Effects Of Remote Ischemic Perconditioning On The Lung Injury Induced By Pulmonary Ischemic Reperfusion In Rats

Chapter 1:A modified way to prepare acute lung ischemia-reperfusion injury model in ratsObjectives:To explore a simple surgical technique which was used to make lung ischemia-reperfusion injury models in rats.Methods:Twenty-four male Sprague-Dawley rats weighting 250-300g were used in this study. After an intraperitoneal injection of 2% sodium pentobarbital (60mg/kg) as previously described, a 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and then connected to a small animal volume-controlled ventilator, which delivered room air at a breath rate of 70 times per min and a tidal volume of 15mL/kg with an inspiratory/expiratory ratio of 12.24 SD rats randomly divided into 2 groups:I/R group and S group.Sham group (Sham, n= 12). Animals underwent a sham thoracotomy through the sixth intercostal space, the lungs were not rendered ischemic.Lung ischemic reperfusion group (I/R, n= 12). The right carotid arterial was cannulated to draw blood samples. The left lung was mobilized via a left anterolateral thoracotomy in the sixth intercostal space. Then, all animals received 300U/kg of heparin intravenously in saline. After surgical procedures, the rats were stabilized for 20 mins to allow the blood gases to stabilize. Stability was defined as arterial partial pressure of oxygen and carbon dioxide. After the baseline measurements, the left pulmonary hilum was occluded with a noncrushing microvascular clamp under the left lung in an inflated state. During ischemia, the animals were ventilated at a breath rate of 90 times per min and a tidal volume of 10 mL/kg with an inspiratory/expiratory ratio of 12. At the end of the 45 mins ischemic period, the clamp was removed and the lung was allowed to ventilate and reperfuse for as long as 120 mins. For reperfusion, the animals were ventilated at a frequency of 70 breaths/min and a tidal volume of 15 mL/kg with an inspiratory/expiratory ratio of 12. Rats were perfused with normal saline (3 ml/kg/h) through the right jugular vein to prevent dehydration during the study period.Arterial blood sample (0.5 ml) were drawn at 20 mins of mechanical ventilation for stabilization after the left lung was mobilized (baseline) and 120 mins after reperfusion (reperfusion 120 mins) for the measurement of PaO2.At the end of each experiment, the rats were killed by exsanguination. The left lung of rats was then removed. The lower lobe of the lung was dissected and weighted immediately, then dried at a constant temperature of 70℃ for 72 h to obtain a dry weightThe upper lobe of lung was fixed in 4% buffered formalin and embedded in paraffin. Thin sections (4 μm) cut from each paraffin block were stained with hematoxylin and eosin and used for histopathologic examination.The wet to dry weight ratio and arterial blood sample for blood gas analysis were compared between 2 groups, the changes in lung structures were observed under the visual inspection and light microscopyStatistical methods:Analysis was performed using SPSS version 13.0 (SPSS Inc, Chicago, USA). Results are presented as mean ± SE. Means between the different groups were analyzed by independent-samples T test. Statistical significant was defined as P< 0.05.Results:At 120 min of reperfusion, PaO2/FiO2 significantly decreased in the I/R group compared with the Sham group (P<0.01).The wet to dry weight in the I/R group was significantly higher than that in the Sham group (P<0.01). Serious damages in I/R group was visible to naked eye and light microscopy.Conclusions:We successfully used a simple surgical technique to establish a lung ischemia-reperfusion injury model in rats with high success rate and offer a useful model to study the mechanism of lung I/R injury.Chapter 2:Effects of remote ischemic perconditioning on the lung injury induced by pulmonary is chemic reperfusion in ratsObjectives:The aim of this study was to document the effects of a simple and low-risk method, remote ischemic perconditioning (RIP), on the lung injury induced by pulmonary ischemic reperfusion in the rat.Methods:Twenty-four male Sprague-Dawley rats weighting 250-300g were used in this study. After an intraperitoneal injection of 2% sodium pentobarbital (50mg/kg) as previously described, a 14-gauge angiocatheter was inserted into the trachea through a midline neck incision and then connected to a small animal volume-controlled ventilator, which delivered room air at a breath rate of 70 times per min and a tidal volume of 15mL/kg with an inspiratory/expiratory ratio of 12.The rats were allocated randomly to one of three groups, two of which received in situ left lung ischemia (45 mins) by clamping the hilum of the left lung and reperfusion (120 mins).Sham group (S, n=8). This group was not exposed to lung ischemia. The rats underwent a sham thoracotomy were observed for 165 mins after the baseline measurements.Lung ischemic reperfusion group (I/R, n= 8). The left pulmonary hilum was occluded with a noncrushing microvascular clamp under the left lung in an inflated state. At the end of the 45 mins ischemic period, the clamp was removed and the lung was allowed to ventilate and reperfuse for as long as 120 mins.Remote ischemic perconditioning group (RIP, n= 8). Remote ischemic perconditioning (RIP) induced by brief cycles of ischemia and reperfusion of bilateral lower limb was applied during sustained lung ischemia.4 cycles of limbs ischemic perconditioning were performed by applying rubber band tourniquet high around each thigh for 5 mins followed by reperfusion for 5 mins to achieve effective perconditioning.To document the effects of RIP on alleviating lung injury by increasing endogenous antioxidant enzyme activities, preventing oxidative damage in lung tissues, and suppressing pulmonary inflammatory responses and that RIP may be dependent on suppressing IκB-α degradation to exert anti-inflammatory actions.Arterial blood sample (0.5 ml) were drawn at 20 mins of mechanical ventilation for stabilization after the left lung was mobilized (baseline, T1) and 120 mins after reperfusion (reperfusion 120 mins, T2) for the measurement of PaCO2 and PaO2.At the end of each experiment, the rats were killed by exsanguination. The left lung of all rats were then removed and divided into three parts, the middle part of lung tissue was snap frozen at -40℃. The lower lobe of the lung was dissected and weighted immediately, then dried at a constant temperature of 70℃ for 72 h to obtain a dry weight. The wet to dry weight (W/D) ratio was calculated to assess pulmonary edema.The upper lobe of lung was fixed in 4% buffered formalin and embedded in paraffin. Thin sections (4 μm) cut from each paraffin block were stained with hematoxylin and eosin and used for histopathologic examination. These sections were scored blind according to the following four items:hemorrhage, alveolar congestion, aggregation or infiltration of neutrophils in the alveolar or interstitial spaces, and thickness of the alveolar wall/hyaline membrane formation. Each item was graded according to a five-point scale:0=minimal (little) damage,1= mild damage,2= moderate damage,3= severe damage, and 4= maximal damage. Thus, minimum and maximum possible scores were 0 and 16, respectively.Frozen lung tissues were homogenized with isotonic sodium chloride and centrifugated for extracting the supernatant. Pulmonary MDA levels and superoxide dismutase (SOD) activities were measured using commercial kits according to the manufacturer’s instructions. MDA levels and SOD activities were expressed as nmol/mg protein and U/mg protein.Myeloperoxidase (MPO) activities were measured using the MPO kits. MPO activities were expressed as U/g wet tissue weight.Frozen lung tissues were homogenized with isotonic sodium chloride and centrifugated for extracting the supernatant. All cytokine (TNF-a and IL-6) concentrations were measured with enzyme-linked immunosorbent assay kits. The lower limits of detection for TNF-α and IL-6 were 70.7 pg/mL and 91.5 pg/mL.Inhibitor kappaB alpha (IκB-α) in lung tissues was detected by western blotting. Cytoplasmic protein fraction was isolated from the lung tissues according to the protocol of the Cytoplasmic Extraction Reagents Kits.IκB-α were detected using rat anti-IκB-α antibody. Anti-β-actin antibody served as a loading control of cytoplasmic protein. After incubation overnight, the membranes were incubated with Goat-anti-Rabbit antibody for 1 h. Films were analyzed by AlphaEase FC software.Statistical methods:Analysis was performed using SPSS version 13.0 (SPSS Inc, Chicago, USA). Results are presented as mean ± SE or n experiments. Means between the different groups were analyzed by one-way analysis of variance and followed by post hoc Student-Newman-Keuls’ test. Statistical comparisons within groups were compared by paired Student’s t-test. Statistical significant was defined as P<0.05.Results:The effect of RIP on lung function was measured by PaO2/FiO@. No significant differences were noted in baseline PaCO2 and PaO2/FiO2 among three groups (P>0.05). No substantial changes over time in PaCO2 and PaO2/FiO2 were observed in the sham group (P<0.05). At 120 mins of reperfusion, PaO2/FiO2 significantly decreased in the I/R and the RIP groups (P<0.05). However, PaO2/FiO2 in the RIP group was significantly higher than that in the I/R group (P<0.05). There was no difference in the value of PaCO2 among three groups at any point during the experience period (P>0.05).MDA levels and SOD activities were quantified to assess the oxidative stress. MDA levels, an end product of lipid peroxidation, were elevated in lung tissues of the I/R group in comparison with the sham group at 120 mins of reperfusion (P<0.05). MDA levels were significantly reduced in the RIP group compared with the I/R group (P<0.05). SOD activities in lung tissues were the inverse of the MDA levels, with higher levels observed in the RIP group, which differed significantly from lower levels observed in the I/R group (P<0.05).MPO activities in the I/R group were higher than that in the sham group (P<0.05). There was significant reduction of MPO activities in the RIP group compared with the I/R group (P<0.05), which suggested RIP inhibited neutrophilic accumulation in lung tissues. The W/D ratio, a parameter of lung edema, increased significantly in the I/R group compared with the sham group (P<0.05). This increase was markedly reduced in the RIP group (P<0.05).No significant damage was observed in the sham group. Interstitial edema, neutrophilic infiltration, haemorrhage and alveolar distortion appeared in the I/R group. RIP group significantly attenuated these histologic changes in comparison with the I/R group. Lung injury scores were in line with the findings of the pathohistological changes (P<0.05).Enayme-linked immunosorbent assay experiments showed that pulmonary IL-6 and TNF-a concentrations were increased in the I/R and the RIP groups (P<0.05), but those were significantly lower in the RIP group than the I/R group (P<0.05).IκB-αin lung tissues was detected by western blotting. The IκB-α expression significantly decreased in the I/R group compared with the sham group (P<0.05). However, the IκB-α expression in the RIP group was much more than that in the I/R group (P<0.05).Conclusions:Remote ischemic perconditioning significantly attenuated pulmonary edema and improved lung function after lung ischemic reperfusion in the rats, in part, by reducing oxidative stress and inflammatory responses. Remote ischemic perconditioning may be dependent on suppressing IκB-α degradation to exert anti-inflammatory actions. This study suggests that remote ischemic perconditioning may represent a potential therapeutic strategy in preventing the lung injury following ischemic reperfusion.