Experimental Study Of The Effects Of Low-dose Hydrocortisone On Pulmonary Acute Lung Injury In Rats

Objective:1.To establish pulmonary ALI/ARDS animal model in rats infected by Escherichia coli.PaO₂/FiO₂＜300mmHg(39.9kpa) is considered as ALI, PaO₂/FiO₂＜200mmHg(26.6kpa) is considered as ARDS.2.To investigate the effects of low-dose hydrocortisone on pulmonary acute lung injury in rats.3.To investigate the effects of low-dose hydrocortisone on the survival rate in ALI models.Materials and methods:1.To establish ALI/ARDS animal model:It was done by two steps.First,observe the infected rats for 72 hours.Second,72 hours was divided into five time points, PaO₂/FiO₂,blood pressure and dynamic compliance were observed at each time point.1.1 Methods:Male Wistar rats(weight range,220～250g) were used for all experiments.Tracheostomy was performed after rats were anesthetized.They were infected with intratracheal Escherichia coli injection[3ml/kg,O111B4, (4.4-5.6)×10¹²CFU/L)].The incision was closed after the operation.All animals had access to chow and water ad libitum.1.2 A total of 29 rats were randomly divided into two groups:Escherichia coli group (n=21) and normal saline group(n=8).They were challenged with Escherichia coli or normal saline respectively both through intratracheal injection.They were observed continuously for 72 hours.1.3 A total of 64 infected rats were randomly allocated to 5 time points.12 hour(n=10),24 hour(n=13),36 hour(n=13),48 hour(n=14),72 hour(n=14),and there were 8 rats in the normal control group.Animals were anesthetized with pentobarbital sodium(45mg/kg intraperitoneally),and a snugly cannula was introduced into the trachea,a snugly catheter was introduced into the carotic artery.Mechanical ventilation with a frequency of 55 breaths/min,a tidal volume(Vt) of 6～8ml/kg,and a positive end-expiratory pressure of 4～6cmH20 was applied.A pneumotachograph was connected to the tracheal cannula for the measurements of airflow and changes in lung volume.Blood pressure,peak inspiratory pressure(PIP),tidal volume(V_t), positive end-expiratory pressure(PEEP),lung-thorax dynamic compliance[Cdyn= Vt÷(PIP-PEEP)],and blood gas analysis were recorded at 30min after ventilation was applied.After the experiment,lungs were fixed with formalin to perform pathological examination.2.To investigate the effects of low-dose hydrocortisone on pulmonary acute lung injury.Rats were infected with intratracheal Escherichia coli injection,twelve hours later,the survived 58 rats were randomly divided into hydrocortisone group(group HC,n=28) treated with hydrocortisone(5mg/kg,intraperitoneally) and pulmonary ALI control group(group C,n=30) treated with normal saline(intraperitoneally).In the normal control group(group N),saline was administered intratracheally and intraperitoneally.Twelve,twenty-four and thirty-six hours later,mechanical ventilation was applied.The blood gas analysis and blood pressure were monitored, and PIP,PEEP,V_t and Cdyn were recorded.After the experiment,animals were killed, the right lungs were washed for three times with normal saline(20ml/kg) to obtain bronchoalveolar lavage fluid(BALF).TNF-α,IL-8 and IL-10 in serum and BALF were measured with ELISA kits.Total protein concentration in BALF was measured with BCA(bicinchoninic acid) kits.The left lungs were weighed for calculating lung index and then fixed for pathological examination.Lung edema,hyaline membrane formation and inflammatory cell infiltration were assessed and scored.3.The effects of low-dose hydrocortisone on the survival rate of ALI models.Twelve hours after infected with intratracheal Escherichia coli injection,33 survived rats were randomly divided into hydrocortisone group(HC,n=16) treated with hydrocortisone(5mg/kg,intraperitoneally) and pulmonary ALI control group (group C,n=17) treated with normal saline(intraperitoneally).All rats were observed continuously for 72 hours.Results:1.Observations of the animal model:1.1 Tachypnea was observed after rats infected for 6 hours.11 rats died during 8h～40h,mortality rate was 52.38%.72 hours later the infected rats began to recover.1.2 Results at each time point:1.2.1 Observations of lung and heart function after ventilated for 30min:â‘ Blood pressure:12h(15.00±0.50)kpa,24h(14.89±0.90)kpa,36h(14.92±1.39)kpa and 48h (15.32±0.68)kpa,significantly lower than normal control group(19.26±0.82)kpa, (P＜0.01).There was no significant difference between 72h[(16.72±2.38)kpa]and normal control group(P＞0.05).â‘¡PaO₂/FiO₂:normal control group(63.82±3.03)kpa, 12h(30.71±7.95)kpa,24h(21.66±5.34)kpa,36h(21.09±4.75)kpa,48h (25.01±8.78)kpa,72 h(33.82±8.02)kpa,they were significantly lower than normal control group(P＜0.01).â‘¢Cdyn(ml/kg.kpa):12h(4.26±0.13),24h(4.19±0.96),36h (4.28±0.69),48h(4.44±0.62),72h(4.58±0.35),they were significantly lower than normal control group(8.16±0.78)(P＜0.01).â‘£The percentage of ALI/ARDS.12h: 71.4%/28.6%,24h.100.0%/85.7%,36h:100.0%/83.3%,48h:83.3%/66.7%, 72h 57.1%/14.3%.1.2.2 Pathological examinations:There was a predominance of alveolar collapse,fibrinous exudates,and alveolar wall edema.They showed inflammation including thick alveolar septa and recruited inflammatory cells.Hyaline membrane formation could be seen.At 72 hour, inflammation became improved with some of exudates were absorbed.2.Effects of low-dose hydrocortisone on pulmonary acute lung injury in rats:2.1â‘ Blood pressure:At 12,24,36 hour,the blood pressure in group HC [(15.47±1.33) kpa,(16.64±1.14)kpa,(17.21±2.41 )kpa,respectively]were higher than those in group C[(13.96±1.37) kpa,(14.94±1.37) kpa,(14.97±0.93) kpa, respectively],(P＜0.05).â‘¡There were no significant differences in ALI and ARDS incidence between group HC and C(P＞0.05).â‘¢PaO₂/FiO₂:At 12,24 and 36 hour, there were no statistical differences in PaO2/FiO2 between group HC [(31.72±12.06)kpa,(21.72±4.85)kpa,(31.33±8.26)kpa,respectively]and C [(25.02±12.46) kpa,(19.25±8.07)kpa,(23.41±8.96) kpa,respectively],(P＞0.05).â‘£Cdyn(ml/kg.kpa):At 12,24 and 36 hour,there were no statistical difference in Cdyn between group HC[(4.22±0.25),(4.40±0.55),(4.51±0.35),respectively]and C [(3.88±1.18),(4.44±0.80),(4.99±0.76),respectively],(P＞0.05).2.2 lung index:At 12 hour,the lung index in group HC[(0.333±0.082)%]was significantly lower than that in group C[(0.432±0.137)%].2.3 Pathological examinations:At 12 hour,there were significant differences in lung edema,intra-alveolar inflammatory cell infiltration and total lung injury score between group HC[(1.63±0.74),(2.25±0.46),(6.63±1.06),respectively]and C [(2.57±0.54),(2.86±0.38),(9.00±1.00),respectively](P＜0.05).There were no statistical differences in hyaline membrane formation and interstitial inflammatory cell infiltration between group HC and C at each time point(P＞0.05).2.4 Protein concentration in BALF(mg/L):At 24 and 36 hour,total protein in group HC[(874.55±426.97),(735.95±136.15),respectively]were significantly lower than those in group C[(1468.52±433.38),(1350.95±410.55),respectively](P＜0.05).2.5 Levels of IL-8,IL-10 and TNF-αin BALF(ng/L):At 24 and 36 hour,IL-8 in group HC[(69.66±13.07),(61.49±15.27),respectively]were significantly lower than those in group C[(91.37±24.59),(98.97±17.77),respectively](P＜0.05);At 12,24 and 36 hour,there were no statistical differences in IL-10 between group HC [(125.24±24.72),(116.06±12.52),(173.92±38.28),respectively]and C [(127.98±28.03),(118.33±25.62),(136.46±34.00),respectively](P＞0.05),TNF-αin group HC[(343.13±50.19),(277.92±84.51),(305.87±61.59),respectively]were significantly lower than those in group C[(467.51±76.09),(360.12±34.69), (375.26±25.79),respectively],(P＜0.05).2.6 Levels of IL-8,IL-10 and TNF-αin serum(ng/L):At 24 and 36 hour,IL-8 in group HC[(164.18±22.20),(163.72±48.69),respectively]were significantly lower than those in group C[(213.35±46.32),(238.86±73.68),respectively](P＜0.05);At 12,24 and 36 hour,there were no statistical differences in IL-10 between group HC [(179.28±69.73),(126.38±34.99),(134.76±56.02),respectively]and group C [(154.75±45.43),(148.21±38.57),(172.46±51.77),respectively](P＞0.05),TNF-αin group HC[(78.13±14.64),(79.15±8.42),(83.25±23.64),respectively]were significantly lower than those in group C[(120.39±22.53),(107.02±34.18), (140.94±33.82),respectively],(P＜0.05).3.The effects of low-dose hydrocortisone on the survival rate in ALI models:There was no statistical difference in survival rate between group HC(68.8%) and group C(64.7%),(Log Rank P=0.014).Conclusions:1.We successfully established pulmonary ALI/ARDS model in rats induced by intratracheal Escherichia coli injection,and acquired some useful information of these models by observing the cardiopulmonary parameters and morphological changes at 5 time points.2.Low dose of hydrocortisone can relieve pulmonary edema and inflammatory cell infiltration,decrease the production of pro-inflammatory cytokines and maintain blood pressure in rats with Escherichia coli induced ALI.3.There was no obvious beneficial effect on survival rate about the use of low-dose hydrocortisone in acute lung injury induced by Escherichia coli in rats.