Effects Of Angiotensin â…¡ System On Neurogenic Pulmonary Edema In Rabbit

Part1. Replication of neurogenic pulmonary edema in rabbitBackgroundNeurogenic pulmonary edema (NPE) is an acute life-threatening complication following central nervous system (CNS) injury, which usually appears within minutes to hours after a severe CNS insult. Epidemiological data of NPE are rare. NPE remains an uncommon but often underdiagnosed clinical problem in the pediatric population. In recent years, hand, foot, and mouth disease caused by enterovirus71(EV71) infections has been an important public health issue. The severe infection cases are often associated with CNS involvement; once occurring NPE and cardiopulmonary failure, they will have poor prognosis, high fatality and disability.The exact mechanism for neurogenic pulmonary edema is unclear, which includes blast injury theory and hemodynamic theory, penetration defect theory and pulmonary capillary permeability doctrine. It is generally considered to be the result of the interaction of these two mechanisms, in which sympathetic activity plays an important role in the development of NPE. Over the years, domestic and international researchers have established various preparation methods of NPE animal model in the process of further studying of pathogenesis and intervention. At present, through the method of EV71infection, animal models with the CNS involvement can be replicated successfully, but NPE has not been obtained. The aim of this part in the study is to choose another method except EV71infection for replicating NPE model, and to observe sympathetic activity and hemodynamic changes.Objectives1. To replicate the neurogenic pulmonary edema in rabbits.2. To observe the sympathetic activity and hemodynamic changes in the process of neurogenic pulmonary edema.Methods1.18healthy adult New Zealand rabbits were randomly divided into three groups: normal group (n=6), NPE group (n=6) and saline (NS) group (n=6).2. NPE models were induced by intracisternal injection of fibrinogen and thrombin.3. Rabbits were given the different treatment by grouping after anesthesia:(1) normal group was anesthesiaed without other treatments, as a blank control.(2) NPE group was made surgical operation (endotracheal intubation, PiCCO arterial and venous catheterization), punctured the cisterna magna and injected fibrinogen and thrombin intracisternally.(3) NS group was made the same surgical operations as NPE group, punctured the cisterna magna and injected equivalent saline intracisternally.4. During the experiment, normal group was observed respiratory and heart rate changes at the time of Oh,1h,2h,3h,4h,5h,6h after anesthesia; NPE group and NS group were observed respiratory rate (RR), heart rate (RR) and mean arterial blood pressure (MABP) changes before and after intracisternal injection of drugs, and the dynamic changes of MABP at the different time points (base state BO and1min,5min,10min,15min,30min,1h,2h,3h,4h,5h,6h after intracisternal injection of drugs). At the time of baseline (B0),15min after intracisternal injection (P15min) and at the end of experiment (P6h,6h after intracisternal injection), the venous samples were taken for the analysis of plasma epinephrine (EPI) and norepinephrine (NE) concentration.5. At the end of the experiment, animals were sacrificed by overdose of10%potassium chloride, the chest were opened immediately, and then we observed whether there was edema fluid spontaneously appearing in the tracheal tubes or not and the level of pulmonary subpleural bleeding macroscopically for evaluation of lung edema. Lower lobes of left lungs were taken for histopathological examination and right lung tissues were collected and stored for using in the study of part2.ResultsIntracisternal injection of fibrinogen and thrombin induced neurogenic pulmonary edema model in rabbits. The observation and test items chaged as follows:1. Respiratory rate (RR):RR had no significant differences among three groups at baseline (P>0.05). The respiratory rate of normal group had no significant change at the time points of observation (P>0.05). The respiratory rate of NPE group and NS group after intracisternal injection immediately (at Plmin) increased significantly when compared with baseline (P<0.01). The increase amplitude of respiratory rate (ΔRR) between the two groups had no significant difference.2. Mean arterial blood pressure and heart rate changes:HR had no significant difference among three groups at baseline (P>0.05).The heart rate of normal group had no significant changes at the time points of observation (P>0.05). When compared with BO, at the time of intracisternal injection immediately (Plmin), MABP in both NPE group and NS group increased significantly (P<0.01) and HR also increased (P<0.05). The increase amplitude of MABP (ΔMABP) between the two groups had significant difference (P<0.01), but the increase amplitude of heart rate (ΔHR) between the two groups had no significant difference (P>0.05). In NPE group, MABP had a downward trend over time and in a short time declined to a basic level, even to very low numerical value. In contrast, MABP in NS group declined to and maintained in a basic level following the time change.3. The level of epinephrine (EPI) and norepinephrine (NE) in plasma:The level of EPI, NE had no significant difference between NPE group and NS group at baseline (P>0.05). In the NS group, when compared with B0, the level of EPI and NE had no significant difference at the time of P15min or P6h (P>0.05). In the NPE group, when compared with B0, the level of EPI increased significantly at the time of P15min and P6h (P<0.01); the level of NE increased significantly at the time of P15min (P<0.01) and P6h (P<0.05). When NPE group was compared with NS group, the level of EPI increased significantly at the time of P15min and P6h (P<0.01); the level of NE increased significantly at the time of P15min (P<0.01) and P6h (P<0.05).4. The edema fluid in tracheal tubes:At the end of experiment, when chest were opened immediately without compressing the lung, pink foamy edema fluid spontaneously appeared in the tracheal tubes in all rabbits of NPE group, but edema fluid did not appear in NS group or normal group.5. Macroscopical lung tissues:The lung tissues in NPE group were markedly swollen, which manifested enlarged size, blunted edge, dark red appearance, large areas of congestion, lung section visible pink foamy fluid. But lung tissues in NS group and normal group had no obvious swelling and showed sharp edge, pink appearance, no congestion, no lung section foamy fluid.6. The level of pulmonary subpleural bleeding:The lungs of NPE group had severe lung edema, pulmonary visible Ⅱ～Ⅲ grade pulmonary subpleural bleeding, of which2/12graded Ⅱ,10/12graded Ⅲ. NS group and normal group had not been seen pulmonary subpleural bleeding, of which the degree of pulmonary subpleural bleeding was0.7. Pulmonary pathomorphology:The lung tissue alveolar septa of rabbits in NPE group were widened significantly, of which some alveolar septa ruptured, alveolar space integrated, blood cells infiltrated to the pulmonary interstitial or alveolar space, and some alveolar spaces were seen homogeneous light dyed pink exudation. The lung tissue alveolar septa of rabbits in NS group and normal group were normal, of which alveolar septa maintained integrity, no red blood cells infiltrated, and alveolar spaces were dry without exudation.Conclusions1. After intracisternal injection of fibrinogen and thrombin, the rabbits appeared sympathetic activity changes.2. After intracisternal injection of fibrinogen and thrombin, rabbits were seen edema fluid appearing in the tracheal tubes and pathomorphological pulmonary edema.3. The method of cisterna magna injection of fibrinogen and thrombin can be used to prepare rabbit neurogenic pulmonary edema model. Part2. Effects of angiotensin Ⅱ system on neurogenic pulmonary edema in rabbitBackgroundRecent studies have found that organism not only exists a systemic renin-angiotensin system (RAS), of which many organs and tissues are also exist independent local RAS. It has been found that RAS can be activated during acute lung injury, such as acute respiratory distress syndrome, meconium aspiration syndrome, asthma and severe acute respiratory syndrome, so we want to study whether RAS is actived during neurogenic pulmonary edema (NPE) or not, and observe the effects of drugs interventing RAS on NPE, which may provide a new breakthrough for the NPE prevention and treatment.Objectives1. To investigate whether RAS is actived during fibrin-induced NPE in rabbits.2. To observe the effects of an angiotensin converting enzyme inhibitor on NPE.Methods1. On the basis of confirming NPE model, the NPE intervention group was added, that was enalaprilat group (Ena goup), which included6healthy adult New Zealand rabbits. After preparing model, each rabbit was given intravenous enalaprilat0.5mg/kg. The groups included in the experiment were normal group, NPE group and Ena goup. 2. During the experiment, we observed the changes of respiratory rate (RR), mean arterial blood pressure (MABP) and heart rate (HR) of Ena group in different time-point including baseline, and1min,5min,10min,15min,30min,1h,2h,3h,4h,5h,6h after intracisternal injection of fibrinogen and thrombin.3. At the end of the experiment, animals were sacrificed. The chest were opened immediately, and then we observed whether there was edema fluid spontaneously appearing in the tracheal tubes or not and the level of pulmonary subpleural bleeding macroscopically for evaluation of pulmonary edema. Lower lobes of left lungs were taken for histopathology by conventional HE staining and right lung tissues were collected to measure the Ang Ⅱ concentration and the mRNA expression of ACE, ACE2and AT1R.Results1. Lung Ang Ⅱ level of NPE group was significantly higher than that of normal group (539.7±146.6vs253.4±37.2pg/ml) and Ena group (539.7±146.6vs308.7±35.4pg/ml)(P<0.05), while there was no significant difference between Ena goup and normal group in lung Ang Ⅱ level (P>0.05).2. The mRNA expression levels of ACE, ACE2and ATI R in lung:1) The ACE mRNA expression level:There was no significant difference among three groups, but the expression level trend was NPE group> normal group> Ena group.2) The ACE2mRNA expression level:Compared with the normal group, the ACE2mRNA expression levels of NPE group and Ena group were both downregulated (P<0.05). There was no significant difference between NPE group and Ena group.3) The AT1R mRNA expression level:There were no statistically significant differences among three groups, and the expression level trend was also not obvious.4) ACE/ACE2mRNA expression level:There was no significant difference among three groups, but the ratio trend was NPE group> Ena group> normal group.3. Evaluation of pulmonary edema1) The edema fluid in tracheal tubes:Pink foamy edema fluid did not spontaneously appear in the tracheal tubes in normal group or Ena group, but appeared in all rabbits of NPE group.2) The level of pulmonary subpleural bleeding:In normal group,12/12graded0; in NPE group,2/12graded Ⅱ,10/12graded Ⅲ; in Ena group,2/12graded Ⅰ,8/12graded Ⅱ,2/12graded Ⅲ.4. Histopathological lung injury scoreThe alveolar overdistension score in the Ena group and NPE group respectively was1.2±0.1vs2.8±0.4, and there was statistically significance between the two groups (P<0.01). The septal/interstitial edema score in the two groups respectively was1.3±0.2vs2.3±0.5, of which had significant difference (P<0.01). The alveolar exsudation score in the two groups respectively was1.5±0.2vs1.8±0.3, but there was no statistically significance between the two groups (P>0.05). The total lung score in the two groups respectively was1.3±0.1vs2.3±0.4, of which had significant difference (P<0.01). Alveolar overdistension, septal/interstitial edema or alveolar exsudation were not seen in the normal group, so the lung injury score was0.Conclusions1. During NPE, lung Ang Ⅱ concentration increased, the ACE2mRNA expression level was downregulated, and lung local RAS was activated.2. Enalaprilat might attenuate the degree of lung injury by interventing NPE, which decreased lung Ang Ⅱ concentration and histopathological lung injury score.