The Research On The Mechanism Of Acute Lung Injury Induced By Endogenous And Exogenous Factors Of Lungs

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS) are some kinds of symptom complex,which can be induced by endogenous and exogenous causes of lungs and manifest themselves in acute,progressive and hy-poxemic respiratory failure.The pathogen of ALI/ARDS can be divided into two sorts;endogenous factors of lungs(such as aspiration pneumonia)and exoge-nous factors(such as serious shock and fat embolism).Domestic and overseas researchers thought the difference of ALI induced by two sorts of factors might bring far-reaching influence on the research and treatment of the disease.In this research,two kinds of ALI models were made,pneumonia evoked by an in-tratracheal instillation of Bacillus coli as endogenous factors,and changes resul-ted from intravenous injection of oleic acid as exogenous factors.Comparing the changes in these two kinds of ALI models,we made note of the differences of the changes of pathology,pathophysiology,pro-inflammatory,anti-inflammatory mediator and nitricoxide synthase,which provided the experimental and theoreti-cal basis for furthering the study of the treatment of ALI.Methods1 Material and MethodsTwenty-seven albino rabbits of New Zealand in regardless of male or fe-male were chosen(from the Experimental Animal Division of China Medical U-niversity),whose weights ranged from 2.5 to 3.0 kg.Bacillus coli(25922 type strain)were provided by the bacterium division of the No.1 Hospital of China Medical University.Other equipments and materials included a blood gas analyzer(AVL OMNI V of Switzerland),a mobile monitor made by HP,a ventilator of Bear 1000 t/es,a electronic turbidity kit,a SUNRIS auto micro-plate reader,Skelaxin(vecuronim bromide),narcotic(20%ethyl carba-mate),radioimmunity kits of TNF and IL-6,a ELISA kit of IL-10,immuno-histochemistry kits of iNOS and eNOS,and a SABC kit.2 Preparation of bacterial suspension(1)The strains of Bacillus coli were inoculated in a bouillon agar culture, cultivated on 37℃for 24 hours.Then colonies were filled into different germfree tubes,and were saved in a refrigerator in -20℃for later use.(2)The stand-by strains of Bacillus coli in refrigerator in -20℃were inoculated in the bouil-lon agar culture and cultivated for 24 hours.Then the colonies were mixed in stroke-physiological saline solution.The concentration of the bacterium liquid was adjusted to 0.5 McB turbidity(this equals 1×10⁸ cfu/ml)by an electronic turbidity kit.Then 12 ml of the bacterium liquid was put into waterbath on 37℃for later use.3.Grouping in the experimentAlbino rabbits were randomly divided into four groups:Groupâ… with the in-tratracheal instillation of Bacillus coli(n=8);Groupâ…¡with the vein intrave, nous injection of oleic acid(n=8);Groupâ…¢with the intratracheal instillation of normal saline(n=6);Groupâ…£as control group(n=5).4.Animal Model Building4.1 The model building of the intratracheal instillation of Bacillus coliAlbino rabbits weighed were anesthetized with 20%ethyl carbamate,fixed in dorsal position on the bench.Their tracheas were disconnected and tracheoto-mies,in which the tracheal intubation on inner diameter 4mm was inserted so as to connect with the Ventilator;4F cardiac catheter was inserted into the external jugular veins to establish liquid passage and connected with the monitor by a pressure pickup.Meanwhile,measurement of central venous pressure was recor-ded and venous blood preparation was taken.The artery intubation was inserted into the arteria carotis externa and connected with the monitor by a pressure pickup.Blood pressure,heart rate and cardiac rhythm were monitored while ar- terial blood sample was taken.Groupâ… :The bacterium liquid mentioned above was instilled into the trachea through the tracheal intubation for four times in 20 minutes,3ml for each time,in four body positions respectively(namely dorsal position,left-lateral position;right arm reclining and prone position)of rab-bits.Arterial blood gas was taken every 2 hour until oxygenation index(PaO₂/ FiO₂)lessened to 300mmHg.Then ALI model was made successfully.The rele-vant indexes were detected and recorded and the experiment ended.Group: Normal saline in water bath on 37℃replaced the bacterium liquid.The other operations referred to Groupâ… .The relevant indexes were detected when the ex-periment lasted 7 hours.Group:Nothing was instilled into the trachea.The other operations referred to Groupâ… .The relevant indexes were detected when the experiment lasted 7 hours.4.2 The contrast research of ALI induced by intratracheal instillation of Ba-cillus coli and by oleic acid injectionAlbino rabbits weighed were anesthetized in 20%ethyl carbamate and fixed on the bench.The cannulas of the trachea,the jugular vein and the arteria caro-tis were placed and connected with a mobile monitor by a pressure pickup for the detection of invasive arterial pressure and heart rate.Groupâ… :The bacterium liquid was instilled into the trachea through the tracheal intubation for four times in 20 minutes,3ml for each time,in four body positions of rabbits respectively. Arterial blood gas was taken every 2 hour until oxygenation index(PaO₂/FiO₂) lessened to 300mmHg.Then ALI model was made successfully.The relevant in-dexes were detected and recorded while the blood preparations were kept. Group:Oleic acid was injected slowly into the external jugular vein(0.08ml/kg) in 20 minutes.The other operations referred to Groupâ… .Group:Nothing was instilled into the trachea.The other operations referred to Groupâ… :The experi-ment lasted 7 hours.5.Indexes of MonitoringThe operative procedure and the conjunction of equipments were firstly fin-ished.After 0.5 hour,the underlying parameters,including systolic blood pres-sure(SBP),diastolic blood pressure(DBP),central venous pressure,blood gas analysis of artery and admixture vein,and the index of respiratory mechanics, were detected.After underlying parameters were recorded,the bacterial liquid or the normal sodium was injected.The indexes of hemodynamics,namely SBP, DBP,HP and cardiac rhythm,were continuously detected.The arterial blood gas was taken every 1 hour for blood gas analysis.The respiratory mechanics were detected before the end of experiment.(1)The detection of respiratory mechanics:The esophageal aerocyst was placed after the Skelaxin was injected into vein and the spontaneously breathing of rabbits was inhibited.The tidal vol-ume was added up to 15 ml/kg,the respiratory rate dropped to 10 b/min,the peak of flow rate descended to 5 L/min,so as to keep the ratio of I to E equal to one to two.At this time the curve of pressure-volume was similar to the curve of quiet state.According to this curve,lung compliance(C)_L,peak inspiratory pressure(PIP)could be obtained from the respiratory machine.(2)The gas ex-change Of lungs:The blood gas analysis including PaO₂ and PaCO₂ was carried out with arterial blood taken from the cannula of arteria carotis.The calculation of interior shut of lungs:the indexes of PaO₂,PaCO₂,SvO₂ and hemoglobin(Hb) could be obtained from the arterial blood gas analysis;the indexes of oxygen partial pressure of the mixed venous blood(PvO₂),venous carbon dioxide par-tial pressure(PvCO₂)and oxygen saturation(SvO₂)came from the detection of the mixed venous blood taken from cardiac catheter.The formula of the calcula-tion is Qs/Qt:Qs/Qt=(CcO₂-CaO₂)/(CcO₂-CvO₂),CcO₂ standing for oxy-gen content of blood capillary of pulmonary alveoli,and CaO₂ for the oxygen content of arterial blood,CvO₂ for oxygen content of mixed venous blood,and when 100%oxygen given,CcO₂=PaO₂×0.0031+Hb×1.34×SaO₂.6.Sample ProcessingThe albino rabbits were executed by blood letting atpreset time and the hearts and lungs taken from the thoracic cavity were observed from the gastro and dorsum.The bronchoalveolar lavage of the right lower lobe was undertaken for 3 times,4ml for each time.The amount of the reclaim douche was calculated after the Filtration with three-layer absorbent gauzes.The cell counting was possible after a drop of the reclaim douche was observed under the microscope.Other samples were centrifuged for 10 minutes at 1200rpm in 4℃and the clear super-natant liquid was kept in -70℃.It could be used for the determination of pro- tein level and cytokine(including TNF,IL-6 and IL-10).The inferior lobes of left lungs were filled and fixed with 4%paraform and were soaked for 24 hours,routinely dewatered,embedded and made into the paraffin blocks.The paraffin blocks were consecutively sliced at 4μm for the dyeing with hematoxylin -eosin and immunohistochemistry.The middle lobes of right lungs were cut, the wet weight of lungs was taken by the electronic balance,Then the lungs were kept in an attemperator in 60℃.After 48 hours,the dry weight was taken. Then the ratio of the wet weight to the dry weight(W/D)was calculated.The se-rum samples at the beginning and the end kept in -70℃were used for the de-termination of TNF,IL-6 and IL-10.7.Methods of Detection7.1 The protein level of BALF:the methods of biuret7.2 The detection inflammatory mediator:The detection of TNF and IL-6 in the way of radioimmunity while the detection of IL-10 in the way of ELISA.7.3 The handing of the pathological samplesThe chips of HE dyeing were observed under the 400 light microscope.Re-ferring to Kendra half- quantitated standard,the leukocytic infiltrate and the e-dema in the alveoli and matrix of lungs were scored.7.4 The detection of iNOS and eNOS of lung tissue:The pathological samples were processed in the way of the polyclonal anti-body of mouse anti rabbit,the staining method of SABC and the coloration of DAB.The samples were observed under the 400 light microscope.According to the standard of Elia N,the scores could be obtained from two aspects:the per-centage of positive cells and the intensity of positive cells dyeing.8.Statistical analysisThe results of the data expressed as the average plus or minus the standard deviation((?)±s),which were analyzed by the statistics software of SPSS12.0. The comparison of the data of the same group at the beginning and the end a-dopted to pairing-t test,the comparison of the means between the groups to One-way analysis of variance and SNK q test,the correlation analysis between two factors to bivariate analysis.Statistical significance was accepted as p＜0.05. 1.1.The typical pathologic and physiopathologic changes of the albino rabbits were induced around 6 hours after an intratracheal instillation of 12×10⁸cfu Ba-cillus coli.2 The hypoxemia was induced in the earlier period of the intratracheal in-stillation of normal saline,but soon disappeared.Neither the hypoxemia nor the pathologic and physiopathologic changes of ALI showed up Within 7 hours.3 The hyperemia and edema of lung tissue appeared in the group of bacteri-um instillation.The symptoms of inferior lobes were more obvious than those of the superior lobes.It seemed that the phenomena were associated with gravity dependence.Leukocytic infiltrate was dominant among pathologic changes;Not only the asystematic hyperemia and edema of lung tissue but also the obvious e-dema of alveoli and alveolar septum appeared in the group of oleic acid injec-tion.The descend of compliance,the increasing of pulmonary shunt and of peak inspiratory pressure(PIP)in the group of bacterium instillation were more serious than the changes in the group of oleic acid injection.4.The cytokine level of TNF and IL-6.heightened in the groups of both bacterium instillation and oleic acid injection.The TNF had positivecorrelation with the IL-6.Both of these cytokines contributed to the tissue damage of lungs.This phenomena was obvious in the group of bacterium instillation.The ratio of BALF to serum suggested that inflammatory reaction of pulmonary region was dominant in the symptoms induced by intratracheal instillation of Bacillus coli and the systemic inflammatory reaction was dominant in the symptoms in-duced by oleic acid injection.5.The cytokine level of the IL-10 heightened in the groups of both bacte-rium instillation and oleie acid injection.The IL-10 had positive correlation with the TNF.It suggested that both mediated the development of inflammation. Camparing these two groups,the increasing of IL-10 in the former group was higher than that in the latter one.6.The iNOS expression of lungs tissue increased obviously in the ALI in- duced both bacterium instillation and oleic acid injection.The increasing of the iNOS expression.In the former was more significant than that in the latter.7.The eNOS expression of lungs tissue descended indistinctively in the ALI induced by bacterium instillation and oleic acid injection.Conclusions1.ALI of albino rabbits could be induced about 6 hours after an intratra-cheal instillation of 12×10⁸ cfu Bacillus coli.2.Compared ALI induced by intratracheal instillation of Bacillus coli with ALI by oleic acid injection,the level of thecytokines of inflammation,the pul-monary tissue infiltration of inflammation cells and the iNOS expression of lungs tissue were more obvious in the former.