| Objective:In recent years, there is considerable experimental and clinical evidence that pro- and anti-inflammatory cytokines play a major role in the pathogenesis of inflammatory-induced lung injury from sea water drowning. Previous studies have confirmed TNF-αis an initiator cytokine in mediating, amplifying, and perpetuating the lung injury process. However, there is very little evidence on the the value of giving Dexamethasone in cases of sea. Therefore, this study aimed to observe the dynamic TNF-αmRNA expression in alveolar macrophage (AM) of in rabbits drowned by sea water, and explore the effect of Dexamethasone injection on TNF-αmRNA expression.Methods:1. Study groups40 rabbits weighting 2.50– 0.36 kg were purchased from Experimental Animal Center of Forth Military Medical University, Shaan′xi Province , China. To determine the dynamic changes of expression of TNF-αmRNA in AM of rabbits after sea water drowning, 25 rabbits were randomly divided into five groups : 0 min control group and 30 , 60, 90, and 120 min seawater drowning groups , 5 rabbits in each group. To observe the effect of Dexamethasone on expression of TNF-αmRNA in AM , the rabbits were randomly divided into3 groups (5 of each) : (CG )normal control group, in which the rabbits were not instillated, just dealed with tracheal intubation; (MG)Model group, in which the rabbits were prepared SW-RDS models according to the below methods. and injected with equal volume of NS; (TG)Treatment group, in which the rabbits were instillated, and injected with 4ml/kg Dexamethasone , and additional intravenous Dexamethasone was provided at 1mg·kg - 1·h– 1 to maintain, and the other treatment conditions were the same as the MG′s.2. Experimental protocolsThe New Zealand rabbits were maintained in the supine position, and anesthetized with 20% glutaral (1.2g/kg) injected through an ear vein. A small median incision was made into the neck, an plastic endotracheal tube (external diameter=4.5mm, internal diameter=3.8mm) was gentlely inserted into the trachea until it was placed approximately 1 cm above the carina . catheter was inserted in the right carotid artery to monitor systemic blood pressure and to obtain blood samples. Then using a 20ml syringe, 4ml/kg body weight of seawater was instilled over 1min into both lungs. Sea water was obtained from Jiao zhou Bay ,Shan dong. Cyanosis, ecphysesis rapidly occured, bubble-like liquid blowed off the intubation, hygro-rales covered both lungs. Arterial blood gas showed PaO2< 50 mmHg (1 mmHg= 0. 133 kPa), SaO2< 45%, PaO2/FiO2 (oxygenation index)<150mmHg, suggested that the model successful established.Arterial blood gas analyses were performed 0min(control group as 0 min) before and 30min , 60min ,90min after seawater instillation . At these time points, 25 rabbits were heparineize and then sacrificed with bloodletting respectively, the thorax was opened rapidly, the lung were processed as the following descripion .To determine the lung dry /wet weight ratio ,the right middle lung was removed and dried to a constant weight(480C for 72h). The dried lung was weighed and the lung dry/wet radio was then calculated. Specimens from the right lower lung for pathological examinationg were fixed in 10 formalin, processed and stained with haematoxylin and cosin, others were frozen in liquid nitrogen, and stored at -700C. 3. Isolation and culture of rabbit alveolar macrophage (AM)The 15 rabbits of CG, MG,TG were heparineize and then executed by bleeding at 100min. the thorax was opened rapidly, occlusion the trachea. and the left lung was rinsed with 5 ml normal saline twice aseptically, recovery rate was 70%.The rinsed normal saline was collected and centrifuged at 1000 g for 5 min. The cell pellet was re-suspended with complete RPMI 1640 medium containing 10% fetal bovine serum (Gibco-BRL, USA) before counting and were incubated in glass cell culture plates for 4 hours at 370C, 5% CO2 to allow the macrophages to adhere. The non-adherent cells were removed by washing the plate twice with warm serum-free medium, and more than 90% of the adherent cell population determined by morphology and Wright's staining was AM.4. Reverse transcription-polymerase chain reaction (RT-PCR)Primers were based on published nucleotide sequence for rabbit TNF-a (forward primer: 5′-GCTGAGCCAGCGTGCGAACG-3′, reverse primer5′-GAGGTACTCAGGCTGGTTGA-3′), with an amplified product of 267 bp, andβ-actin (forward primer: 5′-ACGATGCTCCAAGAGCTGTT-3′, reverse primer: 5′-TCAGGCAGCTCATAGCTCTT-3′), with an amplified product of 367 bp, were evaluated by RT-PCR as described previously with slight modifications.Briefly, total RNA was isolated using Trizol reagent. 2 microliters of 20 ml total RNA reaction was used as template DNA for PCR. PCR was performed by 45 s denaturation at 94℃, 45 s renaturation with specific primers at 55℃, 60s extension at 72℃for 35 cycles, and this was followed by an additional extension step at 72℃for 5 min. Followed by ethidium bromide staining ,they were electrophoresed in 20g/L agarose gel, the PCR products were photography, and scanned and relative intensity of the signals was determined by Band intensities were quantified using Gel-Proanalyzer version 3.0 imaging system.Results 1. Arterial blood gas analyses:Seawater instillation caused a marked decrease in the PaO2, at 60min cut down the lowest point(31.6±4.98mmHg, 1mmHg=0.133kPa), then gradually increased, at 120min cut down again, but significantly lower than the 0min(contol normal group)( P< 0.01).; At 30min PCO2 rised the highest (62.2±3.49mmHg), then gradually cut down , at 120min is 40.6±2.40mmHg, which partly related to the comp hyperventilation of the rabbits, and significantly higer than the 0min(contol normal group)( P< 0.01) ; pH and dry/wet ratio gradually decreased.2. HistopathologyLight microscopic finding in the MG demonstrated a marked lung injury, which represented by hemorrhage, edema, thickened alveolar septum , formation of hyaline membranes,telangiectasis in alveolar wall and the infiltration of inflammatory cells(mainly was polymorphonuclear neutrophils, PMN) in alveolar spares and mesenchymal, but these changes were less pronounced in the TG.3. TNF-αmRNA expressionIncreased expression TNF-αmRNA is very important for proinflammation media acute lung injury. TNF-αmRNA expression sustained a baseline level in normal rabbits(0.142±0.556), seawater instillation caused a marked increase, at 90min heightened the peak(1.204±0.130), which significantly higher than the 0min (contol normal group)( P< 0.01,).then lightly decreased, but significantly higher than the 0min (P<0.01), the decrease possibly related to sequential anti-inflammatory cytokines in vivo.Conclusion:1. Our study confirmed the existence of ALI because the histology section of lung from the seawater drowning rabbits showed marked cellular infiltration, edema, congestion and thickening of alveolar-capillary membrane, which are the typical histological changes found in ALI/ARDS. Simultaneously, the arterial blood oxygenation (PaO2/FiO2< 300) were confirmed ALI/ARDS induced by seawater drowning.2. We found that expression of TNF-αmRNA in AM of rabbits began to increase , and reached a peak level at 90 min , then began to reduce ,but at all time points tested was significantly higher than that of the control. It suggested that sea water drowning could significantly increase the expression of TNF-αmRNA in AM, and the following decreasion possibly was related to the secretion of anti-inflammatory cytokines in the later stage. .3. The dymamic expression of TNF-αmRNA in AM indicated that TNF-αmainly released from activated alveolar macropage, and indicating the lung injury may be related to high expression of TNF-αmRNA . In this rabbit model of intra-trachea instillation of sea water-induced ALI, histology section of lung indicatied that the pneumonedema, hemorrhage, and the infiltration of inflammatory cell in alveolar spaces were less pronounced in TG, meanwhile the TNF-αmRNA expression in AM was significant decreased in the rabbits that received dexamethasone treatment(P<0.05), which showed Dexamethasone could significantly decrease of the high expression of TNF-αmRNA in AM of rabbits drowned by sea water . So we suggested that the treatment of Dexamethasone may be related to that repression of inflammation cascade reaction from spring-head, and the sequential entering of plethoric TNF-αinto blood caused the pathophysiological changes of multiple organs and systems.. Our results were beneficial for clinical use of Dexamethasone in SW-RDS. The contribusion of corticoids was remained further research..
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