/kg body weight). A longitudinal incision was made on the thigh, extending from the inguinal ligament to the proximal knee. Bilateral common femoral arteries were identified and ligated using bulldog clamps to reproduce ALI. After two hours of ligation, the clamps were released to restore blood flow and mimic reperfusion. For Groups B and C, selenium (0.2 mg/kg body weight) was administered via intravenous injection into the femoral vein immediately before and after releasing the ligation. This selenium dosage was chosen as it falls within the pharmacological dose range and has been used in previous studies investigating the effect of selenium on reperfusion injuries in other organs. After three hours of reperfusion, the chest cavity was opened to harvest lung tissue from each of the five lung lobes, resulting in five lung tissue samples per SD rat. Every lung tissue sample was placed in a 10% buffered formalin container. Each sample was sent for histopathologic analyses using a hematoxylin-eosin coloring agent by one anatomical pathology specialist, blinded to the group allocations. A microscope with 100× magnification was used to determine the degree and spread of lung edema. A microscope with 400× magnification was used to count the number of polymorphonuclear (PMN) cells on five fields of view from each sample.

To determine the degree of lung injury, we employed a modified version of the method originally described by Greca et al. [13] (Table 1). Each SD rat produced five lung tissue samples. The mean score of the degree and spread of lung edema from the five samples were calculated, then the mean values were multiplied to determine the edema score of each rat. A mean result of 0.5 or more was rounded up and vice versa. PMN scores were determined by the average number of neutrophils found in alveolar septa under 400× magnification. The edema and PMN scores were multiplied, and the results were grouped into three degrees of lung injury: mild, moderate, and severe.

Table 1 . Scoring method of lung injury modified from Greca et al..

Degree of lung edema	Spread of lung edema	Edema score (degree×spread)	PMN score	Degree of lung injury (Edema×PMN)
1: slight dilation of interstitial lung tissue compared to normal lung tissue	1: focal	Score 1 or mild: result score of 1	Score 1: <50 PMN cells	Mild (1): total score of 1-2
2: dilation between mild and severe	2: diffuse	Score 2 or moderate: result score of 2	Score 2: 50-100 PMN cells	Moderate (2): total score of 3-4
3: clearly visible dilation of interstitial tissue		Score 3 or severe: result score of >2	Score 3: >100 cells	Severe (3): total score of >4

PMN, polymorphonuclear..

Data from the article of Greca et al. (Acta Cir Bras 2008;23:149-156) [13]..

The results of this study were analyzed using the SPSS version 22 (IBM Corp.). The degree of lung injury was obtained as ordinal data, therefore was presented in mean form if the Shapiro-Wilk test revealed that the data was normally distributed (P>0.05), or median form if otherwise. Analysis of variance or the Kruskal-Wallis test was employed to compare the results among the three groups. Additionally, post hoc analysis was conducted using Mann-Whitney test for between-group comparisons. P-values <0.05 were considered statistically significant.

No experimental SD rats passed away during the study period. The results were presented as medians, with minimum and maximum values since the Shapiro-Wilk test revealed that the data were not normally distributed (P<0.05). Group A, the control group, did not receive selenium and exhibited moderate lung injury in two rats and severe lung injury in three. Therefore, the median value for this group was 3, indicating severe lung injury (Table 2). In Group B, selenium was administered prior to reperfusion. Only one rat showed moderate lung injury, while the rest had mild lung injury, resulting in a median value of 1 (mild lung injury). In Group C, selenium administration after reperfusion resulted in moderate lung injury in three rats and mild lung injury in two, with a median value of 2 (moderate lung injury). Microscopic findings of mild, moderate, and severe lung injury are presented in Fig. 1-3, respectively.

Figure 1. Microscopic findings of mild lung injury under ×400 magnification and hematoxylin-eosin staining reveal slight dilation of focal interstitial lung tissue with polymorphonuclear cells <50.

Figure 2. Microscopic findings of moderate lung injury under ×400 magnification and hematoxylin-eosin staining reveal moderate dilation of the focal interstitial lung tissue with polymorphonuclear cells of 50-100.

Figure 3. Microscopic findings of severe lung injury under ×400 magnification and hematoxylin-eosin staining revealing clearly visible dilation of diffuse interstitial lung tissue with polymorphonuclear cells >100.

Table 2 . Descriptive results of the degree of lung injury from the three groups.

Groups	SD Rats	Edema score	PMN score	Degree of lung injury (grade)
A (control group)	A1	3	3	3
	A2	2	3	3
	A3	2	3	3
	A4	2	2	2
	A5	2	2	2
	Median (minimum-maximum)	2 (2-3)	3 (2-3)	3 (2-3)
B (selenium administration before reperfusion)	B1	1	1	1
	B2	2	2	2
	B3	1	1	1
	B4	2	1	1
	B5	1	1	1
	Median (minimum-maximum)	1 (1-2)	1 (1-2)	1 (1-2)
C (selenium administration after reperfusion)	C1	2	2	2
	C2	2	2	2
	C3	2	2	2
	C4	2	1	1
	C5	1	1	1
	Median (minimum-maximum)	2 (1-2)	2 (1-2)	2 (1-2)

The Kruskal-Wallis test was performed to compare the edema score, PMN score, and degree of lung injury among the three groups (Table 3). Both edema and PMN scores were significantly different across the three groups, with P-values of 0.02 and 0.01, respectively. The degree of lung injury also differed significantly between the three groups, with a P-value of 0.01. Post hoc analysis with the Mann-Whitney test, as shown in Table 4, Supplementary Table 1, 2, was used to analyze further and compare the study outcomes between each group. For the edema score, only Groups A and B differed significantly (P=0.03). The same result was observed for the PMN score, where only Groups A and B exhibited a significant difference (P=0.01). For the final outcome, which was the degree of lung injury, Groups A and B differed significantly (P=0.01). However, no significant difference was found between Groups A and C (P=0.06) or between Groups B and C (P=0.31).

Table 3 . Comparison of the degree of lung injury between the groups.

	Group A (n=5)	Group B (n=5)	Group C (n=5)	P-value (KW)
Edema score median (minimum-maximum)	2 (2-3)	1 (1-2)	2 (1-2)	0.02
PMN score median (minimum-maximum)	3 (2-3)	1 (1-2)	2 (1-2)	0.01
Degree of lung injury median (minimum-maximum)	3 (2-3)	1 (1-2)	2 (1-2)	0.01

Group A: control group; Group B: selenium before reperfusion; Group C: selenium after reperfusion..

KW, Kruskal-Wallis test; PMN, polymorphonuclear..

Table 4 . Post hoc analysis to compare the degree of lung injury between the groups.

Group	Degree of lung injury (grade)	P-value (MW)
A (control group)	3 (2-3)	0.01
B (selenium administration before reperfusion)	1 (1-2)
A (control group)	3 (2-3)	0.06
C (selenium administration after reperfusion)	2 (1-2)
B (selenium administration before reperfusion)	1 (1-2)	0.31
C (selenium administration after reperfusion)	2 (1-2)

Grade 1: mild lung injury; Grade 2: moderate lung injury; Grade 3: severe lung injury..

MW, Mann-Whitney test..

Lungs are vulnerable to insults from reperfusion injury associated with ALI due to a complex mechanism involving neutrophil activation by ROS and resulting metabolites [14]. On histopathological analysis, lung injury is characterized by edema, alveolar congestion, and infiltration or aggregation of PMN cells in the alveoli, alveolar septum, or hyaline membrane [10]. These injuries can clinically manifest as acute respiratory distress syndrome [3,14].

In this study, all three groups underwent skeletal muscle ischemia for two hours, followed by reperfusion for three hours before obtaining lung tissue samples. Skeletal muscle can survive for more than two hours of ischemia; however, the cellular damage increases as the duration of ischemia increases. Catabolism of adenosine diphosphate into hypoxanthine occurs after two hours of ischemia, and it is further metabolized into xanthin and free radicals after reperfusion. During the first hour of reperfusion, neutrophils are the first cells recruited to the injury site [15]. As a result of reperfusion, neutrophils, free radicals, and inflammatory mediators from the ischemic lower limbs will enter the systemic circulation and subsequently affect the lungs and other distant organs [16]. Inflammatory mediators, such as Tumor Necrosis Factor-alpha and Interleukin-1, have been associated with lung injury [15].

Group A, the control group, revealed severe lung injury characterized by severe interstitial edema and ample infiltration of PMN cells in the alveolar septum, consistent with findings by Sun et al. [10]. This is attributed to inflammation and oxidative stress. Consequently, the formed ROS synergistically damaged the microvascular endothelial cells of the lungs [10]. Pontes et al. [17] and Dorsa et al. [18] also reported severe lung injury resulting from IRI, albeit with a shorter duration of ischemia and reperfusion. Similar histopathological changes in the lung tissue as a result of IRI were also documented by Sotoudeh et al. [19].

One strategy to counteract the damage associated with IRI is selenium administration, which functions as an antioxidant [4]. In the present study, tissue edema, PMN infiltration, and degree of lung injury were reduced in groups treated with selenium administration (Groups B and C). This finding demonstrated that selenium had a protective effect on the lungs against damage from IRI. Various experimental studies have presented the protective effect of selenium from IRI on other organs. For instance, Bozkurt et al. [12] found that selenium prevented further IRI-induced damage in ovarian torsion. Zendedel et al. [20] also reported a reduced degree of sciatic nerve injury as a side effect of IRI in animals treated with selenium. Reduced edema and mast cell infiltration of muscle tissue attributed to selenium administration were also reported by Gholami et al. [11].

Selenium serves as an enzyme cofactor of various selenium-dependent enzymes (selenoenzymes), including glutathione peroxidase. There are eight types of glutathione peroxidase distributed in different cellular compartments. Both glutathione peroxidase-1 and glutathione peroxidase-4 are located in the cytosol and mitochondria, and they work together to bind complex lipid hydroperoxides. ROS formed after reperfusion are converted into hydrogen peroxides by superoxide dismutase enzyme. Accumulated hydrogen peroxides will damage the cells and lead to tissue injuries. To prevent this, hydrogen peroxide needs to be converted into water and oxygen by glutathione peroxidase. This enzyme also transforms lipid hydroperoxide and peroxynitrite radicals produced by IRI into harmless substances, thereby reducing cellular damage and lung injury [12,21,22]. The findings from this study align with this theory, as groups receiving selenium experienced less tissue edema and PMN infiltration.

This study further analyzed the difference in the timing of selenium administration. Although the degree of lung injury in Group B, which received selenium before reperfusion, was less severe than in Group C, where selenium was administered after reperfusion, this difference was not statistically significant (P=0.31). However, this histopathological difference could produce a clinically different outcome. In the group that received selenium administration before reperfusion, selenium had a more extended period to optimally function in decreasing the number of free radicals, thereby resulting in less lung injury.

Despite the small sample size, our results revealed a significant difference between the groups. Furthermore, this study attempted to analyze how the differences in the timing of selenium administration affected the results. This study differed from similar studies by obtaining tissue samples from all five lung lobules to be histopathologically analyzed [17,18]. Using samples from all lung lobules contributed to higher coverage of assessable lung tissue. It might have better represented lung injury in all lobules rather than only reflecting samples from one or two lung lobules.

The limitation of this study was the unavailability of pulse oximetry as an objective evaluation. Pulse oximetry could have been used to evaluate distal tissue perfusion before ligation and after reperfusion. Successful ligation or reperfusion was evaluated only subjectively by the researcher via observation of the artery pulsation distal to the ligation. Further studies, especially clinical studies, are needed to determine the clinical effect of selenium in preventing lung injury by IRI. Different timings of selenium administration should also be evaluated because this study found better histopathological results in groups treated with selenium before reperfusion despite the non-significant difference.

Selenium administration before reperfusion significantly decreases the degree of lung injury as a result of lower limb IRI in SD rats. Although statistical analyses revealed no significant difference between different timings of selenium administration, further studies should evaluate the optimal dosage and timing.

Supplementary Tables can be found via https://doi.org/10.5758/vsi.230065

None.

The authors have nothing to disclose.

Concept and design: DAH, CZT. Analysis and interpretation: DAH, CZT, GAW, WTS, S. Data collection: CZT, LR, GAW. Writing the article: GAW, WTS, S. Critical revision of the article: DAH, LR, CZT. Final approval of the article: all authors. Statistical analysis: CZT, GAW. Obtained funding: none. Overall responsibility: DAH, CZT.