| BackgroundVentilator Induced Lung Injury (VILI) refers to the problems of lung tissue damages and other serious complications in the process of mechanical ventilation due to improper operation or immune reaction. The main clinical manifestations were sudden respiratory distress, blood pressure lowering, restlessness or facial bruising. Researches show that the clinical incidence rate is about 20～35%, and severe complication or mortality rate is about 18～32%.More attention is drawn to the idea that inflammatory cytokine should participate and regulate the injury. Inflammation is an important cytokine in the human immune response in the process of synthesis and secretion. In terms of function, these cytokine can be divided into two categories:anti-inflammatory and pro-inflammatory. TNF-a refers to pro-inflammatory cytokines in inflammatory mediators in the earliest time with the most obvious effect and IL-10 is the most typical and multi-functional negative regulatory factor in the inflammatory cytokines. There are lot of studies targeting the effects of different tidal volumes of ventilation on lung injury, but the studies on the effects of different tidal volume ventilation on pro-inflammatory and anti-inflammatory cytokine expression are still relatively rare. This study also analyses the changes of ventilation and inflammatory factor concentration, examine the correlation between them.Part One:The preparation of experimental model of mechanical ventilation with different tidal volumes and the observation of lung tissue damagesObjective:To establish the experimental model of mechanical ventilation with different tidal volumes.Materials and Methods:Select 24 SD rats from Sun Yat-Sen University animal test center as the subjects. The rats are from the same batch in terms of cleanness. Half of them are male and the other half are female, with the body weight between 220-250g. The experiment time is 2012 October -2013 June. The 24 SD rats were randomly divided into three groups with 8 rats in each group (n=8):F group (intubation without gas), L group (low tidal volume ventilation) and H group (high tidal volume ventilation) in three groups.After experiment the rats were placed in an environment with the constant temperature of 25℃, and they were fed again in the next 24 hours. After the resumption of feeding,10% chloral hydrate anesthesia, with 0.35g/100g standard, was injected into the rats. When the rats lost consciousness, they were killed by cutting the carotid artery. By means of thoracic separation, the appearance of the intact lung tissues on the left and right sides were observed.Results:1. Observe the behavior of ratsThe rats in group F without ventilation were restored to normal activities after 24 hour feeding, and there were no abnormal symptoms in terms of their respiration and behavior. Low tidal volume ventilation was done in the rats of L group. Their activeness was decreased, and they were tired and lied down, with furs erect. Some of these rats even had dreadful breaths. Cyanosis occurs in the skin of Group H with high tidal volume ventilation, and these rats were tired and tended to lie down, with chaotic furs. Some of these rats even appeared groggy and other neurological symptoms also emerged.2. Observe the lung tissues of ratsThe appearance of lungs of the rats in Group F without ventilation was normal. The lung was red, without bleeding or swelling. The lungs of the rats in group L had a similar color with group F, but the lung tissues were a bit swollen, with some visible petechial hemorrhage. Rats in Group H had bloody fluid in lung, and the lung was obviously swellen, with petechial hemorrhage and splinter hemorrhage.3. Rats’W/D value changeWhen the mechanical ventilation was increased, the W/D value in the three groups of rats had an obvious increase. In group F, the average value of W/D was 5.14. In group L and H, the values were 6.54 and 9.41 respectively. There were significant differences when comparing group L and group H with the normal group F. Therefore the results were statistically significant (P<0.05). The ratio of group H was significantly higher than that of group L, so there was statistically significant correlation (P<0.05).4. Score of rat lung tissue injuryAs the mechanical ventilation increased, the score of the rats’lung injury significantly increased. There were significant changes in the comparison between Groups H and L and the normal group F, so the results were statistically significant (P <0.05); There was no statistical significance (P>0.05) in the comparison between group L and group F.5. Histopathologist observation in ratsIn the process of observing the pathologic changes in lung of rats under optical microscope, the structures of the bronchial epithelial cells and alveolar in group F were found to be complete; the alveolar septum is normal, and there was no inflammatory cell infiltration. The pulmonary septum in group L with low tidal volume of mechanical ventilation was thickening; Slight edema of pulmonary interstitium was found, with a small amount of leakage in alveolar space and a small amount of inflammatory infiltration of inflammatory cells. The alveolar septum in group H with high tidal volume of ventilation was thickening, with some alveolar rupture and visible hemorrhage in alveolar space. Interstitial pulmonary edema was found, small vascular rupture could be seen, and large numbers of inflammatory cells were accumulated.Conclusion:1. The changes of the rats’behaviors, the appearance of their lung tissues, and the W/D value indicate the successful establishment of mechanical ventilation model. Such changes also indicate that the changes of the mechanical ventilation volume are closely related to the degree of lung tissue injury, especially in the way that over mechanical ventilation would cause the severity of lung tissue injury in rats.2. Compared with Group H with the high tidal volume of mechanical ventilation, the lung tissue injury in group L with the low tidal volume of ventilation is less severe, without worsening the rats’ lung tissue injury or accelerating the inflammatory reaction in tissue cells.Part Two:Effects of different tidal volumes of ventilation on the expression of TNF-α and IL-10 in rat lung tissue homogenate.Objective:To observe the effects of different tidal volumes of ventilation on the expression of TNF-αand IL-10 in rat lung tissue homogenate.Methods:1. Animal grouping and the establishment of modelThe preparation of animal model has been completed in the first part. The difference here lies in the preparation of specimens and the measurement of indexes.2. Lung tissue homogenate preparationThe right lung tissues obtained in the first part was taken away from liquid nitrogen. Two grams of tissues were thoroughly rinsed with physiological saline. The tissues were chopped and homogenized in the homogenizer. Then the prepared homogenate was put into the centrifuge for separation, wherein the centrifugal time is 20min, the centrifugal speed was 12000r/min. Finally the supernatant was obtained and preserved with the temperature of -70℃ for further test.3. Determination of inflammatory factorsDetermination of inflammatory factors must be taken from the above specimens, and the detection method is the ELISA method. The overall research was done in accordance with the detailed instruction in the ELISA kit.Results:1. Expression of TNF-α in the rats’ pulmonary homogenateThe experimental results show that, the expression of TNF-α content increased significantly in group H than in group F and the results were statistically significant (P <0.05). When compared with group F, the TNF-α protein content in the lung tissue homogenates increased slightly in group L; but the results were not statistically significant (P>0.05). The content of TNF-α in the lung tissue homogenate in group H was significantly higher than that in group L, so the results were statistically significant (P<0.05).2. Expression of IL-10 in rat lung tissue homogenateThe experimental results show that, the content of IL-10 in the rats’ lung tissue homogenate in group H was significantly higher than that in group F; the results were statistically significant (P<0.05).Compared with group F, the expression of IL-10 in lung homogenates in group L was increased, but there was no statistical significance (P>0.05). The content of IL-10 in the rats’ lung tissue homogenate in group H was significantly higher than that in group L, and the results were statistically significant (P<0.05).3. Correlation between tidal volume and TNF-α and IL-10 expression [Interior]Between H group and L group the terms of the content of TNF-α (r=0.543, P=0.105) in lung homogenates was no significant correlation. And the content of IL-10 (r=0.393, P=0.261) was no significant correlation too.4. Correlation of TNF-α and IL-10 content[Inter]For group L with low tidal volume of ventilation, a moderate positive correlation in expression of TNF-α and IL-10 in lung homogenates can be found (r=0.754, P=0.012). For group H with high tidal volume of ventilation, there was a positive correlation between the expression of TNF-α and the expression of IL-10 in lung homogenates (r=0.915, P=0.000).Conclusion:1. High tidal volume of mechanical ventilation not only increases the lung tissue injury in rats, but also causes significant increase of the inflammatory factors (TNF-α and IL-10) in the pulmonary tissue cells.2. Compared with high tidal volume, the low tidal volume reduce the rat lung tissue injury, also decreased the expression of TNF-α and IL-10 in lung homogenates.3. There was no significant correlation between the expression of TNF-α and IL-10 and the changes of the tidal volumes of mechanical ventilation, which means that the changes of tidal volume cannot be used as an index to indicate the changes of the expression of TNF-α and IL-10 in lung homogenate.4. Under the conditions with the same tidal volume of ventilation, the changes in the TNF-α are highly correlated with the changes in the IL-10, which means, when the expression of TNF-α increases, the expression of IL-10 increases at the same time. |
PDF Full Text Request