Experimental Studies Of Lung Reparation And Life Support In Acute Lung Injury

IntroductionRespiratory failure remains a major cause of morbidity and mortality in infantsand children. One of the most severe forms of acute respiratory failure is acute lunginjury and acute respiratory distress syndrome (ARDS), which, in China, has amortality of 60-70%, twice or three times as much as those in the industrializedcountries. Patients with ARDS may respond favorably to advanced methods ofintensive care, which include, but are not limited to, various forms of lung-protectiveventilation strategy, such as tidal volume restriction, lung volume recruitment, proneposition, high-frequency oscillatory ventilation, inhaled nitric oxide, surfactant.There remains, however, a substantial proportion of ARDS patients do not respondwell to above mentioned therapies and subjected to die or develop chronic lungdisease, such as lung fibrosis.Inflammation and fibroproliferation are the most important pathophysiologicalprocess of ARDS. The lung fibroproliferation of ARDS has traditionally beenregarded as a late event as reparative phase, but recent studies suggest that this phaseis initiated earliest at 24 h of diagnosis of ARDS, as evidenced by lung fibroblastsgrow and collagen turnover, which suggests that firbroproliferation is an earlyresponse to lung injury and an important therapeutic target as well asanti-inflammation.Bone marrow derived stem cells have great therapeutic potential on injuredlungs, which have the capability to localize themselves in the lungs and differentiateinto specific cell types, such as pneumocytes, endothelial cells, and other cell types. Lung reparation after injurious assault includes specific signals that cause bonemarrow derived stem cells to proliferate and migrate to the site of injury. It protectsthe lungs from injury and fibrosis through suppression of inflammation andenhancement of production of reparative growth factors. We sought to determinewhether bone marrow stromal cells (BMSCs) could localize in injured lungs,differentiating into pneumocytes, and to evaluate their therapeutic potential on lunginjury.In contrast to cell transplantation techniques, in our daily clinical practice,treatment of ARDS using advanced respiratory therapies, especially conventionalmechanical ventilation remain the mainstay in current pediatric intensive care.Although mortality from ARDS is improving, approximately 40% patients withARDS fail to improve or are deteriorating after ventilation. Identifying the keyintervention on inflammation and fibroproliferation in ARDS is an important task atpresent. When conventional strategies fail to improve ARDS, extracorporealmembrane oxygenation (ECMO) may then be intervened as the last but mostimportant therapeutic option during acute phase of severe respiratory failure orARDS. The application of ECMO allows one to reduce overall adverse effects ofmechanical ventilation, thereby minimizing iatrogenic injury from high mechanicalventilation pressures and high fraction of inspired oxygen. ECMO is not a treatmentbut rather a means to support the patients and optimize all aspects of care untilfunctional and histological recovery of the lungs.Partâ… Experimental Study on Differentiation of Murine BoneMarrow Cells to PneumoeytesBackgroundBMSCs have potential of self-renewal and multilineage differentiation. Recentreports suggest that BMSCs may also differentiate into nonstromal tissues, includinglung epithelial cells, which provides a strong rationale to use MSCs as a therapeutictool for intractable lung diseases. We sought to determine whether BMSCs couldlocalize in injured lungs, differentiating into pneumocytes, thereby involving in areparative and regenerative process, especially as a promotor for pulmonaryepithelial cell recovery, with or without lipopolysaccharides (LPS)-induced inflammation.Objective1. To isolate, culture and characterize BMSCs;2. To establish a model of inflammatory lung injury in mature (adult) mice inducedby LPS, and to establish hematopoiesis reconstitutes with BMSCs in vivo;3. To observe migration and differentiation of transplanted BMSCs towardsalveolar epithelial cells (AEC), its efficiency in reparation of injured AECMethods1. BMSCs were isolated from whole bone marrow of C57BL/6J female donor miceby adherence selecting in DMEM/F12 medium with 15% fetal bovine serum,incubating at 37℃with 5% CO₂ in fully humidified atmosphere. Once adherentcells were more than 90% confluent, all colonies were trypsinized and passaged. Bymorphological means, cell growth curve and phenotype were evaluated. Itsmultilineage potency into osteoblasts and neurons was also investigated.2. Mice (n=8 per group) were anesthetized, and received intratracheal 1 mg/kg LPSdissolved in sterile 0.9% NaCl via a canula, followed by 0.15 ml of air, whereascontrol mice received 0.9% NaCl. The mice were sacrificed at 1, 3, 7, 14 d afterinstillation of LPS, total proteins (TP) and WBC count in bronchoalveolar lavagefluid (BALF) were analyzed, lung morphology was determined at the end of theexperiment.3. Primary BMSCs cultured from male C57BL/6 mice were prepared as donor cells,and female C57BL/6J mice as recipients of bone marrow transplantation wererandomly allocated to five groups: (1) control; (2) TBI, total body irradiation (lethaldosage); (3) LI, LPS plus TBI; (4) IM, TBI plus BMSCs; (5) LIM, LPS plus TBIplus BMSCs. Bone marrow reconstituion after transplantation was proved by bloodroutine and bone marrow nucleated cell count analysis, morphology of lung, spleenand liver evaluated by histopathology.4. Recipients of bone marrow transplantation, C57BL/6J mice, received BMSCsfrom donors of the same species as green fluorescent protein (GFP) transgenic mice,were divided into four groups: (1) CM, healthy mice with BMSCs injection; (2) LM, LPS plus BMSCs; (3) IM, TBI plus BMSCs (4) LIM, LPS plus TBI plus BMSCs.Distribution of green fluorescence in lung epithelia was observed by fluorescencecytochemistry of GFP and cytokeratin. To examine whether typeâ…¡AEC werederived from circulation, primary cultures of pneumocytes and detection ofGFP-positive cells by flow cytometric analysis were performed. Meanwhile,expression of surfactant protein (SP)-A, SP-C and aquaporin (AQP)-5 mRNA of thelung tissue was evaluated by real time PCR.Results1. BMSCs are fibroblast-like and grew well in vivo. Detection of cell phenotype byusing fluorescence cytochemistry revealed that, both primary and first passaged cellsshowed CD34 negative, but CD59 and CD90.1 positive characteristics. With theinductive reagents in vitro, BMSCs differentiated into osteoblast cells and neurons.2. Intratracheal LPS administration resulted in increased TP concentration and WBCcounts in BALF at 1 d after instillation, but these phenomena decreased gradually,and the results were consistent with lung histological changes.3. After lethally irradiation, allmice in TBI and LI groups died in 5-10 days. Beforedeath, cytopenia was observed in peripheral blood and bone marrow, indicating allmice died of bone marrow failure. A survival rate after transplantation at 60-70% inIM and LIM groups, along with recovery in WBC, hemoglobin, platelet andnucleated cells in bone marrow. The recovery was gradual after BMSCstransplantation. Irradiation injury in the lungs was seen by histopathology on day 3,with mild leukocytes infiltration and hemorrhage. Much more severe damage wasobserved in LIM. On day 7, the lung injury was gradually alleviated. On day 28, thelungs returned to nearly normal.4. On day 14 after transplantation, evidence of the donor cells derived from BMSCswas seen as differentiated into AEC with co-expression of both GFP and cytokeratinin MI group. Cell counts of GFP-positive AECâ…¡increased significantly in LI andLIM groups compare to the CM. Up-regulated SP-C mRNA expression wasobserved in LIM groupConclusion 1. BMSCs was successfully isolated and cultured in vitro, which showed stablecharacteristics of biological markers and multilineage differentiation.2. BMSCs may proliferate and localize in injured mice lungs, and differentiate intopneumocytes at day 14 after transplantation with a differentiation approximate rateof 10%, presumably participating the reparation of lung injury. The recipients have atendency of higher differentiation potential when LPS-induced inflammatory lunginjury exists.Partâ…¡Experimental Study of Oleie Acid-induced AcuteRespiratory Distress Syndrome with Extraeorporeal MembraneOxygenationBackgroundARDS affects both children and adults, and its mortality in children may behigher than 60% according to recent multicenter clinical epidemiologic study inChina. ECMO is a novel life support technique, or a modified artificial heart-lungmachine, exerting a long term support for patients with severe cardio-pulmonaryfailure. As conventional mechanical ventilation therapy fails, it may be used asultimate life support means, and it may save more than 50% adults, 70% childrenand 80% neonates in the past 2 decades. However, this technique requiresspecialized expert and is at high costs and risk of adverse outcome in multiple organdamage. In this experimental study, we sought efficacy and safety of ECMO innormal lung and oleic acid-induced ARDS model.Objective1. Establish an oleic acid-induced acute lung injury and respiratory failure model inyoung piglets for testing efficacy and safety of ECMO;2. To delineate ECMO effects in lung mechanics, gas exchange, hemodynamics, pathology of major organs, brain oxygenation and perfusion, and inflammation andendogenous nitric oxide (NO) production as assessment in that model.MethodsTwenty-one young piglets of 5-6 weeks old, weighing 9-14 kg wasanaesthetized, intratracheally intubated and subjected to mechanical ventilation.After 30 min of stabilization, oleic acid was injected via central venous line toinduce clinical ARDS. After isolation of right carotid artery and left external jugularvein, catheters were placed respectively for monitoring hemodynamic parametersusing Picco device. Right external jugular vein and right femoral vein were intubatedfor drainage and re-infusion as a circuit for ECMO operation. The animals weredevided into 4 groups: normal control (CON, n=5) with mechanical ventilation onlyfor 24 h; ECMO (n=6), normal animals for ECMO operation of 24 h followed by 6 hrecovery; ARDS (n=5), after induction of ARDS mechanical ventilation for 24 h;AEC (n=5), ARDS plus ECMO for 24 h followed by 6 h recovery. Parameters oflung mechanics, gas exchange and hemodynamics were taken at baseline, ARDSestablishment, and hourly during various treatment periods. At the end of theexperiment, animal lungs were processed for measurement of histopathology,biochemical and biophysical alterations in bronchoalveolar lavage fluid or lungtissue. Special analyses were made for proinflammatory cytokines, apoptosis in lungand heart, and infrared oxygenation of brain tissue, etc. Constitutive and induciblenitric oxide synthases were detected to correlate with lung injury and interventions.Results1. Establishment of ARDS: In 4-6 h of OA, ARDS was induced as evidenced byPaO₂/FiO₂＜200 mmHg, a more than 50% reduction of dynamic compliance (Cdyn),severe and widespread neutrophil infiltration, edema, hemorrhage and alveolaratelectasis in the lungs.2. Surival rate: All animals in both CON and ECMO groups survived theexperiments, whereas one animal in AEC group died at 22 h of ECMO, but allanimals inARDS group died after 14-24h of treatment.3. Management of ECMO system: Blood flow rate during ECMO bypass was 70-80ml/kg body weight, with an activated clotting time (ACT) at 180-220 s throughout, while SvO₂ 68-75% was maintained.4. Hemodynamics: There was a trend in increment of cardiac output (CO) withexperimental time, in contrast to a decrease of systemic vascular resistance (SVR) inECMO group; compared to ARDS, AEC group had certain level of improvement inmean systemic arterial blood pressure (MABP), CO and SVR.5. Lung mechanics: There were decreases of Cdyn in all the groups, especially inECMO group at 16 and 24h and post-ECMO period compared to that at baseline(p＜0.01). At time 0h, values of Vd/Vt in both ARDS and AEC groups weresignificantly higher than the baseline level (p＜0.05), whereas in the treatment period,these groups diversed. AEC group had improvement in PaCO₂ comparable to theCON group.6. Gas exchange: There was a significant improvement in PaO₂/FiO₂ in AEC groupafter ECMO was initiated in contrast to ARDS group in which oxygenation wasdeteriorated all the time. Both CON and ECMO groups had moderate deteriorationin PaO₂/FiO₂. This was consistent with A-aDO₂, oxygenation index, ventilationindex and intrapulmonary shunting (Qs/Qt) increment.7. There were tendencies in peripheral WBC in ARDS and AEC groups. Hb and Hctwere also decreased with ECMO time prolonged. However, no statistical differenceswere found within and between the groups, nor any significant differences in thebiochemical parameters.8. Compared to CON, both ARDS and AEC groups had significantly decreased totalphospholipids and elevated minimum surface tension in BALF (p＜0.01), while therewas no such difference between ECMO and CON groups.9. Lung inflammation and injury: Compared to CON, ARDS and AEC groups hadhigher total proteins, lower disaturated phosphatidylcholine to total protein ratio inbronchoalveolar lavage fluid, increased wet-todry lung weight ratio,myeloperoxidase activity, expression of interleukin 6 and 8 mRNA in lung tissue(p＜0.01).10. Histopathology: Compared to CON group, mild morphological alterations werefound in the lungs, heart, liver, intestine, kidney and brain in ECMO group.11. Brain oxygenation and perfusion: With NIRS technique, no significant differencewas detected across the groups. 12. Endogenous NO: In ECMO, contents of nitrite/nitrate were decreased, and lungtissue expression of eNOS mRNA attenuated in ECMO group.ConclusionsAfter successful establishment of OA-induced ARDS in piglets, we comparedin parallel the effects of mechanical ventilation or ECMO in the normal and ARDSanimals. In particular there were no specific differences of hemodynamics, braintissue oxygenation and perfusion between the normal control and ARDS-ECMOgroups. ECMO improved global effects in ARDS animals, and alleviated their lunginjury severity without impose extra risk and impairment in extrapulmonary organsystems. Inflammation initiated in healthy animals with evidence of mildhistological alteration in lungs, heart, liver, intestine, kidney and brain. After ECMOthere was a reduced production of endogenous NO with attenuated expression ofendothelial NO synthase in the lungs.