Experimental Study Of Acute Lung Injury Induced By Mechanical Ventilation In Rats

Mechanical ventilation (MV) which has unique curing effects on the serious illnesses, can't be replaced by other therapies. MV itself, however, may also act as a damaging factor to induce or aggravate lung injury which is called ventilator-induced lung injury (VILI). VILI is one of the serious complications in the process of mechanical ventilation, and is also an important factor in deteriorating patient's health, even to death. Therefore, mechanical ventilation, if misused, will fail in any supposed therapeutic effects and also subject people's life and property to a heavy loss.Many research have proved that high airway pressure or large volume ventilation can initiate or aggravate lung injury. Of course, clinically it is impossible for us to setting such high tidal volume which is high enough to induce lung injury. However, it remains controversial whether conventional tidal volume ventilation is absolutely safe without initiating inflammatory lung injury.Animal experiment studies have indicated that MV with high PIP and large V_T may activate neutrophils (PMN). After activation, PMN can release not only various kinds of cytokines and inflammatory mediators but also a lot of oxyradicals and proteinases, which are the chief causes of the inflammatory lung injury. However, PMN is neither the only cell nor the starting cell to evoke inflammatory lung injury in the process of VILI and its activation depends on the release of other preinflammatory factors.Macrophage inflammatory proteins (MIP) which were discovered first by Wolpe and colleagues in 1988 in the supernatant derived from irritating macrophagic system (RAW 264.7) with LPS are new proteins including MIP-1, MIP-2, MIP-3, MIP-4 and MIP-5. MIP-1 and MIP-2 have extensive biological activities and are related to respiratory disease closely. They not only take part in inflammatory and immunological reactions, but also have effects on tumor resisting, hematogenesis promoting and impairment restoring. By executing bronchoalveolar lavage for 30 ARDS children, Murch and colleagues found that the concentration of MlP-1α in BALF supernatant was significantly higher than that of the control group and was obviously decreased after being treated with Dexamethasone for 12 to 24 hours. However, whether MIP-1α has similar effects on the process of VILI, so far there are not related articles published.NF-κB regulating the transcription of target genes is a kind of specific DNA binding protein discovered in recent years. As a dimer compounds, NF-κB is composed of two subunits from Rel protein-family, among which the heterodimer composed of p65 and p50 is of the most representative. Normally, NF-κB binding with I-κB resides in cytoplasm in the state of deactivation. After being stimulated by various etiological factors I-κB can be phosphorylated and separated from NF-κB, thus NF-κB is activated. The latter enters the nucleus, binds with the specific site in DNA and regulates the transcription of target genes. It has been proved that NF-κB can participate in the immune and inflammatory reactions in the lungs by regulating the genetic expression of numerous cytokines and adhesion molecules, such as IL-1β, TNF-α, IL-6, IL-8, ICAM-1, selectin E etc. However, whether NF-κB takes part in regulating MIP-1α gene transcription there are still no final conclusions and it needs to be investigated further.Alveolar macrophages (AM) are the major starting cells of the local imflammation in the lungs. They not only possess powerful phagocytosis but also can synthesize and secrete many kinds of inflammatory mediators and cytokines when activated, which leads to local imflammation in the lungs losing control and is an important cause of ALL Studying with in vivo rabbit lung injury models, Takata and colleagues discovered that AM activated and released proinflammatory cytokines, such as TNF-α, IL-1β and IL-8 etc, might play an important role in activating PMN. Hirani and colleagues demonstrated that among numerous cytokines participating in ALI/ARDS, IL-8 was the strongest chemotatic factor causing PMN to recruit towards lung tissues. IL-8_could be produced by AM or other cells and could be adjusted by local oxygenation state. However, what roles AM has played in the pathogenesis of VILI has not been understood so far.In view of the present researches on VILI, this study will observe the lung injury induced by different volume ventilation in rats under macroscopy, light and electron microscope, then test their arterial blood gas, activity of MPO and the level of MIP-1α, protein content and PMN count in BALF. Simultaneously, the genetic and their protein expression of MIP-1α and NF-κB in lung tissue and alveolar macrophage will be detected with immunohistochemistry and in situ molecular hybridization technique, so as to explore the effects of different tidal volume ventilation on acute lung injury in rats at molecular level and provide a new clue to the prevention and treatment of VILI.CONTENTSThe study includes three parts:Part 1: Pathology of acute lung injury induced by ventilator in rats.Part 2: Effects of neutrophil's activation on acute lung injury induced by ventilator in rats.Part 3: Effects of macrophage inflammatory protein-1α (MIP-1α) on acute lung injury induced by ventilator in rats.METHODS1. Thirty-two healthy male Wistar rats (provided by Experimental Animal Center ofShanxi Medical University) weighing 300-310g were randomly divided into 4 groups: â‘  control group: without mechanical ventilation; â‘¡ low tidal volume group (L-V_T) : V_T 7ml/kg; â‘¢ conventional tidal volume group (C-V_T) : V_T 12ml/kg; â‘£ high tidal volume group (H-V_T) :V_T40ml/kg.2. Observe the pathologic alteration of lung injury induced by different volume ventilation in rats under macroscopy, light and electron microscope.3. Test lung wet/dry weight ratio (W/D).4. Carry out blood gas analysis by automatic blood gas analyzer.5. Count the white blood cells (WBC) and PMN in BALF with light microscope.6. Measure the activities of myeloperoxidase (MPO) in BALF with the method of Luo Wusheng.. 7. Measure the levels of protein in plasma with biuret detection and measure the levels of protein in BALF with Coomassie brilliant blue G-250 detection respectively.8. Detect the content of MIP-α in plasma and BALF with ELISA9. Detect the expression of MIP-1α protein in lung tissue with immunohistochemical staining.10. Detect the expression of MIP-1α mRNA in lung tissue with situ hybridization technique.11. Detect the expression of NF-κB p65 protein in lung tissue with immunohistochemical staining.12. Detect the expression of MIP-1α and NF-κB p65 protein in alveolar macrophage with immunohistochemical staining.RESULTS 1. Pathological changes of the rat lungsOf the low tidal volume group, the appearance of the lungs was normal under macroscopy. Though there were a few neutrophils infiltration under light microscope, the lung tissue structures were not obviously different from that of control group under transmission electron microscope. Of the conventional tidal volume group, the appearance of the lungs was swollen slightly, but their colour and lustre were almost the same as the control group. Under light microscope the interstitial edema and neutrophils infiltration could be found, and under electron microscope there were a great quantity of low electronic density liquid materials congregated in interalveolar septum. Of the high tidal volume group, the lungs were swollen obviously and the spotty hemorrhages could be found on their surface. There were widespread interstitial edema and massive neutrophils infiltration under light microscope. Under electron microscope, the conjunctions between the alveolar epithelial cells and between the capillary endothelial cells were injured with gaps. The neutrophils were in activated state protruding many spine-like processes and their granules contents released with many vacuoles left.2. Results of lung wet/dry weight ratio (W/D) and blood gas analysisThe results showed that W/D of the high and conventional tidal volume group was significantly higher than those of the low tidal volume group and the control group (P<0.01), PaO₂ of the high and conventional tidal volume group were significantly lower than those of the low tidal volume group and the control group (P<0.01,P<0.05). However, no statistical difference could be found between the low tidal volume group and the control group (P>0.05).3. Results of WBC and PMN in BALFThe results showed that WBC and PMN counts in BALF were significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.001), but no significant difference could be found between low tidal volume group and control group (P>0.05).4. Results of MPO in plasma and BALFThe results showed that the activity of MPO in BALF was significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.01, P<0.05). The activity of MPO in BALF of the high tidal volume group was significantly higher than that of the conventional tidal volume group (P<0.01), but no significant difference could be seen between low tidal volume group and control group (P>0.05). Of the activity of MPO in plasma there was no statistical difference existed among all rat groups (P>0.05).5. Results of protein levels in plasma and BALFThe results showed that the protein levels in BALF were significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.01), the protein levels of the high tidal volume group were significantly higher than that of the conventional tidal volume group (P<0.01). However, no significant difference could be found between the low tidal volume group and the control group (P>0.05). Of the protein levels in plasma there was no statistical difference existed among all rat groups (P>0.05).6. Results of MlP-1α content in plasma and BALFThe results showed that MlP-1α in BALF were significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.01). No significant difference could be found either between the low tidal volume group and the control group or between the conventional tidal volume group and the high tidal volume group in BALF (P>0.05). Of the MIP-1α content in plasma no statistical difference could be found among all rat groups (P>0.05). Correlation study showed that there was a positive correlation existed either between MIP-1α content and MPO or between MIP-1α content and PMN in BALF respectively (r=0.454, P<0.05; r=0.431, P<0.05).7. Result of MIP-1α expression in lung tissue detected with immunohistochemical stainingThe results showed that MIP-α expression in bronchiole and alveolar epithelial cells were significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.01), but no significant difference could be found between the low tidal volume group and the control group (P>0.05). Besides the bronchiole and alveolar epithelial cells a little MlP-1α protein was also expressed in vascular endothelial cells and smooth muscle cells.8. Results of MIP-1α-mRNA detected with hybridization in situ and NF-κB p65 detected with immunohistochemical staining in lung tissueThe results showed that both MIP-1α mRNA and NF-κB p65 protein positive bronchiole epithelial cell percentage of rats ventilated with high and conventional tidal volume were significantly higher than those ventilated with low tidal volume and control group (P<0.01), but no significant difference could be found between the low tidal volume group and the control group (P>0.05). Correlation study showed that there was a positive correlation existed between the MIP-1α mRNA positive bronchiole epithelial cell percentage and the NF-κB p65 protein positive bronchiole epithelial cell percentage (r=0.482, P<0.05).9. Results of MIP-1α and NF-κB p65 of alveolar macrophage in BALF detected with immunohistochemical stainingThe results showed that the positive alveolar macrophage percentage of MIP-1α and NF-κB p65 in BALF were significantly higher in rats ventilated with high and conventional tidal volume than in those ventilated with low tidal volume and control group (P<0.01), but no significant difference could be found between the low tidal volume group and the control group (P>0.05). Correlation study showed that there was a positive correlation existed between the MIP-1α positive alveolar macrophage percentage and the NF-κB p65 positive alveolar macrophage percentage (r=0.457, P<0.05).CONCLUSIONS1. Low tidal volume ventilation does not have obvious damaging effects on the normal lung tissues. Of the conventional tidal volume ventilation, although no obvious lung injuries can be seen under macroscopy they can be displayed under light and electron microscope. Injuries caused by high tidal volume ventilation not only are obvious under macroscopy and light microscope, but also have special alterations in ultramicrostructure. All of these indicate that high and conventional tidal volume ventilation without any protecting procedures can produce injuries to the normal lung tissue. Although the injuries caused by conventional tidal volume ventilation is not as serious as those caused by high tidal volume ventilation, it shall not be ignored absolutly.2. Recruitment and activation of the neutrophils in the lung tissues might play an important role in the pathogenesis of VILI. The more the tidal volume is, the more the number and activity of neutrophils are, and the more the lung injury happens. Measuring the protein levels in plasma and BALF and calculating the permeation index of alveolar membrane can reflect the degree of lung injury to some extent, which is not only simple and reliable, but also useful as a practical method. Of the MPO activity and the protein levels in plasma there is no statistical difference existed between any groups, which indicates that VILI chiefly occurs in local lung tissues and has few effects on the whole body.3. MIP-1α is related closely to VILI. MIP-1α evokes PMN to be recruited and activated in the lungs through chemical agitation and chemical chemotaxis, which may be one of the important accesses to VILI. In the process of VILI, the sources of MIP-1α are multiple. Except for AM, other cells in the lung tissues can also express MlP-la.4. In the process of VILI, MIP-1α delivery from lung tissues may be regulated by NF-κB to some extent. The chain of mechanical stimulation →NF-κB→cytokines may be an important pathway in the occurrence of VILI. Therefore, it may be viewed as a new direction to prevent and treat VILI by multiple interventions, such as blocking NF-κB activation, reducing MIP-1α delivery and suppressing PMN activation and so on.5. Alveolar macrophage (AM) is one of the starting cells to VILI. AM's activating and releasing MIP-1α is an important factor leading to VILI, which may be regulated by NF-κB. AM can receive the signals from the mechanical stimulation directly or indirectly and make NF-κB in the cells activated. The activation of intra-cellular NF-κB is both the critical step and the important mark for AM's activation.