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Intravenous and Intratracheal Mesenchymal Stromal Cell Injection in a Mouse Model of Pulmonary Emphysema

The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.


Supplementary Methods
Non-invasive lung function measurements: Animals were each placed in an individual plethysmograph and a 30 min interval was used for the animal to adjust to their new environment before the measurements were started. Measurements were continuously recorded for 1 h after which animals were returned to their cages.
Invasive lung function measurements: Animals were anesthetized with an ip injection of Ketamine (150 mg/kg) and Xylazine (10 mg/kg) before tracheostomy. A 25G endotracheal tube was inserted and sutured to prevent leakage. Following intraperitoneal injection of Pancuronium (5mg/kg), the animal was ventilated at 150 breaths/min with a tidal volume of 10mL/kg, and lung function was measured using the Flexivent ventilator (SCIREQ, Montreal, QC, Canada). The flexivent measures flowvolume relationships of the respiratory system, and uses forced oscillations to discriminate between the airways and the alveolar compartment. The "Total Lung Capacity" (TLC), "Snapshot" (based on single-compartment model), and "Primewave-8" (based on constant phase model) perturbations were used to measure lung function.
For the "TLC" maneuver, the lung is inflated to +30 cmH 2 O and deflated afterwards. For the "Snapshot" maneuver, three sinusoidal waves of in-and expiration provide the resistance, elastance and compliance parameters. For the "prime-wave 8", multiple frequency forced oscillations were applied to measure tissue elasticity and airway resistance properties. Volume-driven pressure-volume loops were also obtained and provided TLC, inspiratory capacity from zero pressure and static and dynamic compliance. All measurements were repeated until 4 successful recordings, with a coefficient of determination of >0.95, were obtained. Animals also underwent forced expirations by inflating to TLC and then rapidly applying a negative pressure of -55 cmH 2 O to the expiratory valve. In the current state of small animal ventilator technology, this technique is the closest to mimicking forced expiratory spirometry in humans.
Parameters obtained from this maneuver were the forced vital capacity, mean forced expiratory flow, peak forced expiratory flow and forced expiratory flow at 0.05, 0.1 and 0.2 seconds.
Histology and morphometry: Following lung function measurements, the animals were exsanguinated by cutting the abdominal aorta while still under anesthesia. The thoracic cavity was opened and heparin dissolved in phosphate buffered saline (PBS) was injected into the right ventricle of the heart, and the lungs were flushed free of blood. A catheter was inserted into the trachea to allow 4% (w/v) paraformaldehyde (PFA) to inflate the tissue at a constant pressure of 20 cm H 2 O. After 5 minutes, the catheter was removed and the trachea tied off. The lungs were removed from the thorax as a single block and fixed in 4% PFA overnight. Fixed lungs were embedded in paraffin at 60 o C.
Randomly orientated tissue blocks were cut into 7 µm sections, stained with hematoxylin-eosin and Hart's elastic stain, and morphometry was performed on digital images taken at x200. Images were captured from 5 non-overlapping fields from each slide, with 4 slides per animal. Mean linear intercepts (Lm) was measured and calculated(1). For alveolar number determination, 20 randomly selected nonoverlapping fields from sections obtained from 4 animals per group were examined.  During invasive lung function measurements, an average for each individual mouse was calculated from 4 accepted (coefficient of determination:

Supplemental
COD>0.95) measurements for each parameter. *p<0.05, **p<0.001 when compared to control animals at the same time point. During whole body plethysmography (WBP), mice were allowed to adjust to the measuring chambers for 30 minutes, afterwards measurements were taken continuously for 60 minutes, and an average was calculated for each mouse individually. *p<0.05, **p<0.001 when compared to control animals. BAL fluid from 6 mice per group was collected and processed using Luminex xMAP technology. Values for a subset of the measured cytokines (IL-1β, IL-10,IL-13,GM-CSF) were below the detectable range. *p<0.05, **p<0.001 when compared to control animals at the same time-point. During invasive lung function measurements, an average for each individual mouse was calculated from 4 accepted (coefficient of determination: COD>0.95) measurements for each parameter. *p<0.05, **p<0.001 when compared to control animals within the same experiment (intratracheal or jugular vein).