Influence Of Ventilation With Lower Tidal Volume And Positive End-expiratory Pressure On The Development Of Ventilator-associated Pneumonia

PartⅠInfluence of ventilation with lower tidal volume and positive end-expiratory pressure on the pulmonary and systemic inflammation in health ratsObjective To investigate the influence of ventilation with low tidal volume and positive end-expiratory pressure(PEEP) on the pulmonary and systemic inflammation in health rats and its clinical relevance.Methods Specific pathogen-free male Sprague-Dawley rats, weighing 242-286g, were ventilated for Omin(C group, n=8),60min(P60 group, n=8),120min(P120group, n=8)and 240min(P240 group, n=8) using the following ventilation settings:tidal volume(VT) 6 ml/kg, PEEP 5 cmH20, respiratory rate(RR) 88breaths/min, and fraction of inspired oxygen(FIO2) 21%. Rats in R group(n=8) were allowed to recover for 2 days after 240min ventilation. Rats in H group(n=8) were ventilated for 240min using the following ventilation settings:VT 40 ml/kg, PEEP 5 cmH2O, RR 88breaths/min, and FIO2 21%. Hemodynamic parameters were recorded and blood analysis was done during ventilation with lower tidal volume and PEEP in other 8 rats. Histopathologic changes were examinated. Lung wet to dry weight ratio(W/D) and myeloperoxidase(MPO) activity were measured. Apoptosis of pneumonocytes were detected by terminal deoxynucleotidyl transferase mediated nick end labeling(TUNEL). Expression of interleukin-1β(IL-1β) mRNA, tumor necrosis factor-a(TNF-a) mRNA and macrophage inflammatory protein-2(MIP-2) mRNA was assessed by reverse transcript-polymerase chain reaction(RT-PCR). Plasma concentrations of IL-1β, TNF-αand MIP-2 were measured by enzyme linked immunosorbent assay (ELISA).Results Histopathologic changes in C and P60 groups were absent. P120, P240 and R groups showed moderately increased inflammatory cellular infiltration and thickness of the alveolar wall. In H240 group the alveolar structure was severely damaged and there were high levels of intra-alveolar exudates and interstitial edema. MPO activity, Expression of IL-1βmRNA, TNF-a mRNA and MIP-2 mRNA, plasma concentrations of TNF-a and MIP-2 in P120, P240 and H240 groups, W/D values in P240 and H240 groups, MPO activity in R group, apoptosis index and plasma concentration of IL-1βin H240 group and expression of IL-1βmRNA in P60 group were significantly higher than those in C group(P<0.05 or 0.01). All above indexes in H240 group were significantly higher than those in P240 group(P<0.01). All above indexes in R group except apoptosis index and plasma concentration of IL-1βwere significantly lower than those in P240 group(P<0.05). Hemodynamics were stable and PaCO2 was less than 55 mmHg during ventilation with lower tidal volume and PEEP.Conclusions Ventilation with lower tidal volume and PEEP can induce reversible pulmonary and systemic inflammation in health rats. This may make the host more vulnerable to a "second hit". PartⅡA new model of methicillin-resistant Staphylococcus aureus ventilator-associated pneumonia in ratsObjective To develope a new model of methicillin-resistant Staphylococcus aureus (MRSA)ventilator-associated pneumonia(VAP) in rats and evaluate the influence of ventilation with lower tidal volume and positive end-expiratory pressure(PEEP) on the development of pneumonia.Methods One hundred pathogen-free male Sprague-Dawley rats were randomly assigned to the following experimental groups:(1) N(nonventilated pneumonia) group(n=44):rats were infected intrabronchially with 0.2 ml of 1010 cfu/ml MRSA inoculum;(2)V(ventilator-associated pneumonia) group(n=44):rats were ventilated for 4 h using the protective ventilation settings:tidal volume (VT)6 ml/kg, PEEP 5 cmH2O, respiratory rate(RR) 88 breaths/min, and fraction of inspired oxygen (FiO2)=0.21. After ventilation, rats were inoculated intrabronchially with the same amount of MRSA as in N group; (3) P (protective mechanical ventilation) group (n=6):0.2 ml of normal saline were instilled into lungs of rats after 4 h of ventilation as in V group; (4)C(control) group(n=6):Only 0.2 ml of normal saline were instilled into lungs of rats. Rats from both P and C groups were killed 48 h after instillation of normal saline. Rats from other two groups were killed 3,6,12,24,48 h after inoculation. Every ten rats from both N and V groups were used to observe five-day survival rate. Histopathologic changes were examinated. Macroscopic score, microscopic score, bacteremia occurrence were recorded. Lung wet weight to body weight(WW/BW) and lung bacterial concentration were measured.Results WW/BW Values of WW/BW were not different between C and P groups. They were baseline levels. Compared with C group, values of WW/BW in both N and V groups at 48th significantly increased. The latter was significantly higher than the former. In both C and P groups, no pneumonia was observed. Both N and V groups developed histologically pneumonia 24 h after inoculation. In V group, pneumonia was most often bilateral, different aged, multifocal, and predominantly affected lower lobes, especially on the right side. Lung bacterial concentrations in V group at each time point were significantly higher than those of N group. It was 12.2±0.9 log10 cfu per gram of tissue at the 48th h in V group, and 8.7±0.4 in N group. Bacteremia occurred in nine of ten rats at the 48th h in V group, while two of ten rats in N group. Five-day survival rate in V group was significantly lower than that of N group(20% VS 20%).Conclusions Rat model of MRSA VAP was obtained by intrabronchially innoculating rats with 2×109 cfu MRSA after 4 h of ventilation with lower tidal volume and PEEP.VAP was more severe than nonventilated pneumonia in terms of histopathologic and microbiologic criteria, especially systemic spread. Ventilation with lower tidal volume and PEEP reduces bacterial lung cleanse and promotes the spread of bronchial and systemic infection. PartⅢInfluence of ventilation with lower tidal volume and positive end-expiratory pressure on the expression ofβ-defensin-3 in lungs of rats with methicillin-resistant Staphylococcus aureus ventilator-associated pneumoniaObjective To develope a new model of methicillin-resistant Staphylococcus aureus (MRSA)ventilator-associated pneumonia(VAP) in rats and evaluate the influence of ventilation with lower tidal volume and positive end-expiratory pressure(PEEP) on the expression ofβ-defensin-3 (BD-3) in lungs of rats.Methods One hundred pathogen-free male Sprague-Dawley rats were randomly assigned to the following experimental groups:(1) N(nonventilated pneumonia) group(n=44):rats were infected intrabronchially with 0.2 ml of 1010 cfu/ml MRSA inoculum;(2)V(ventilator-associated pneumonia) group(n=44):rats were ventilated for 4 h using the protective ventilation settings:tidal volume (VT)6 ml/kg, PEEP 5 cmH2O respiratory rate(RR) 88 breaths/min, and fraction of inspired oxygen (FiO2)=0.21. After ventilation, rats were inoculated intrabronchially with the same amount of MRSA as in N group; (3) P (protective mechanical ventilation) group (n=6):0.2 ml of normal saline were instilled into lungs of rats after 4 h of ventilation as in V group; (4)C(control) group(n=6):Only 0.2 ml of normal saline were instilled into lungs of rats. Rats from both P and C groups were killed 48 h after instillation of normal saline. Rats from other two groups were killed 3,6,12,24,48 h after inoculation. Every ten rats from both N and V groups were used to observe five-day survival rate. Expression of BD-3 was detected by immunohistochemistry staining and Western blot(WB). Concentrations of interferon-y (IFN-γ) in lung homogenate were detected by enzyme linked immunosorbent assay(ELISA).Results BD-3 mainly expressed in bronchiole epithelium and less in alveolar epithelium. Expression of BD-3 in both N and V groups increased 6 h after inoculation and reached peak 12 h after inoculation. Expression of BD-3 in N group 12 h,24 h and 48 h after inoculation was significantly higher than that of P group. Concentrations of IFN-y in lung homogenate of both N and V group increased 6 h after inoculation and until 48 h after inoculation, but there was no difference in concentrations of IFN-y at each time point between N and V groups.Conclusions Expression of BD-3 in lungs of rats with MRSA VAP is significantly lower than that of rats with MRSA pneumonia. This maybe associated with the reduced bacterial lung cleanse and promotion the spread of bronchial and systemic infection by ventilation with lower tidal volume and PEEP.Part IV Effect of mechanical stretch on the expression of humanβ-defensin-3 in alveolar epithelial cells elicited by interferon-yObjective To study the effect of mechanical stretch on the expression of human beta-defensin-3(HBD-3) in alveolar epithelial cells(A549 cells) elicited by interferon-y(IFN-γ) and to investigate the role of HBD-3 in the pathogenesis of ventilator-associated pneumonia(VAP).Methods A549 cells were treated with mechanical stretch (group S),10 ng/ml IFN-y (group I),10 ng/ml IFN-y and mechanical stretch (group IS), respectively. Cells with no treatment served as controls (group C). Cells were stretched by 20% at a rate of 30 cycles/min by Flexercell-4000TTM Unit and treatments lasted for 2 h,4 h and 6 h. HBD-3 mRNA expression was determined by real time RT-PCR after treatment; After 6 hour' treatment, cells were cultured until 24 h after the start of treatment and the expression of HBD-3 was examined by laser scanning confocal microscope. Apoptosis of cells stretched for 0h,2h,4h and 6h was detected by flow cytometer(FC).The experimental data were statistically analyzed using one-way ANOVA analysis and q test.Results 2h,4h or 6h stretch significantly increased cell apoptosis. Only mechanical stretch could not significantly change the expression of HBD-3 mRNA in A549 cells. Compared with group C, HBD-3 mRNA expression after 2,4 and 6 hour' treatment of 10 ng/ml IFN-y increased significantly by 2.63±0.60,8.02±1.63 and 12.21±2.35 fold respectively. Compared with group C, HBD-3 mRNA expression after 2,4 and 6 hour' treatment of 10 ng/ml IFN-γand mechanical stretch increased by 1.54±0.16,4.37±0.87 and 6.99±1.32 fold, respectively. However, they were significantly lower than those of groupⅠ(P<0.01). HBD-3 expression in group IS after 6 hour' mechanical stretch was significantly less than that of groupⅠ(P<0.01), with no significant difference from that of group C.Conclusions Mechanical stretch can significantly inhibit the up regulation of HBD-3 in alveolar epithelial cells elicited by IFN-γ, this may be one of the reasons that patients under mechanical ventilation(MV) have a higher risk of VAP.