The Effect Of High Tidal Volume Ventilation On Lung Tissue Apoptosis And Endothelial Cell Permeability

With the widespread use of ventilators in clinical practice, the morbility of ventilation-induced lung injury (VILI) increases accordingly, hitting 15% among the ventilated patients from a recent report, So, it is important to study the mechanism of VILI and drug screening for its prevention and treatment.The research of VILI has undergone a series of phases, barotraumas, volutrauma, atelectrauma and biotrauma. At present it has been accepted that VILI results from multiple factors including both direct cell injury from the mechanical force and indirect one from mechanical force induced the release of a variety of cytokines, pro-inflammation mediators. Different factors may play different roles at different VILI phases. Section OneThe injurious mechanical ventilation may result in alveolar cell death and vascular endothelial cell death in lung tissue by way of the above-mentioned mechanism. It is believed recently that apoptosis plays an essential role in cell death in acute lung injury. But the relationship between the mechanical ventilation and apoptosis remain unknown, So it is important to study the effect of the injurious ventilation on early apoptosis of lung tissue cells. We will investigate this action inthe first section of this research.Object: To investigate the effect of different tidal volume ventilations on early apoptosis of lung tissue cells.Method: Thirty healthy male Sprague-Dawley rats were randomly divided into three groups, A, B and C. Tracheotomy was performed after anesthesia in each group. Rats in each group received a distinct ventilation with different tidal volume for 4 hours (group A, VT=10ml/kg; group B, VT=20ml/kg; group C, VT=40ml/kg). After 4 hours, lung histopathological changes were assessed and compared among these three groups. The severity of pneumonedema was evaluated by W/D, and transferase d-UTP end labeling assay (TUNEL) was used to define the extent and distribution of apoptosis in bronchus and lung tissues. The expression of caspase-3 was determined by immunohistochemistry.Result: After different tidal volume ventilation, as the increasing of the tidal volume, the lung hyperaemia is more remarkable .the structure is more confused on the pathological section, the inflammatory cell is increasing, the alveolocapillary membrane become thicker, cellular nucleus vaculization is more remarkable, the apoptosis body is increased, the ratio of W/D(wet/dry of lung tissue)is higher, TUNEL assay also exhibited the increase of apoptosis index on both alveolar epithelial cell and bronchial epithelial cells in sections. Immunohistochemistry study showed caspase-3 protein, a key role in apoptosis, was expressed in the cytoplasm and nucleus of alveolar epithelial cells and vascular endothelial cells, especially in bronchial epithelium, the dependabity of W/D and apoptosis index as well as caspase-3 is significant.Conclusion: As the increasing, the injurious degree of pathogical and ultrastructural change is aggravated, the number of apoptosis cell is increased, the expression of caspase-3 is enhanced, the dependabity of W/D and apoptosis index as well ascaspase-3 is significant. We conclude that the apoptosis plays an important role in the ventilation-induced lung injury, which was performed through caspase-3. Section TwoInjurious ventilation may cause overexpressions of certain genes that synthesize some effective proteins to stimulate the release of certain inflammatory factors produced by inflammatory cells, which eventually results in injuries to alveolar epithelium and microvascular endothelium,that is so called biotrauma. An important pathological characteristics of biotrauma is the lung edema which induced by microvascular hyperpermeability. But the mechanisms of ventilation-induced hyperpermeability remains unknown, and the effect of inflammatory mediators released into blood by injurious ventilation on vasopermeability is unclear. In addition, as to the diversity, multiplicity, and interactions of the cytokines and pro-inflammatory mediators, inhibition of one single factor cannot effectively prevent the occurrence of biotrauma-induced lung edema. So, in the second section of this research, we studied the effects of the inflammatory mediators in VILI (high tidal ventilation induced) rat serum and/or Ulinastatin (a protease inhibitor) on endothelial cytoskeleton and monolayer cell permeability in vitro, attempting to explore the cellular and molecular mechanisms of biotrauma-induced lung edema and establish the basis of drug to biotrauma.Object: To investigate the effects of VILI rat serum on endothelial cytoskeleton and monolayer cell permeability, as well as the therapeutic effect of Ulinastatin. Method: Thirty healthy male Sprague-Dawley rats were divided into three groups: group A (normal tidal volume ventilation), group B (high tidal volume ventilation) and group C (high tidal volume ventilation plus Ulinastatin). Rat serum from each animal was added to endothelial cell line ECV-304 medium for two hours to observe the effects of serum and/or Ulinastain on endothelial F-actin, one of the maincomponents of cytoskeleton, and monolayer cell permeability.Results: As compared to that from normal tidal volume ventilated rats, serum from high tidal volume ventilated rats caused a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity at the peripheral filament bands and formation of the long and thick stress fibers in the center, which resulted in endothelial contraction and higher permeability, whereas, injection with Ulinastain could inhibits the above-mentioned changes significantly.Conclusion: Serum from high tidal volume ventilated rats increases endothelial permeability by reorganizing actin cytoskeleton. the result prove that high tidal volume ventilation can release the cytokines into blood,which can induced the hyperpermeability of endothelial cells. But the hyperpermeability can be blocked by pretreatment with Ulinastain through the inhibition of pro-inflammatory mediators, the research establishes the theoretical basis of Ulinastain treating the biotrauma induced lung edema.