Edaravone attenuates lung injury in a hind limb ischemia-reperfusion rat model: A histological, immunohistochemical and biochemical study

Journal Pre-proof Edaravone Attenuates Lung Injury in A Hind Limb Ischemia-Reperfusion Rat model: A Histological, Immunohistochemical and Biochemical Study Amira Adly Kassab, Adel Mohamed Aboregela, Amany Mohamed Shalaby

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S0940-9602(19)30137-2

DOI:

https://doi.org/10.1016/j.aanat.2019.151433

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AANAT 151433

To appear in:

Annals of Anatomy

Received Date:

30 May 2019

Revised Date:

29 September 2019

Accepted Date:

7 October 2019

Please cite this article as: Adly Kassab A, Aboregela AM, Shalaby AM, Edaravone Attenuates Lung Injury in A Hind Limb Ischemia-Reperfusion Rat model: A Histological, Immunohistochemical and Biochemical Study, Annals of Anatomy (2019), doi: https://doi.org/10.1016/j.aanat.2019.151433

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Edaravone Attenuates Lung Injury in A Hind Limb Ischemia-Reperfusion Rat model: A Histological, Immunohistochemical and Biochemical Study

Amira Adly Kassab a and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, 31527,

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aHistology

Egypt.

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Email: [email protected]

Anatomy and Embryology Department, Faculty of Medicine, Zagazig University,

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bHuman

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Zagazig, 44519, Egypt. cBasic

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Adel Mohamed Aboregelab,c (author)

Medical Sciences Department, College of Medicine, Bisha University, Kingdom of Saudi

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Arabia.

Email: [email protected], [email protected]

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Amany Mohamed Shalaby a* (corresponding author) aHistology

and Cell Biology Department, Faculty of Medicine, Tanta University, Tanta, 31527,

Egypt.

Email: [email protected] [email protected]

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Abstract Edaravone is a potent free radical scavenger that has a promising role in combating many acute lung injuries. Ischemia/reperfusion process is a serious condition that may lead to multiple organ dysfunctions. This work was designed to investigate novel mechanisms underlying ischemia/reperfusion-induced lung injury and to evaluate the protective role of edaravone. Thirty

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adult male rats were divided into three experimental groups; operated with no ischemia (Shamgroup), ischemia/reperfusion (I/R) group and edaravone-I/R group. Hind limb ischemia was

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carried out by clamping the femoral artery. After two hours of ischemia for the hind limb, the rat

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underwent 24 hours of reperfusion. Rats in the edaravone-I/R group received edaravone (3 mg/kg), 30 minutes before induction of ischemia. At the end of the I/R trial, specimens from the lungs were

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processed for histological, immunohistochemical, enzyme assay, and RT-qPCR studies. Specimens from I/R group showed focal disruption of the alveolar architecture. Extensive

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mononuclear cellular infiltration particularly with neutrophils and dilated congested blood capillaries were observed. A significant increase in iNOS, NF-κB, and COX-2 immunoreaction

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was detected and confirmed by RT-qPCR. Ultrastructural examination showed RBCs and fluid inside alveoli, cellular infiltration, and vacuolations of the inter-alveolar septum. In addition to the presence of extravasated neutrophils and RBCs within the inter-alveolar septum. In contrast,

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minimal changes were observed in rats which received edaravone before the onset of the ischemia. It could be concluded that edaravone exerted a potent protective effect against lung injury induced by a hind limb I/R in rats through its antioxidant and anti-inflammatory activities. Keywords: edaravone; ischemia/reperfusion; lung; electron microscopy; RT-qPCR 1. Introduction

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Ischemia-reperfusion (I/R) injury is a process of tissue damage caused by the arrival of the blood supply to the tissue after a period of interruption of the arterial blood supply (Hori et al., 2013). Ischemia causes damage to a certain organ which is exacerbated by reperfusion. So reperfusion is considered to be more harmful than the ischemia itself due to enhanced generation of oxygen radicals (Yildirimi et al., 2014). In addition to the damage that occurs in the tissue which was subjected to the ischemia and reperfusion process, distant organs are also affected by this process

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(Dorsa et al., 2015). So, I/R process is a critical state that can, in the worst case, lead to multiple

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organ dysfunction syndromes. The extent of tissue injury usually relates to the extent of reduction in blood flow and to the length of the ischemic period, which influence the cellular level of ATP

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production and the reduction of intracellular pH (Kalogeris et al., 2017). The lung is one of the

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distant organs most vulnerable for I/R processes (Wang et al., 2013).

The ischemia-reperfusion process occurs in several clinical conditions such as major surgical

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procedures (Ferrari RS and Andrade, 2015). Acute lung injury may occur due to skeletal muscle ischemia-reperfusion resulting from trauma, limb revascularization, orthopedic surgery, and free

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flap reconstruction in which peripheral arterial clamping is routinely used and can lead to not only local damage but also severe destruction of the lung (Sotoudeh et al., 2012; Takhtfooladi et al., 2015).

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Previous studies revealed that ischemia-reperfusion injury of the lung usually resulting in alveolar and interstitial edema with the injury of the blood-air barrier as well as fragmentation of the epithelium lining the alveoli and denudation of the basement membrane, in addition to marked dysfunctions of the intra-alveolar surfactant (Knudsen et al., 2011).

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Reactive oxygen species (ROS) and the inflammatory cytokines play an important role in the pathogenesis of I/R induced lung injury (Yildirimi et al., 2014; Takhtfooladi et al., 2015). Oxidative stress promotes activation and nuclear localization of pro-inflammatory transcription factor; nuclear factor-κB (NF-κB), as well as several inflammatory mediators as inducible nitric oxide synthase (iNOS) which play a crucial role in several critical respiratory disorders. NF-κB is a major inducer for classically activated pro-inflammatory macrophages and cyclooxygenase-2

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(COX-2) which regulates pro-inflammatory eicosanoids formation promoting lung inflammation.

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Moreover, the correlation between NF-κB activation and the pathogenesis of acute lung injury has

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been strongly suggested (Sunil et al., 2014).

Several protective agents have been introduced to minimize I/R induced-lung injury such as

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caffeic acid phenethyl ester or erdosteine (Calikoglu et al., 2003; Mehmet et al., 2008), Zinc aspartate (Turut et al., 2009), N-acetylcysteine (Sotoudeh et al., 2012), tramadol (Takhtfooladi et

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al., 2013), melatonin (Takhtfooladi et al., 2015) and sevoflurane (Lou et al., 2015). Yet, all demonstrated agents showed limited successful effects, thus encouraging the search for a more

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efficient pulmonary protective agent for this life-threatening dilemma. Edaravone is a potent antioxidant drug and oxygen-free radical synthetic scavenger (Jiao et al., 2011; Yu et al., 2015). It has a protective effect on various cells in the body such as endothelial

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cells, neurons, myocardial cells, skeletal muscle, and hepatocytes preventing oxidative stressinduced cellular damage and enhancing the mitochondrial functions (Hori et al., 2013; Zhang et al., 2013; Shokrzadeh et al., 2014). It is widely used in cerebrovascular, cardiovascular, digestive, and endocrine diseases (Kikuchi et al., 2012). Moreover, edaravone was proposed to have a promising role in combating many acute lung injuries. Reactive oxygen species (ROS) scavenging is the most accepted mechanism for edaravone's protective effect (Ferrari and Andrade, 2015).

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Nevertheless, edaravone has been strongly suggested to suppress NF-κB activation that may lead to lung injury (Kikuchi et al., 2013). Thus, the present study was designed to investigate novel mechanisms underlying I/R-induced lung injury and to evaluate the potential protective role of edaravone against lung injury induced by a hind limb ischemia-reperfusion in the adult male albino rat, by employing different

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histological and immunohistochemical techniques.

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2. Materials and methods

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2.1. Experimental animals

The experiment was carried out on thirty adult male Wistar albino rats weighing 170-200 grams.

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They were housed in suitable, clean, properly ventilated cages under controlled conditions of humidity, temperature, and a 12-hour light/dark cycle and were fed on a similar commercial

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laboratory diet and water. The animals were acclimatized to their environment at least one week before starting the experiment. All animal work was conducted under the guidelines for the use of

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animals in research established by the local ethical committee of the Faculty of Medicine, Tanta University, Egypt (Approval number: 32703/11/18). 2.2. Chemicals

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Edaravone (Aravon) was purchased from Sun Pharma Laboratories Ltd. in the form of 20 ml

glass ampule that contains 1.5 mg edaravone/ml. 2.3. Experimental Design The rats were randomly divided into three equal groups:

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Group I (Sham-operated group-with no I/R): Rats were subjected to all operative procedures for femoral artery dissection and were handled like group II but without arterial occlusion and reperfusion. Group II (I/R group): Rats of this group were subjected to two hours ischemia by occlusion of the femoral artery by a vascular clamp followed by 24 hours reperfusion after removal of the

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clamp.

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Group III (edaravone-I/R group): Rats of this group received an intraperitoneal injection of edaravone at a dose 3 mg/kg body weight 30 minutes before induction of ischemia (for two hours)

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followed by reperfusion (for 24 hours) (Hori et al., 2013).

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2.4. The technique of hind limb I/R

The rats were anesthetized using ketamine plus xylazine (10 mg/kg and 50 mg/kg) by

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intramuscular injection, respectively. The left hind limb was completely clipped using an electric shaver. After clipping and disinfecting, a skin incision was done on the medial surface of the left

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hind limb. The femoral artery and vein were isolated from the surrounding structures. The femoral artery was dissected and clamped. All rats were subjected to 2 hours of ischemia by occlusion of the femoral artery with a vascular clamp followed by 24 hours of reperfusion by removal of the

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clamp. Rats were kept anesthetized throughout the ischemic period. Body temperature was maintained using a heating pad that covered the surgical site. Following the ischemic period, the vascular clamp was removed and then the surgical site was routinely closed. During the reperfusion period, rats were returned to their cages with food and water ad libitum (Sotoudeh et al., 2012; Takhtfooladi et al., 2015). 2.5. Specimen collection

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After 24 hours of reperfusion, the rats were euthanized using an overdose (300 mg/kg) of intraperitoneal pentobarbital injection (Sotoudeh et al., 2012). Thoracotomy was done and the thoracic contents were removed rapidly en block as one unit. The left lungs were excised, and the upper left lobes were frozen in liquid nitrogen then divided into pieces and stored at −80 °C until the preparation of tissue homogenates and mRNA extraction. While the lower lobes were

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processed for light and electron microscopic studies.

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2.6. Determination of lung myeloperoxidase (MPO) activity

Myeloperoxidase is an enzyme present in neutrophils and was used as a marker of infiltration

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of the neutrophils (Carden and Granger, 2000). Lung myeloperoxidase activity was determined according to Reyes et al. (2006). 50 mg of lung tissue was homogenized in 0.5 %

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hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (pH 6.0) at a ratio of

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1:10 (w/v) for 20 seconds, then centrifuged at 17,000 rpm for 20 minutes. The pellet was resuspended in HTAB, freeze-thawed (20 minutes at 80 °C), homogenized for 60 seconds,

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sonicated three times for 30 seconds, and centrifuged at 17,000 rpm for 20 minutes. The sample was then mixed with 100 mM phosphate buffer (pH 6.0) containing 1 mM of o-dianisidine dihydrochloride and 0.005 % of H2O2. Myeloperoxidase product was measured by spectrophotometry at 650 nm absorbance.

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2.7. Determination of the levels of the oxidative stress Lung homogenates were prepared according to Wang et al. (2017). Briefly, pieces of lung tissue

were homogenized in potassium phosphate buffer (pH 7.4) and centrifuged at 4800 rpm for 30 minutes at 4 °C. The supernatants were used for estimation of malondialdehyde (MDA),

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glutathione peroxidase (GSH- Px), and superoxide dismutase (SOD). Their levels were measured by using commercial assay kits (Nangjin Jiancheng Bioengineering Institute, Nangjin, China). 2.8. Light microscopic studies The lower left lobe from each animal in each group was cut into 5 mm3 pieces, fixed in 10 % neutral-buffered formalin for 24 hours, washed, dehydrated, cleared, and embedded in paraffin.

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Then, five micrometers (μm) sections were cut and subjected to the following techniques:

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• Hematoxylin and Eosin staining (Gamble, 2008): To study the histological structure of rat lung.

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(Ten slides per group)

• Immunohistochemical staining using the streptavidin-biotin-peroxidase technique for iNOS, NF-

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κB, and COX-2 (markers of inflammation) according to Ramos-Vara et al. (2008).

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Briefly, ten sections per group were deparaffinized, rehydrated, and rinsed in tap water, treated with 3 % hydrogen peroxide for 10 minutes then immersed in antigen retrieval solution.

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Nonspecific protein binding was blocked by incubating the sections in 10 % normal goat serum in phosphate buffer solution (PBS). sections were then incubated in a humid chamber at 4 °C with the primary anti-iNOS antibody (rabbit polyclonal antibody, 1:100 dilution, ab15323, Abcam, Cambridge, Massachusetts, USA), anti-NF-κB antibody (rabbit polyclonal anti-rat antibody

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against the P65 subunit of NF-κB; 1:20 dilution, ab86299, Abcam, Cambridge, Massachusetts, USA) and anti-COX-2 (rabbit polyclonal antibody, 1:100 dilution, ab15191, Abcam, Cambridge, Massachusetts, USA) overnight. After washing in phosphate-buffered saline (PBS), the corresponding biotinylated secondary antibody was added to lung sections for one hour at the room temperature then washed in PBS. Streptavidin peroxidase was added for 10 minutes and then washed in PBS. 3, 3’diaminobenzidine (DAB)-hydrogen peroxide was applied as a chromogen to

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visualize the immunoreaction site. Finally, the immunostained lung sections were counterstained by Mayer's hematoxylin. For negative control sections, the primary antibodies were excluded. All the slides were assessed in triplicates to confirm the accuracy of the obtained results. 2.9. Morphometric study Images were obtained using a Leica microscope (DM3000; Leica Microsystems, Wetzlar,

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Germany) coupled to a CCD camera (DFC-290; Leica, Heerbrugg, Switzerland). Image analysis

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was performed using a Leica QWin 500C image analyzer computer system (Leica Imaging System Ltd, Cambridge, UK) at the Central Research Lab, Faculty of Medicine, Tanta University (Tanta,

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rat group were examined to quantitatively evaluate:

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Egypt). Ten different non-overlapping randomly selected fields from each lung specimen in each

1) The thickness of inter-alveolar septum [in H&E stained sections at a magnification of 400].

following equation:

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2) The mean percentage of iNOS, NF-κB, and COX-2 were determind according to the

Number of positive cells

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Percentage of positive cells =

Total counted cells

x 100 [in the DAB-stained slides at a

magnification of 400].

2.10. Quantitative real-time PCR (RT-qPCR)

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Total RNA from the ten frozen lung specimens per group was extracted using the RNA Mini kit according to the manufacturer's protocol (Qiagen RNeasy, Germany). RNA in each sample was quantified using Nanodrop spectrophotometer. 1000 ng total RNA/sample was used to synthesize cDNA by reverse transcription with a Super-Script ™cDNA synthesis kit (Invitrogen, CA, USA), according to the manufacturer's instructions. mRNA levels expressions were determined by

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Stratagene, MX3000P quantitative PCR System (Agilent Technologies) and analyzed using MxPro QPCR Software (Agilent Technologies). 12.5 µl 2x QuantiFast PCR Master Mix, 1µM of each primer and 5µl cDNA were added in the mixture then the volume was completed to 25 µl with nuclease-free water in the following conditions: 95 °C for five minutes, then 40 cycles at 94 °C for 30 seconds, and combined annealing and extension 60 °C for 60 seconds. All kits were supplied by (QIAGEN, Valencia, CA, USA). The annealing temperatures for different genes were

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set as following: 62 °C for iNOS and GAPDH (house-keeping gene), 60 °C for NF-ĸB and 58 °C

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for COX-2. The primers were designed according to Inam et al. (2017) and Zhang et al. (2017). (Table 1). All the samples were assessed in triplicates to confirm the reproducibility and accuracy

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of the obtained results.

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2.11. Electron microscopic studies

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Processing the lung samples for transmission electron microscope was done according to (Woods and Stirling, 2008). Very small pieces of lung tissues (1mm3) were fixed in 2.5 %

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phosphate-buffered glutaraldehyde for two hours at 4 °C. Then rinsed in PBS. After that, the specimens were post-fixed in prepared 1 % phosphate buffer osmium tetroxide for one hour at 4 °C. Then, the specimens were dehydrated in ascending grades of alcohol at 4 °C, immersed in propylene oxide, and embedded in the epoxy resin mixture. Semithin sections (0.5–1 μm thick)

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were cut by the automatic LKB ultramicrotome, mounted on glass slides, stained with toluidine blue, and examined under the light microscope for selection of the suitable areas. Ultrathin sections (80–100 nm thick) were cut and contrasted with uranyl acetate and lead citrate for examination under JEOL-JEM-100 transmission electron microscope (JEOL, Tokyo, Japan). Electron microscopy sample processing, examination and photographing were done in the electron microscopy unit, Faculty of Medicine, Tanta University, Egypt.

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2.12. Statistical analysis Obtained data were statistically analyzed using one-way analysis of variance followed by Tukey’s procedure for comparison between all groups using the statistical package of the social sciences software (version 11.5; SPSS Inc., Chicago, Illinois, USA). The values were expressed as mean ± SD. Differences were regarded as significant if the probability (P) value was less than

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0.05 (Dawson-Saunders and Trapp, 2001).

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3. Results

All experimental rats tolerated all operative procedures and survived until the final experimental

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period.

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3.1. Myeloperoxidase (MPO) activity assessment in lung tissue

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The I/R group (group II) showed a significant increase in the MPO activity (5.72±1.52 U/mg) as compared to the sham group (group I) (1.88±0.58 U/mg). While the edaravone-I/R group (group

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III) showed no significant change in the MPO activity (2.32± 0.29 U/mg) as compared to the sham group (group I) (table 2).

3.2. Oxidative stress assessment

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The I/R group (group II) showed a significant increase in MDA level (3.18 ± 0.32 nmol/mg) and a significant decrease in the levels of GSH-Px (32.14 ± 1.34 U/mg) and SOD (22.33 ± 2.54 U/mg) as compared to the sham group (group I) (0.96 ± 0.13 nmol/mg, 88.35 ± 2.43 U/mg, 50.12 ± 3.24 U/mg respectively). On the other hand, edaravone-I/R group (group III) showed no significant change in the levels of MDA (1.12 ± 0.18 nmol/mg), GSH-Px (79.43 ± 3.57 U/mg) and SOD (43.47 ± 3.34 U/mg) as compared to the sham group (group I) (table 2).

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3.3. Lung histopathology The hematoxylin and eosin-stained lung sections of the sham group showed normal alveolar histological architecture giving the characteristic spongy structure of the lung. Normal clear alveoli were separated by a thin inter-alveolar septum (1.9±0.52). The alveoli were lined by flat type I and cuboidal type II pneumocytes (Figs. 1A and B). On the other hand, lung sections of I/R group

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showed variable structural changes. Focal areas showed loss of the normal architecture with marked narrowing of many alveolar spaces. Markedly thickened inter-alveolar septum (7.6±1.4)

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with extensive mononuclear cellular infiltration particularly with neutrophils and dilated congested

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blood capillaries were observed (Figs. 1C and D). Moreover, extravasation of blood was seen in some alveolar spaces (Fig. 1E). Other areas contained aggregates of large cells with a high

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nuclear/cytoplasmic ratio and vacuolated deeply blue-stained cytoplasm occurred in association with alveolar macrophages. These cells appeared larger than the alveolar macrophage (Figs. 1E

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and F). While lung sections of edaravone-I/R group revealed alveolar architecture almost similar to that of the sham group. Nevertheless, mild mononuclear cellular infiltration within the inter-

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alveolar septum was noticed in some focal areas increasing the septal thickness to (2.8±0.29) (Figs. 1G and H).

3.4. Immunohistochemical results

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3.4.1. Immunohistochemistry of iNOS Inducible NOS-immunostained lung sections of the group I (sham group) showed a positive

brown cytoplasmic reaction in a few alveolar cells (Fig. 2A). In group II (I/R group), a positive cytoplasmic reaction for iNOS was observed in many alveolar cells (Fig. 2B), whereas in group III (edaravone-I/R group) a positive cytoplasmic reaction for iNOS was detected only in some

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alveolar cells (Fig. 2C). These findings were further confirmed by the morphometrical and statistical analysis that showed a significant increase in the mean percentage of iNOS immunoexpression in I/R group (group II) (33.75±3.14) as compared to the sham group (group I) (15.29±0.715). Whereas, edaravone-I/R group (group III) showed no significant change in the mean percentage (17.39±1.849) as compared to the sham group (group I) (Histogram 1A).

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3.4.2. Immunohistochemistry of NF-κB

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The NF-κB-immunostained lung sections of the sham-operated group (group I) showed few alveolar cells exhibited a positive brown nuclear and/or perinuclear cytoplasmic immunoreaction

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for NF-κB (Fig. 2D). In I/R group (group II), many cells expressed positive nuclear and/or perinuclear cytoplasmic immunoreaction for NF-κB (Fig. 2E), whereas edaravone-I/R group

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(group III) showed a positive nuclear and/or perinuclear cytoplasmic immunoreaction for NF-κB

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only in some alveolar cells (Fig. 2F). The morphometrical and statistical analysis showed a significant increase in the mean percentage of NF-κB immunoexpression in I/R group (group II)

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(41.333±3.58) as compared to sham group (group I) (20.37±1.95), whereas edaravone-I/R group (group III) showed no significant change (21.795±2.47) as compared to the sham group (group I) (Histogram 1A).

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3.4.3. Immunohistochemistry of COX-2 The COX-2-immunostained lung sections of the sham-group (group I) showed a positive brown

cytoplasmic reaction in a few cells within the alveolar wall (Fig. 2G). In I/R group (group II), a positive cytoplasmic reaction for COX-2 was observed in many cells within the alveolar wall (Fig. 2H), whereas in (group III) edaravone-I/R group (group III) a positive cytoplasmic reaction for COX-2 was detected only in some cells of the alveolar wall (Fig. 2I). These findings were further

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confirmed by the morphometrical and statistical analysis that showed a significant increase in the mean percentage of COX-2 immunoexpression in I/R group (group II) (37.419±2.712) as compared to the sham group (group I) (18.75±1.354). Whereas, edaravone-I/R group (group III) showed no significant change in the mean percentage (19.13±1.942) as compared to the sham group (group I) (Histogram 1A).

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3.5. Quantitative real-time PCR (RT-qPCR)

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The results of the current study revealed enhancement in the levels of mRNA expression of iNOS, NF-ĸB, and COX-2 genes (5.37±0.73, 7.536±0.285 and 4.674±0.175 respectively) after ischemia

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and reperfusion with (P < 0.001) as compared to the control (GAPDH). Also, the data demonstrated significantly improved conditions regarding the mRNA expression of all the tested

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genes in the edaravone-I/R group as compared to the I/R group. As well as, mRNA expression

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levels of iNOS, NF-ĸB and COX-2 genes in edaravone-I/R group (1.97±0.581, 1.36±0.465 and 1.53±0.547 respectively) displayed no significant difference as compared to the control (GAPDH).

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As regards iNOS it showed a mild difference (Histogram 1B). 3.6. Lung ultrastructure

The transmission electron microscopic analysis of the lung ultrathin sections of the sham group

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revealed patent alveoli lined by type I pneumocyte, and type II pneumocyte with a rounded euchromatic nucleus and prominent nucleolus, multiple lamellar bodies and short microvilli on its surface. The inter-alveolar septum containing blood capillaries and an interstitial cell (Fig. 3A). On the other hand, lung ultrathin sections of the I/R group showed type II pneumocytes with few lamellar bodies, in addition to the presence of RBCs inside alveoli (Fig. 3B). Multiple blood capillaries appeared congested with RBCs with vacuolation of the cytoplasm of pneumocyte type

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I (Fig. 3C). Others contained neutrophils with marked thinning of their blood-air barrier (Fig. 3D). Moreover, Fluid inside alveoli, cellular infiltration and vacuolations of inter-alveolar septum were also detected (Fig. 3E). In addition to the presence of extravasated neutrophils and RBCs within interalveolar septum (Fig. 3F). As regards edaravone-I/R group, the alveoli were patent and type II pneumocytes appeared similar to sham-group with their multiple lamellar bodies, euchromatic

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nucleus and short microvilli on their surfaces (Fig. 3G). While some blood capillaries appeared with the minor discontinuity of the blood-air barrier (Fig. 3H).

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4. Discussion

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Peripheral arterial clamping is a routine surgical step procedure in orthopedic surgery and emergency cases as in trauma resulting in acute lung injury due to transient arterial occlusion and

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the subsequent skeletal muscle ischemia-reperfusion (I/R) (Sotoudeh et al., 2012). This is a fatal

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condition ending in non-cardiopulmonary edema or adult respiratory distress syndrome (Blaisdell, 2002). Accordingly, this study was performed to evaluate the possible protective role of edaravone

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against lung injury induced by hind limb ischemia-reperfusion in adult male albino rats by using light and electron microscopy.

In skeletal muscle, I/R, the enhanced generation of oxygen radicals and subsequent release of a significant amount of ROS from the reperfused tissue into the circulation has been suggested

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(Takhtfooladi et al., 2015). Oxidative stress induced by ROS is one of the important mediators of lung injury. ROS are highly reactive molecules that can cause direct oxidative damage and also can mediate many inflammatory responses. Oxidative stress usually leads to activation of the NFκB as well as various inflammatory mediators as iNOS with subsequent induction for COX-2 and classically activated pro-inflammatory macrophages which play an important role in lung

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inflammation. Moreover, these toxic molecules alter the cellular protein, lipid, and RNA causing cell dysfunction or death (Reyes et al., 2006; Sunil et al., 2014; Ferrari and Andrade, 2015). A further contribution to lung injury occurs by activation of phospholipase A2 by ROS formation resulting in eicosanoids and platelet-activating factor (PAF) formation (Jiang et al., 2012). It was reported that PAF promotes mobilization of arachidonic acid from the cell membrane

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phospholipids which is metabolized in the lung producing inflammatory lipid mediators (Boilard et al., 2010).

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Results of the present study revealed a significant increase in the MPO activity in I/R group.

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This finding is in agreement with previous research of Takhtfooladi et al. (2013) who stated that MPO is considered as an indicator of activated leukocytes accumulation in the tissues and is

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usually associated with an overproduction of ROS with subsequent depletion of the antioxidant capacity of the body. Also, it was stated that MPO was a local mediator of tissue damage (Aratani,

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2018).

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A significant increase in MDA level and a significant decrease in the levels of GSH-Px and SOD in the I/R group were detected in this study. These findings were in line with the previous of Takhtfooladi et al. (2016) who indicated that I/R process promoted oxidative stress in the lung with overproduction of ROS that might directly damage cellular membranes by lipid peroxidation

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(Wang et al., 2017). MDA is an end product of peroxidation of polyunsaturated fatty acids and is considered as an indicator for lipid peroxidation (Gaweł et al., 2004). Furthermore, the depletion of the antioxidant capacity of the body usually occurred as a result of the overproduction of ROS (Takhtfooladi et al., 2013).

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The hind limb I/R induced severe changes in the lung of the albino rats. These changes were in the form of disturbed alveolar architecture, inflammatory cellular infiltration especially with neutrophils and macrophages, an apparent increase in the thickness of the alveolar walls, congestion of interstitial capillaries and intra-alveolar hemorrhage. This was confirmed in this study by electron microscopic examination that revealed fluid inside alveoli associated cellular infiltration and vacuolations of the inter-alveolar septum. In addition to the presence of

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extravasated neutrophils and RBCs within the inter-alveolar septum. All these changes were

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suggested by some investigators to indicate acute lung injury which is characterized mainly by significant neutrophil infiltration and subsequently acute respiratory distress syndrome with

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generalized cellular dysfunction (Li et al., 2007; Huang et al., 2012; Wang et al., 2013). It was

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reported that I/R of hind limb leads to overproduction of oxygen radicals in the skeletal muscle, causing the attack of polyunsaturated fatty acid in the cytomembrane in a process known as lipid

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peroxidation. This increases the pulmonary vascular endothelium permeability to fluids and inflammatory cells, resulting in pulmonary edema and neutrophil sequestration in lung tissue

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(Wang et al., 2018). The present results supported the previous studies which reported that ischemia-reperfusion is associated with not only local injury, but also severe damage in remote organs like the lungs (Huang et al., 2012; Takhtfooladi et al., 2015).

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In this work, I/R induced a disturbance in the histological structure of the alveoli. Large cells with a high nuclear/cytoplasmic ratio and vacuolated deeply blue-stained cytoplasm occurred in aggregates or cohesive cell groups in some focal areas. These cells were associated with alveolar macrophages and their size was larger than that of the alveolar macrophages. The morphological appearance of these cells in our H&E stained slides was in line with previous researchers description who reported that these cells represent an intermediate stage in the differentiation of

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type II pneumocytes to type I epithelial cells (Linssen et al., 2004). These cells are hyperplastic or reactive type II pneumocytes which are activated and begin to proliferate early in response to acute I/R lung injury. A recent study reported that the lung contains alveolar epithelial progenitor lineage within the alveolar type II cell population that are recruited after injury for tissue repair. Recently, it was suggested that neutrophils promote the proliferation of type II cells for the resolution of

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acute lung injury. So, these large cells may play a role in the regeneration of the damaged lung area (Paris et al., 2016; Zacharias et al., 2018).

lamellar bodies within type II

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induced by the I/R process. These changes include fewer

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In this study, using the electron microscope showed further ultrastructural changes in the lung

pneumocytes. This is in agreement with the previous results of Knudsen et al. (2011) who

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attributed this result to the increased exocytosis of lamellar bodies in the lungs subjected to I/R

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process with a subsequent decrease in their number.

Results from this study demonstrated that edaravone offered an evident improvement in the lung

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biochemical and structural changes. This could be attributed to the antioxidant capacity of edaravone as many studies reported that edaravone enhanced the antioxidant defense mechanisms and reduced the oxidative stress. Uchiyama et al., (2015) stated that edaravone administration before reperfusion of the ischemic liver attenuates oxidative stress by decreasing MDA production

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in the reperfused liver and the subsequent lung injury. Thus, it has a beneficial role in preventing lung injury induced by hepatic I/R. A previous study of Yu et al. (2015) showed that edaravone is a potent free-radical scavenger and could inhibit the bleomycin-induced pulmonary fibrosis through adjusting the oxidant/antioxidant imbalance. In dogs, edaravone attenuated I/R-induced pulmonary dysfunction, focal hyaline membrane formation, neutrophil infiltration, interstitial edema, and oxidative stress markers, as MDA (Akao et al., 2006).

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It was reported that edaravone reduced the production of MDA and increased the activities of glutathione peroxidase and superoxide dismutase (SOD) in I/R lung injury in rabbits (Qiu et al., 2008). Moreover, it also suppressed the pulmonary MPO activity, phospholipase A2 activation, which causes edema formation, and neutrophil extravasation in an isolated rat lung model (Reyes et al., 2006). These are in line with our results.

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The immunohistochemical results of this study revealed a significant increase in the expression of NF-κB and COX-2 in lung tissue of I/R group. ROS generated by I/R can provoke activation

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of various genes coding for proteins involved in the inflammation and cell damage as NF-κB

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(Zhouyang et al., 2014). NF-κB activation is responsible for the induction of pro-inflammatory cytokines and chemokines (Ferrari and Andrade, 2015). Moreover, NF-κB activation promotes the

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expression of macrophage with subsequent overexpression of the pro-inflammatory biomarker COX-2 (Beigh et al., 2017). COX-2 is an inducible enzyme involved in the formation of pro-

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inflammatory eicosanoids resulting in lung damage and a marker of classically activated macrophages. So, NF-κB is a major inducer for classically activated pro-inflammatory

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macrophages and COX-2, leading to acute lung injury (Sunil et al., 2014). Moreover, there was also a significant increase in iNOS expression in lung tissue of I/R group. This was in agreement with the findings of some investigators who reported a significant up-

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regulation in the expression of iNOS in I/R lung tissues. Inducible NOS indicates the levels of free radical in vivo. NF-κB activation is responsible for the increased expression of iNOS. The increased iNOS expression could induce a sustained release of NO, which could further react with the superoxide released by the sequestered white cells to form peroxynitrite and hydroxyl radicals, leading to damage of the alveolar-capillary membranes (Bayomya et al., 2014; Zhouyang et al., 2014).

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This was confirmed by quantifying the expression of mRNA for iNOS, NF-ĸB, and COX-2 genes that showed a significant increase in the injured lung after ligation and reperfusion. This coincides with Yoshidome et al. (1999) who reported pulmonary NF-kB activation in the lung injury induced by hepatic ischemia-reperfusion. Moreover, lung tissue iNOS activity was significantly increased in the injured lung tissue due to ruptured abdominal aortic aneurysm

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(Harkin et al., 2004). Also, Kohmoto et al. (2006) reported an increase in the mRNA expression of iNOS and COX-2 after ischemia/reperfusion injury of the transplanted rat lung grafts.

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In this study, pretreatment with edaravone decreased the inflammatory cellular infiltration. This

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anti-inflammatory effect of edaravone has been attributed to its suppressive effect on NF-κB and COX-2 pathway as observed from the immunohistochemical and RT-qPCR results of the current

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work. Though, other investigators explained the anti-inflammatory activity of edaravone to be through suppression of iNOS activity and neutrophil activation in addition to its anti-cytokine

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effects (Kikuchi et al., 2013; Zhouyang et al., 2014). Moreover, Hori et al. (2013) stated that pretreatment with edaravone decreased the level of iNOS expression in ischemia-reperfusion

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injury of skeletal muscle in the murine hind limb. A previous study revealed that edaravone prevented lung injury and attenuated inflammatory cells and pro-inflammatory cytokine production in lipopolysaccharide-induced lung injury in mice (Tajima et al., 2008). In another

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study, edaravone attenuated inflammatory cells and interstitial fibrosis in rabbits with bleomycininduced pulmonary injury (Asai et al., 2007). 5. Conclusion

The current study suggests that edaravone might be beneficial in minimizing the lung structural changes induced by skeletal muscle I/R in rats most probably through its anti-oxidant and anti-

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inflammatory effects. Therefore, edaravone may be a useful therapeutic agent for the patient undergoing orthopedic surgery to minimize I/R complications.

Conflict of interest

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The authors confirm that this article content has no conflict of interest.

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Ethical statement.

All animal work was conducted under the guidelines for the use of animals in

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research established by the local ethical committee of the Faculty of Medicine, Tanta

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University, Egypt (Approval number: 32703/11/18).

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Legends of Figures Fig.1. Representative photomicrographs of hematoxylin and eosin-stained lung sections of the sham group showing; [A] Normal alveolar architecture (*) with a thin inter-alveolar septum (arrows). [B] Thin inter-alveolar septa (arrow), flat type I pneumocytes (arrowhead) and few

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cuboidal type II pneumocytes (wavy arrow). Sections of the I/R group showing; [C] Loss of the normal architecture with marked narrowing of many alveolar spaces (wavy arrow), markedly thickened inter-alveolar septum (arrows) with extensive mononuclear cellular infiltration (curved arrow) and dilated congested blood capillaries (arrowheads). [D] Extensive inflammatory cellular infiltration of the alveolar septum especially with neutrophils (arrows). [E] Extravasation of blood in the alveolar spaces (arrowheads) with the presence of large cells with high nuclear/cytoplasmic

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ratio and deeply stained cytoplasm (arrows). [F] Aggregates of large cells with a high

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nuclear/cytoplasmic ratio and vacuolated deeply blue-stained cytoplasm (arrows) occurred in association with alveolar macrophage (arrowheads). Sections of the edaravone-I/R group showing;

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[G] Alveolar architecture nearly similar to the control group (*). Notice mild mononuclear cellular

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infiltration within the alveolar septum (arrows). [H] Thin inter-alveolar septa (arrow), type I

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(arrowhead) and type II pneumocytes (wavy arrow) almost similar to the control.

Fig.2. Representative photomicrographs of immunohistochemically stained lung sections with anti- iNOS antibody; [A] The sham group shows a positive brown cytoplasmic immunoreaction

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for iNOS in a few alveolar cells (arrows). [B] The I/R group shows a positive cytoplasmic immunoreaction for iNOS in many alveolar cells (arrows). [C] The edaravone-I/R group shows a positive cytoplasmic immunoreaction for iNOS in some alveolar cells (arrows). The immunohistochemically stained lung sections with anti-NF-κB antibody; [D] The sham group shows a positive brown nuclear and/or perinuclear immunoreaction for NF-κB in a few alveolar cells (arrows). [E] The I/R group shows a positive nuclear and/or perinuclear immunoreaction for

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NF-κB in many alveolar cells (arrows). [F] The edaravone-I/R group shows a positive nuclear

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and/or perinuclear immunoreaction for NF-κB in some alveolar cells (arrows). The immunohistochemically stained lung sections with anti- COX-2 antibody; [G] The sham group

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shows a positive brown cytoplasmic COX-2 immunoexpression in a few cells within the alveolar

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wall (arrows). [H] The I/R group shows a positive cytoplasmic COX-2 immunoexpression in many cells within the alveolar wall (arrows). [I] The edaravone-I/R group shows a positive cytoplasmic

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COX-2 immunoexpression in some cells within the alveolar wall (arrows).

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Fig.3. Representative transmission electron photomicrographs of lung ultrathin sections of the experimental groups showing; [A] Section from sham group revealing patent alveoli (A), type I pneumocyte (PI), and type II pneumocyte (PII) with rounded euchromatic nucleus (N) and prominent nucleolus (Nu), multiple lamellar bodies (L) and short microvilli on its surface

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(arrowhead). The interalveolar septum containing blood capillaries (B) and an interstitial cell (IC). Section from I/R group showing; [B] Two type II pneumocytes (PII) with few lamellar bodies (L), notice, the presence of RBCs inside alveoli (A). [C] Multiple congested blood capillaries (B) containing RBCs with vacuolation of the cytoplasm of pneumocyte type I (asterisks). [D] A blood capillary (B) containing neutrophil cell (N) with marked thinning of the blood-air barrier (arrow). [E] Fluid inside alveolus (F), cellular infiltration (curved arrows) and vacuolations of the inter-

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alveolar septum (asterisks). [F] Extravasated neutrophil cell (N) and RBCs within the interalveolar septum. Sections from edaravone-I/R group showing; [G] Patent alveolus (A), apparent normal pneumocyte type II with euchromatic nucleus (N) and prominent nucleolus (Nu), multiple lamellar bodies (L) and short microvilli on its surface (arrowhead). [H] A blood capillary (B)

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containing RBCs with a minor discontinuity of blood-air barrier (arrow).

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Histogram 1: Morphometrical and statistical analysis of [A] Mean percentage (%) of iNOS, NFκB, and COX-2. [B] mRNA expression of iNOS, NF-ĸB, and COX-2 genes in lung tissue of all studied groups. ** indicates moderate significance vs control

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*** indicates high significance vs control

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Table 1: Primers sequences of iNOS, NF-κB, COX-2 and GAPDH genes. Target iNOS

Sequence Forward 5′-ACCAGAGGACCCAGAGACAA-3′ Reverse 5′-CCTGGCCAGATGTTCCTCTA-3′

NF-ĸB

Forward 5′-GAGGCGTGTATTAGGGGCTA-3′ Reverse 5′-ACGCTCAGGTCCATCTCCTT-3′ Forward 5'-TGAAACCCACTCCAAACACA-3'

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COX-2

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Reverse 5'-TGGAACAACTGCTCATCACC-3'

Forward 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'

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Reverse 5' CGGAGTCAACGGATTTGGTCGTAT-3'

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GAPDH

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Table 2: Mean ± SD of laboratory parameters (MPO, MDA, GSH-Px and SOD) Groups

MPO (U/mg)

GSH-Px (U/mg) SOD (U/mg) 88.35 ± 2.43 32.14 ± 1.34 aP 79.43 ± 3.57

50.12 ± 3.24 22.33 ± 2.54 aP 43.47 ± 3.34

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Sham group (I) 1.88± 0.58 I/R group (II) 5.72± 1.52 aP edaravone-I/R group 2.32± 0.29 (III) a P (P<0.05) vs. group I.

MDA (nmol/mg) 0.96 ± 0.13 3.18 ± 0.32 aP 1.12 ± 0.18

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