Imaging of stem cell therapy in Acute Respiratory Distress Syndrome in sheep and its relationship with histopathology

Methods: 10 healthy sheep were separated as the control (PBS) and treatment(BM-MSCs). The BM-samples were aspirated in the treatment group and isolation and expansion of BM-MSCs were achieved. 24h after ARDS-induction by E.coli LPS (400 µg/kg), 5×10 7 BM-MSCs and 1 ml PBS were intrapulmonary infused in the treatment and control groups, respectively. The total lung volume and the Hounseld unit by CT-scan and the cardiac parameters by echocardiography were calculated before of ARDS (time -24) and cells/PBS infusion time(time 0) and then for 6,12,24,48,72,168h after cells/PBS infusion. At the end, the sheep were sacriced and the hearts and lungs were macroscopically and microscopically checked. Results: The results showed the total lung volume and the Hounseld unit increased at time 0, but BM-MSCs declined their amount, so that the changes were signicant at 168h compared with time 0 and comparisons between the two-groups represented the signicant difference at 72h and 168h. The cardiac parameters did not changes signicantly in the two-groups at different times but the comparison between them demonstrated signicant difference in LVPWs, IVSs,IVSd,%FS,RVOT Vmax ,LA(d1) and AO(d2) at 168 h, LVIDs,ESV and %EF at 72h and 168h and LVPWd at 72h. Also, the histopathology ndings indicated decline of inammatory reactions, edema, hemorrhage and hyperemia in the respiratory system and no observation of damage to the heart compared with the control-group. Conclusions: The results determined BM-MSCs in ARDS decrease edema and inammation of the lungs and increase the alveoli air volume and prevent damage to heart function.

Background ARDS is one of the main and developing reason of respiratory failure. In very cases such as COVID-19, there is a complication by secondary severe ARDS (1). A particular characterize of ARDS is a decrease in aerated respiratory volume and diffuse alveolar damage (2). The clinical symptoms occur resulting from disruption of alveolar-capillary permeability and leakage of in ammatory cells, uid and protein into spaces of interstitial and the alveoli (3). The current medical treatments in ARDS and some lung diseases are just supportive and cannot inhibit the disease progression (4,5). Stem cell therapy is a new approach for ARDS treatment and the MSCs have a role in control of in ammation and tissue regeneration (6).
There are several techniques to evaluate the treatment process for ARDS and one of them is imaging. The diagnostic main landmark of ARDS is presence of bilateral in ltrates on the chest radiograph (bilateral radiographic opacities) (7,8) and according to Berlin de nition for the diagnosis of ARDS a conventional chest x-ray is obligate (9). But determining the exact cause and clinical complications of ARDS are di cult using radiology, only (10). The chest computed tomography (CT-scan) is a known and effective method to con rm the ARDS diagnosis and its role is critical to the disease process and the treatment further understanding and also, is a research technique in the experimental studies (8).The CT-scan is fast, painless, noninvasive, repeatable and accurate technique, which can calculate densities of lung and separated aerated from nonaerated lung zone (11,12). The CT-scan uses to check abnormalities in radiology and etc., and for diagnose the cause of inexplicable cough, dyspnea, lung in ammation and edema and the other signs. Nonetheless, there are some indications for CT-scan in ARDS that consisted of con rmation of diagnosis, calculation of recruitability, identi cation of a pulmonary or extrapulmonary cause, prediction of prognosis, and follow-up (8). Also recently CT-scan is using for diagnosis of the global pandemic COVID-19. COVID imaging pattern is consistent with ARDS and suggests that same physiological mechanism (1). Also according to the Berlin de nition, the echocardiography use is recommended to evaluate function of cardiac at the time of suspected extrapulmonary causes and the absence risk factor of ARDS (7). Therefore, because imaging plays a key role in the diagnosis of ARDS and can provide disease progression and response to treatment, the present study was designed for evaluation of CT-scan and echocardiography in management and monitoring stem cells therapy in repair and regeneration ARDS in sheep and its comparison with evaluation of histopathology.

Study design
Sampling, isolation and characterization of BM-MSCs Ten healthy male Shall sheep were randomly separated into the two groups of control (PBS, n = 5) and treatment (BM-MSCs, n = 5). All animal rights and ethics were performed according to the Animal Research Ethical Committee of University of Tehran (Tehran, Iran). The BM samples were aspirated in the treatment group from anaesthetized animal illiac crest with ketamine 10% 35 mg/kg (Alfasan-Holland), and xylazine 2% 5 mg/kg (Interchemie-Holland). Isolation and expansion of BM-MSCs were achieved by a protocol explained perviosly (13). For characterization, the BM-MSCs were tracked with antibodies of PEconjugated versuse CD44, CD29 and CD31 (Abcam, Inc), CD45 (Biolegend, Inc) and were checked by ow cytometry (BD Bioscience, USA) and ability to differentiate BM-MSCs was evaluated via speci c staining for osteogenic and adipogenic lineages.
Experimental Procedure ARDS experimental model and BM-MSCs autologous transplantation in sheep were carry outed by a protocol explained perviosly (14). Brie y, all sheep in the two group were anesthetized and LPS at dose 400 µg/kg were intrapulmonary injected. Then, according to Berlin de nition, ARDS was proved based on ndings of radiography. 24 h after ARDS induction, 5 × 10 7 BM-MSCs and 1 ml PBS were intrapulmonary infused in the treatment and control groups, respectively. The sheep in the two group were imaged by CTscan for lung evaluation and echocardiography before of ARDS (time − 24) and cells/PBS infusion time (time 0) and after for 6, 12, 24, 48, 72, 168 h after cells/PBS infusion.

Radiography
The chest radiographic examination was done befor starting the study to check the lung and heart health and after to con rm ARDS. The sheep were exited from the study with radiographic evidences of disoreders of pulmonary and cardiac. For these purposes, two standard chest radiographs, ventro-dorsal and lateral, were done with equipment of digital X-ray Kodak carestream directview classic CR (Toshiba), and the factors of techniqal 70 Kv and 3.2 mA/s for every sheep. Computerized Tomography Scan (CT-scan) For examination of CT-scan, rst, each sheep was anaesthetized with xylazine and ketamine and then, placed in sternal recumbency and head toward the gantry. After, the plain CT-scan was done with the equipment of Somatoma Spirit class II (Siemens), and the factors of technical 130 Kv, 75-95 mA, time of rotation: 1, thickness of slice: 1 mm and pitch: 1, from apical thoracic inlet until L2. All obtained images were renewed into windows of pulmonary and soft tissue. Then the total lung volume (intraalveolar space) and the Hons eld unit were calculated for each animal using the Pomona technique in volumetric software on the computer system of Leonardo. Margins of lung parenchymal were manually identi ed, and Houns eld units mean were determined for each section and the pattern of 3D was viewed for more

Histopathology
At the end the study, 7 days after BM-MSC transplantation/PBS infusion, the sheep were killed and for inspection and sampling, the chest was rifted, and the heart and lung were ligatured and dissected and were macroscopically checked and abnormal symptoms such as hyperemia, bleeding, edema, etc. were documented. Then, the histopathological slides were prepared as has been perviosly explained (13). The heart and lung were immersed in formalin 10% and the sections were commonly provided and with method of H&E were stained and then, by Nikon Optical Microscope (E600 Eclipse, Japan) viewed. Finally, a digital camera (u eye 2250) with a software of microbial version 2 provided images from samples.

Statistical analysis
From the SPSS program and the tests of independent samples t-test and repeated measure at the level p < 0.05 were used for statistical analysis of data and obtained information were expressed as mean ± SD.

Sheep BM-MSCs characterizations
The Flow cytometric ndings displayed that BM-MSCs represented cell surface antigen of CD44, 89% and CD29, 91% but did not represent CD45 and CD31, antigens of hematopoietic cells and endothelial cells, respectively. Also, ability to differentiate BM-MSCs via speci c staining showed the MSCs maintained their differentiating potential to osteoblasts and adipocytes. These ndings proved sheep cultured cells were BM-MSCs, which were applied in this study.

Findings of radiography
The radiography ndings demonstrated the lungs were completely normal before the injection of LPS (Fig. 1, A and C), but 24 h after LPS injection, abnormal lung radiographies as mixed pattern with predominance of alveolar pattern observed (Fig. 1, B and D). Most con icts observed in the posterior and dorsal regions of the lung. The ndings con rmed the acute lung disorder and ARDS model. The results of CT-scan showed at day the in ammation (time 0), the Houns eld unit mean and the total lung volume increased which was indicative the enhancement in the production of mucus, edema, and acute in ammation (Fig. 2, II, B). The stem cells transplantation caused decline of the Houns eld unit and the lungs volume mean at different imaging times compared with time 0, indicating an increase in air volume in the lung alveoli and decrease of edema and in ammation (Fig. 2, II, C). These changes (both Houns eld unit and total lung volume) were signi cant in one week after the transplantation (p=0.028 and p=0.013, respectively) and comparisons between the two groups represented the signi cant difference at 72 h (p=0.012 and p=0.030, respectively) and 168 h (p=0.036 and p=0.011, respectively) ( Fig. 2, III). In the control group, the increase in Houns eld unit and total lung volume continued due to edema and in ammation until the end of the study (Fig. 2, III).
Histopathology ndings revealed, transplantation of BM-MSCs was capable to decline edema and in ammation of the lungs and repair damaged structures. The lungs macroscopic evaluation indicated edema, hyperemia, the presence of secretions in airways, hemorrhage and hepatization in the group of control, but slight edema and hyperemia seen in the group of treatment. The lungs microscopic evaluation in the treatment group displayed, the severity of in ammatory reactions, edema, hemorrhage and hyperemia reduced compared with the control group (Fig. 3E-F). In addition, the lesions and severity of in ammation in trachea, bronchi, and bronchioles were much less than the control group. So that, in trachea, the mild edema of the submucosa region and less in ltration of the in ammatory cells and in bronchi and bronchioles, slight hypermia and low number of monoclonal in ammatory cells observed.
But, evaluation of histopathology in the control group demonstrated severe hyperemia and hemorrhage and purulent acute in ammation so that, the pulmonary blood vessels, particularly the pulmonary arterioles and the alveoli capillaries were dilated and accumulated from blood (Fig. 3A). In these capillaries, a large number of leukocytes were predominantly polymorphonuclear (neutrophils and band cells). Neutrophils adhered to the inner surface of the capillary walls and following the marginalization and sequestration, these observed in intraalveolar space (Fig. 3B). In some samples, there were foci of purulent pneumonia with bronchitis and bronchiolitis (Fig. 3C). In the pleura, in ammation and severe edema (Fig. 3D) and in trachea, sever hyperemia and the edema of the submucosa region and the in ltration of single-nucleated in ammatory cells as multifocal observed.
The results of CT-scan and histopathology of the lung determined after in ammation, BM-MSCs transplantation decreased of edema and in ammation and repaired damaged structures and increased air volume in the alveoli. Histopathology ndings displayed, BM-MSCs transplantation was able to prevention of damage to the heart. In the heart microscopic check in the MSCs receiver group viewed much less damage than the control group (Fig. 5C-D). While, in the PBS receiver group determined hyperemia, presence of polymorphonuclear leukocytes in the capillaries and neutrophils penetrate into the heart muscle and caused myocarditis (Fig. 5A) and cardiomyolysis (Fig. 5B). The results of echocardiography and histopathology of the heart showed after in ammation, BM-MSCs transplantation improved cardiac parameters and prevented damage to heart function, while in the control group there was the damage to heart function.

Discussion
ARDS is one of the developing reason of respiratory failure which is characterized by a decrease in aerated respiratory volume and diffuse alveolar damage (15). Also, the main complication in global pandemic COVID-19 is a secondary severe ARDS and the alveolar cells damage is the main reason of ARDS related to COVID-19 (1,16). Stem cells have effects of positive in reducing acute pulmonary in ammation in the experimental models (13,17,18) and the imaging is a technique to evaluate the treatment process in ARDS. The CT-scan is an effective technique to evaluate the MSCs effects in lung and the echocardiography is recommended to assess function of cardiac.
The present study ndings displayed the BM-MSCs caused decline of the Houns eld unit and the lungs volume mean in CT-scan and improve the cardiac function and parameters in echocardiography and also, reduce edema and in ammation of the lungs and prevention of damage to the heart in histopathology.
This study is the rst imaging evaluation to the process of BM-MSCs effects in the treatment of ARDS experimental model in sheep in the world.
CT-scan has been used in the ARDS studies since the 1980s to represent heterogeneous pulmonary patterns (19). The advantage of this method is the close relationship between CT density and physical density of the lung. This feature shows quantitative information from the lung in different conditions based on the varying degrees of air present in the lung (11,12). In CT-scans of ARDS, the alveolar volume of the lungs appears to be less than normal, due to a uniform pulmonary edema and alveolar collapse. Therefore, CT-scans carry outs a good estimate of pulmonary edema (20). In CT-scan surveys, increasing the Houns eld unit and the total volume con rm the alveolar air replacement with mucus and cells of in ammatory and the occurrence of acute in ammation, which, is consistent with the results of this study on day zero. But, BM-MSCs therapy reduced the amount of these factors at different times of the study that indicates an increase in alveolar air volume and a decrease in lung in ammation. Kobayashi et al., in ARDS patients demonstrated relationship of radiology results with histopathology results, so that increasing lung attenuation in images of radiography and alveolitis and thickening of alveolar septal in histopathology observed which, their results are match with relationship the CT-scan and histopathology results of the present study (21). The results of the our previous study on the rabbit ARDS model (13), which are consistent with the results of this study, showed that the Houns eld unit increased, one day after the in ammation, which con rmed the increase in the production of mucus, edema and acute in ammation. Nevertheless, stem cells transplantation led to a decline in the Houns eld unit, which was signi cant at 48, 72, and 168 h, indicating an enhancement in the alveolar air. In addition, according to statistical studies, the mean volume of the lung increased after in ammation, which cell therapy resulted in a decrease in this process that was signi cant at 72 and 168 h (13).
Echocardiography is a procedure of clinical diagnosis and prognostic to evaluate the performance of the heart in the ARDS. We indicated, which lung in ammation leads to alterations in parameters of echocardiography and decreases function of heart, but in the autologous BM-MSCs recipient group there was no remarkable change in the echocardiography parameters and cardiac function. This suggests that stem cells transplantation was able to prevent the occurrence of these changes. The ndings are consistent with our past survey on rabbits (13).

Histopathology evaluations in various studies have shown reduction of in ammation and edema after
MSCs therapy in the pulmonary damage model. As the researchers have indicated decreasing edema, the thickness of the wall between the alveolar and bleeding in mice after treatment (17,22,23), reduce the in ammatory cells in ltration, edema and hemorrhage following the transplantation in rabbit (13)