(Supplementary Table S3, Fig.?2D). The inter-alveolar septal thickness was also measured to evaluate the degree of interruption of air-blood barrier that occurred as a consequence of collagen deposition. were injected intravenously 28? days after induction and rats were sacrificed after another 28?days for assessment. Minute respiratory volume (MRV), forced vital capacity (FVC) and forced expiratory volume 1 (FEV1) were recorded using spirometer (Power lab data alpha-Boswellic acid acquisition system). Histological assessment was performed by light microscopic alpha-Boswellic acid examination of H&E, and Massons trichrome stained sections and was further supported by morphometric studies. In addition, electron microscopic examination to assess ultra-structural changes was done. Confocal Laser microscopy and PCR were used as tools to ensure MSCs homing in the lung. Results: Induction of lung fibrosis was confirmed by histological examination, which revealed disorganized lung architecture, thickened inter-alveolar septa due excessive collagen deposition together with inflammatory cellular infiltration. Moreover, pneumocytes depicted variable degenerative changes. Reduction in MRV, FVC and FEV1 were recorded. BM-MSCs treatment showed marked structural improvement with minimal cellular infiltration and collagen deposition and hence restored lung architecture, together with lung alpha-Boswellic acid functions. Conclusion: MSCs are encouraging potential therapy for lung fibrosis that could restore the normal structure and function of BLM induced lung fibrosis. Electronic supplementary material The online version of this article (10.1007/s13770-020-00294-0) contains supplementary material, which is available to authorized users. animal study) A count of 2 106 BM-MSCs in 1?ml complete media or 1?ml of complete media without cells were injected intravenously in the tail vein of BM-MSCs treated and cell free media treated groups respectively [26C28]. Homing of BM-MSCs into hurt lung tissue Cell labeling Before injection of BM-MSCs, the cells cytoplasmic membranes were labeled with fluorescent probe (chloromethyl – benzamide octadecyl indocarbocyanines (CM-DiI)) (molecular probes, Thermo Fisher Scientific). Labeled alpha-Boswellic acid cells were viewed under confocal laser microscopy (Leica microsystems, DMi8, Wetzlar, Germany) 72?h after injection in the lung tissue of 2 rats [29]. Actual time-quantitative polymerase chain reaction (RQ-PCR) for detection of the Y chromosome The lung tissues were processed for identification of male BM- MSCs which were injected into female rats through identification of Y chromosome; using real-time quantitative polymerase chain reaction [30, 31]. Detection of SRY DNA was performed using the following primers; forward (5-CATCGAAGGGTTAAAGTGCCA-3) and reverse (5-ATAGTGTGTAG- GTTGTTGTCC-3) [32, 33]. Real-time PCR amplification, data acquisition, and analysis were carried out using the Real-Time detection system Software (Applied Biosystems 7500, Foster City, CA, USA). Assessment of lung fibrosis and the effect of BM-MSCs on lung regeneration Lung function assessment Pulmonary function assessments [tidal volume (VT), minute respiratory volume (MRV), forced vital capacity (FVC), forced expiratory volume (FEV1) and FEV1/FVC ratio] were assessed using a Power Lab digital data acquisition system (4/25, AD Instrument, Bella Vista, Australia), 28?days after BM-MSCs or cell free media injection and before sacrifice of rats. The ventilatory alpha-Boswellic acid parameters were recorded using a pneumotachometer MLT1L (Lab chart?8, AD Instruments, Rabbit Polyclonal to PDK1 (phospho-Tyr9) Castle Hill, NSW, Australia) with P1 channel end connected to the store of the NP/Whole Body Plethysmography (WBP). Histological and histochemical assessment At the end of the study, 28?days after treatment, all rats were sacrificed and both lungs were dissected, then each lung was divided into two pieces. One piece was fixed in 10% neutral-buffered formalin, then processed to obtain (6 um) thin sections. Some sections were routinely stained with H&E as well as others with Massons trichrome for light microscopic examination using, (Olympus BX41) equipped with spot digital camera (Olympus DP20). Histomorphometric study was carried out, using NIH Fiji? program (NIH, Bethesda, MD, USA), where the area percentage of collagen fibers in Masson trichrome stained sections inter-alveolar septal thickness and alveolar surface area in H & E stained sections, were measured in five randomly selected sections for each item. Data was offered as mean??standard deviation (SD) of randomly determined ten fields/section (n?=?5/group)[34]. The second piece was cut into small pieces (1/2C1 mm3) and immediately fixed in 3% phosphate buffered glutaraldehyde pH 7.4, then processed to obtain.