Animal Care
Ten male Shall sheep with mean weight 35±4 kg were selected and their health was confirmed with clinical and paraclinical examinations and they were randomly divided into two groups of treatment (BM-MSCs recipient group, n=5) and control (PBS recipient group, n=5). Then, all sheep were kept in a place at the Institute of Biomedical Research, University of Tehran in ventilated conditions and appropriate light and temperature. The rations of sheep during the study period included alfalfa and barley. All protocols of animals and experiments were checked and appropriated by the Animal Research Ethical Committee of University of Tehran (Tehran, Iran).
Sampling from bone marrow (BM) and isolation and culture of MSCs
First, the sheep of the treatment group were off feed for 12 h and then anaesthetized with 35 mg/kg ketamine 10% (Alfasan-Holland), and 5 mg/kg xylazine 2% (Interchemie-Holland) as IM. After that, BM samples (about 5 ml) were collected from anaesthetized animal illiac crest in aseptic condition. Then, under GMP conditions, BM-MSCs were isolated with density gradient method, ultimately the cell precipitation were transfered into 25 cm2 flasks with DMEM- high glucose (Gibco), 20% FBS (Gibco) and 100 U/ml penicillin/streptomycin (Biowest, France) and placed at 37 °C in a humid atmosphere of 5% CO2 (Memert, USA). The medium was replaced every 3–4 days. When confluency of the cells reach more than 80%, the monolayer cells were trypsinized with 0.25% trypsin-EDTA (Gibco, USA) and passaged.
Characterization of BM-MSCs
The BM-MSCs at passage three were harvested and labeled with PE-conjugated antibodies against CD45 (Biolegend, Inc), CD29, CD31 and CD44 (Abcam, Inc.), and then the cells were analyzed using flow cytometry (BD Bioscience, USA) and the Flowjo program version 7.6.1. In addition, to check multi-lineage differentiation ability of BM-MSCs, they were separately treated with osteogenic differentiation medium for 21 days and adipogenic differentiation medium for 14 days. Then the cells were fixed with Paraformaldehyde 4% for 20 min at room temperature and were respectively stained with Alizarin Red (Bioidea-Iran) for 10 min and Oil Red O (Bioidea-Iran) for 15 min at room temperature. After that, cells were washed with distilled water to remove excess stain and were observed using the Olympus IX71 inverted microscope.
ARDS modeling in sheep
The dose calculation of E. coli LPS and ARDS establishment
First, one vial of LPS from E. coli O55: B5, 10 mg (Sigma-Aldrich) was dissolved in 10 ml of sterile PBS and divided into sterilized microtubes, and were stored in freezer at -80 ⁰C. After this, five Shall sheep weighing 30-35 kg were selected and anesthetized and intubated, and doses of 50, 100, 200, 400 and 800 μg/kg diluted in PBS heated to 37 °C were intrapulmonary prescribed. Then, the sheep were clinically and paraclinically investigated and finally, according to the findings, the dose of 400 μg/kg was confirmed for inflammation in this study.
In the next stage, all sheep both in the treatment group and in the control group, were anesthetized and intubated and ARDS experimental model was induced by LPS at 400 µg/kg. The findings of clinical signs, radiography, blood and BAL profiles and blood gases analysis were recorded before and after administration of LPS.
Autologous transplantation of BM-MSCs
In the treatment group, 24 h after induction of ARDS, first, viability and number of BM-MSCs were assessed by trypan blue staining. Then, under general anesthesia, sheep were sternaly placed, trachea was cannulated. Then, autologous transplantation of 5×107 BM-MSCs were intrapulmonary done. Also, in the control group, 1 ml PBS was intrapulmonary infused only, one day after induction of ARDS.
Clinical and laboratory investigations
The sheep both in the treatment group (ARDS+BM-MSCs) and in the control group (ARDS+PBS) were monitored by clinical examination, evaluation of blood and BAL samples consisted of blood gases analysis, hematologic analysis and measurement of cytokines before establishment of ARDS (time -24), time of BM-MSCs transplantation/PBS infusion (times 0) and then for 3, 6, 12, 24, 48, 72, 168 h after BM-MSCs transplantation/PBS infusion.
Clinical examination
The clinical symptoms of RR, HR, RT, breathing sound, cough, nasal discharge, mucosal membrane, appetite and physical condition were determined and documented based on clinical scores for each sheep. The scoring is based on examination of clinical benchmark according to scientific texts and articles 11,12 that were individually specified for each sheep (Table 1).
Table 1: Scored system of clinical signs in the sheep.
|
Signs
|
0
|
1
|
2
|
3
|
4
|
Breathing sound
|
Normal
|
-
|
-
|
Abnormal
(Crackle-Wheeze)
|
-
|
Cough
|
Absent
|
A single cough
|
Involuntary, repetitive coughs
|
Repetitive coughs
|
-
|
Nasal discharge
|
Absent
|
Serous
|
mucus
|
Muco-purulent
|
Purulent
|
Mucosal membrane
|
Normal
|
Mild hyperemia
|
Severe hyperemia
|
Cyanotic
|
-
|
Appetite
|
Normal
|
Decrease (<50%)
|
Decrease (≥50%)
|
Anorexie
|
-
|
Physical condition
|
Normal
(alert)
|
Dull (slowly response)
|
Depressed (acutely slow response)
|
Severe depression (recumbent)
|
-
|
Sampling and assessment of blood and BAL samples
Blood samples were collected from the ear artery and jugular vein into syringes containing the anticoagulation. Arterial samples containing heparin were used for blood gases analyses by Blood Gas Analyzers (OPTI CCA-TS) and the parameters included pH, PO2, PCO2, HCO3, TCO2, SatO2 and anion gap and electrolytes such as Na+, K+, and Cl- were measured.
Venous samples containing EDTA were distributed in two microtubes. One microtube was immediately used for haematologic parameters analysis, and the other was centrifuged at 800 g at 4 °C for 20 min. Then, plasma was separated and frozen at -80 °C for measurement of cytokines. The pro and anti-inflammatory cytokines concentrations including TNF-α, IL-6, and IL-10 were measured with commercially available ELISA kits (Eastbiopharm-USA) following the manufacturer's protocols.
Also, after general anaesthetizing with ketamine and xylazine, sheep were sternaly positioned and lung wash was performed with PBS via endotracheal tube. 10 ml sterile normal saline (room temperature) was instilled into lung and immediately aspirated and samples of BAL were collected. Then, samples were centrifuged at 400 g at 4 °C for 10 min and the supernatants were placed at fireezer at -80 °C for measurement of cytokines and supernatants from pellets were used for complete cell counts. The pro and anti-inflammatory cytokines concentrations including TNF-α, IL-6, and IL-10 and concentrations of Ig M, albumin and total protein to evaluate the pulmonary vascular permeability were measured with commercially available ELISA kits (Eastbiopharm-USA) following the manufacturer's protocols. In addition, smears were prepared from pellets and stained by Giemsa and total cell count, neutrophils and macrophages were counted.
Statistical analysis
The results were analyzed statistically by SPSS software (version 24). Data were assessed by the independent samples t-test, repeated measure, Mann–Whitney U and Friedman tests at the significance level p<0.05.