Ventilation Strategies and Experimental Lung Injury John Baier M.D. University of Manitoba

Role of Mechanical Ventilation in Lung Injury Mechanical ventilation is life saving but it also causes lung injury Evolution of ventilation techniques over the last 20 years Past Practices Current Trends Low or normal arterial CO2 Increasing tolerance for high or very high arterial CO2 Normal acid-base or alkalosis Respiratory acidosis Larger tidal volumes Small tidal volumes No PEEP Optimal PEEP

Ventilator induced Lung Injury Ventilator induced lung injury (VLI) is similar to that induced by other means different etiologies have similar cellular and biochemical mechanisms VLI compounds lung injury induced by other means VLI Membrane damage Alveolar type II cell necrosis Exposure of underlying basement membrane Activation of inflammation Injury to pulmonary vascular bed Increase in vascular permeability Interstitial and alveolar edema Protein leak into alveolar space Intra-alveolar activation of coagulation pathways Activation of inflammatory pathways

Ventilator induced Lung Injury Inflammatory cell influx and activation Neutrophils Alveolar macrophages Increase in cytokines, prostanoids, growth factors Airway Effects Bronchospasm Cilliary dysfunction

Ways by which Ventilation Can Cause release of Inflammatory Mediators Stress failure of plasma membranes (necrosis) Release of preformed mediators Proinflammatory effects of cytosol released from damaged cells Stress failure of endothelial and epithelial barriers Loss of compartmentalization Hemorrhage and accumulation of leukocytes in the lungs Overdistention without tissue destruction Effects on the vasculature independent of stretch and rupture Increased intraluminal pressure Increased shear stress

Mechanotransduction Several studies have show that mechanical stretching of cell layers can induce production of inflammatory mediators Alter Na+ K + ATPase function (lung edema)

Mechanical stretch stimulates MIP-2 in fetal lung cells Mourgeon et al: Am Journal Physiology (lung Cell Mol Physiol) 2000 Primary fetal lung cells isolated from rats Cell grown on gelfoam sponges Stretched by computer controlled solenoids Incubated with or without the presence of LPS

Mechanical stretch enhances LPS induced MIP-2 production Mechanical stretch of the cells induced production of MIP-2 Greatly enhanced the stimulatory effect of LPS on MIP-2 production Cells in an organotypic culture were subjected to 2 or 5% unidirectional stretch for 4 h at 40 cycles/min in the absence (control; no stretch) and presence of LPS (100 ng/ml). MIP-2 concentrations in the culture medium were analyzed by ELISA. P < 0.0001 by 1-way ANOVA. * P < 0.05 compared with all other groups. # P < 0.05 vs. all control and LPS-only groups.

Fetal rat lung cells were subjected to 5% stretch for 4 h at different frequencies in the presence ( ) and absence of LPS (100 ng/ml; ). P < 0.0001 by 1-way ANOVA. * P < 0.05 compared with that of stretch only at 20 or 40 cycles/min

Ventilatory Strategies Hyperventilation Hypocapnea PEEP Hypoventilation Hypercarbia “therapeutic hypercarbia”

Hyperventilation and Lung Injury “Too much of a good thing is bad for you or Stretching it too far!”

Why (or How) does Hyperventilation Cause Lung injury? Airleak Syndromes Excessive stretch Effects of low CO2 Effects of alkalosis HSC Winnipeg Manitoba 1987-1988

Effect of high tidal volumes Hyperventilation Effect of high tidal volumes

Synergistic effect of high tidal volume and hyperoxia on lung injury Quin et al: Journal of Applied Physiology 2002 Ventilated rat model Compared 2 tidal volumes 7 ml vs 20 ml kg-1 Extra dead space added to ETT tube to maintain PaCO2 at 30-40 torr (20 ml kg-1 group) RA vs 100% oxygen

Large Tidal volume Ventilation is synergistic with hyperoxia to induce lung edema Ventilation with large tidal volumes increased lung water Lung water was further increased in the presence of hyperoxia Lung water measured by lung wet-to-dry weight ratio in control, nonventilated rats; rats ventilated for 2 h at tidal volumes (VT) 7 or 20 ml/kg for 2 h and killed immediately after exposure; and rats ventilated for 2 h with VT 20 ml/kg and killed 6 and 24 h after ventilation. Open bars are rats on room air (RA), and solid bars are rats with hyperoxia (means ± SE). *P < 0.05 vs. control; P < 0.05 vs. VT 7 ml/kg RA; P < 0.05 vs. all other groups.

Large Tidal volume Ventilation is synergistic with hyperoxia to induce lung inflammation Ventilation with large tidal volumes increased lung MIP-2 (analogous to IL-8 in the human) and neutrophil influx. Neutrophil influx was further increased in the presence of hyperoxia Measurements of MIP-2 (A) and neutrophils (B) in BAL in control, nonventilated rats and rats ventilated for 2 h at 7 and 20 ml/kg and killed 6 h after ventilation. Open bars are rats on RA, and solid bars are rats on hyperoxia (means ± SE). *P < 0.05 vs. control, nonventilated animals; P < 0.05 vs. all other groups

Hyperventilation Effect of hypocarbia

Hypocapnic alkalosis injures the lung Laffey et al: Am J Resp Crit Care Med 2000 Isolated rabbit lungs Altered the inspired concentration of CO2 to induce hypocapnic alkalosis FiCO2 of 0.06 (control) vs. 0.01 (alkalosis) FiO2 constant (0.75) VT 4 ml kg-1 (PIP changes) 2 experimental protocol 3 hour period of ventilation and perfusion Warm ischemia - reperfusion

Hypocapnic alkalosis induces increased pulmonary permeability Mechanical ventilation (3 hours) increased pulmonary capillary permeability (Kf,c) in both control and hypocapnic groups. Hypocapnic ventilation was associated with a much greater increase in Kf,c Kf,c before and after prolonged ventilation. Final Kf,c was significantly greater than baseline in both groups (*p < 0.05); the magnitude of the increase was significantly greater in the group with hypocapnia versus the control group ( p < 0.05).

Hypocapnic alkalosis reduces lung compliance Peak airway pressures required to maintain tidal volume increased over time in the hypocapnic group compared to controls Peak airway pressure [Paw] at baseline, before and after prolonged ventilation. Paw was significantly elevated in the group with hypocapnia (*p < 0.05) but not in the control group.

Hypocapnic alkalosis results in increased lung weight Increase in lung weight after prolonged ventilation. Lung weight was significantly increased in the group with hypocapnia (*p < 0.05) but not in the control group

Deleterious effects of hypocapnea on pulmonary reperfusion injury Kf,c before and after IR injury. Kf,c was significantly increased following IR in the control group and the group with hypocapnia (*p < 0.05); the magnitude of the increase was significantly greater in the group with hypocapnia versus the control group ( p < 0.05). Peak airway pressure (Paw) measured at baseline, before and immediately after IR injury, and at the end of the experiment. Paw was significantly increased following IR in the group with hypocapnia (*p < 0.05) but not in the control group

Summary thus far (what we have learned) High tidal volume ventilation induces lung injury Alkalosis induces lung injury Synergistic with other factors that induce lung injury

Effects of PEEP on VLI

Effect of CPAP vs IMV with PEEP on lung injury in newborn Jobe et al Pediatr Res 2002 Preterm lamb model Studied spontaneous breathing with CPAP 5 vs IMV with PEEP of 4 Ventilator rate fixed at 40 breaths/min PIP adjusted to give PCO2 of 40 mm Hg Tinsp 0.7 sec Measured indices of lung injury and inflammation

Deflation pressure-volume curves for the CPAP lambs and the ventilated lambs. All volumes between 10 and 40 cm H2O pressure are higher for the CPAP lungs than for the ventilated lungs (p < 0.05). Blood pH and PCO2 values for the cord blood and for 120 min after birth. The pH and PCO2 values were different between the ventilation and CPAP groups at all times except for the cord blood values at 0 time (p < 0.05).

CPAP is associated with decreased lung inflammation Spontaneous breathing on CPAP was associated with decreased neutrophil influx and decreased H2O2 production compared to IMV There were no differences in mononuclear cell numbers or cytokine mRNA expression

Role of PEEP in determining lung injury Naik et al: AM J Resp Crit Care Med 2001 Preterm lamb model Delivered 126-132 days Treated with surfactant with rSP-C 3 treatment groups No PEEP 4 cm H20 PEEP 7 cm H2O PEEP FiO2 and PIP adjusted to keep PaCO2 50-60 mm Hg

Sequential measurements of PaCO2 and the tidal volumes Sequential measurements of PaCO2 and the tidal volumes. The ventilatory goals were to keep PaCO2 between 50 and 60 mm Hg, and the tidal volumes (VT) 10 ml/kg. There were no differences between the groups. The filled symbols indicate combined PaCO2 values and VT for both 2 h and 7 h ventilation groups (A-C ) Ventilatory pressures measured as peak inspiratory pressure minus positive end-expiratory pressure (PIP   PEEP), PaO2/FIO2 ratios, and lung gas volumes at 40 cm H2O (V40) 2 h and 7 h. The ventilatory pressures required to achieve the target PaCO2 were lower for the 4 and 7 PEEP groups. The PaO2/FIO2 ratios were higher for the 4 and 7 PEEP groups. V40 with 0 PEEP had consistently lower lung volumes relative to 4 and 7 PEEP after both 2 h and 7 h ventilation. *p < 0.05 versus 4, 7 PEEP

Effect of PEEP on Inflammatory markers of lung injury Ventilation increases neutrophil influx and H202 activity in the lung. Optimal PEEP moderates neutrophil influx Neutrophil counts (A) and total H2O2 activity (B) in alveolar wash fluid after 2 h ventilation. All ventilated groups had higher neutrophil counts relative to fetal controls. The 0 and 7 PEEP groups had elevated neutrophil counts relative to the 4 PEEP group. Total H2O2 activity in alveolar washes was increased in ventilated groups compared with unventilated fetal control. to < 0.05, control group versus all ventilated groups; *p < 0.05 versus 4 PEEP.

Effect of PEEP on Cytokine Expression IL-1 and IL-6 mRNA levels after 2 h and 7 h ventilation. IL-1 and IL-6 mRNAs were elevated in all ventilated groups relative to fetal control groups. Both IL-1 and IL-6 mRNA were significantly increased in 0 PEEP relative to 4 PEEP after both 2 h and 7 h ventilation. IL-1 and IL-6 mRNA decreased between 2 h and 7 h. tp < 0.05, control groups versus all ventilated groups, *p < 0.05 versus 4 PEEP, and #p < 0.05 versus 7 h ventilation. The inset shows representative RNase protection assay for IL-1 and IL-6 after 2 h ventilation IL-8 and TNF-a mRNA levels after 2 h and 7 h ventilation. IL-8 was increased in all ventilated groups relative to fetal controls. IL-8 was also significantly elevated in 0 PEEP compared with 4 PEEP after 7 h ventilation. TNF- was increased in all ventilated groups after 7 h ventilation relative to fetal control groups. The different PEEP levels did not influence TNF-a mRNA levels. tp < 0.05 control groups versus all ventilated groups. *p < 0.05 versus 4 PEEP. **p < 0.05 control groups versus groups ventilated for 7 h. The insets show representative RNase protection assays for IL-8 and TNF- after 2 h ventilation.

Morphology of the lungs Morphology of the lungs. (A) Representative sections that were scored as 1 = collapsed. 2 = distended, and 3 = overdistended alveoli. All the panels were photographed at the same magnification. Original magnification: ×230. Scale bars = 100 µm. (B) Percentage fractional areas of alveolar inflation. The 0 PEEP group had more collapsed alveoli compared with 4 and 7 PEEP. 7 PEEP showed more overdistended alveoli compared with 0 PEEP. * p < 0.05 versus 4 and 7 PEEP; tp < 0.05 versus 4 and 7 PEEP

Ventilator Strategies and Lung Injury Tremblay et al J Clin. Invest Isolated rat lung model Compared the effects of PEEP and high tidal volumes on cytokine production in the presence of LPS 4 treatment groups Control (C) Moderate volume MV with PEEP (MVHP) Moderate volume MV with zero PEEP (MVZP) High Volume MV with zero PEEP (HVZP)

Static Lung Compliance after 2 hrs of ex vivo ventilation Ventilation without PEEP or with high volumes results in decreased static compliance Static compliance curves of the lungs prior to ex vivo ventilation and following 2 h of ex vivo ventilation. A significant rightward shift developed in both zero PEEP groups (P < 0.005 for MVZP, HVZP), whereas no shift was observed in the presence of 10 cm of PEEP for either saline- or LPS-treated lungs.

Ventilation strategy and Inflammatory mediators Cytokine concentrations were greatest in lungs ventilated without PEEP or with high volumes Effect of ventilation strategy on absolute lung lavage cytokine concentrations for the saline- injected groups. A similar trend was seen for all cytokines with lowest levels in the control group (C) and highest in HVZP. Despite similar end-expiratory distention, MVHP ventilation had significantly lower BAL cytokine concentrations than HVZP ventilation. *P < 0.05 vs. Control, MVHP, MVZP; P < 0.05 vs. Control, MVHP; §P < 0.05 vs. Control.

Ventilation strategy and Inflammatory mediators Cytokine concentrations were greatest in lungs ventilated without PEEP or with high volumes The pattern of lavage cytokines seen in response to ventilation strategy was similar to the saline-treated groups except for MIP-2, in which the control group (C) had comparable levels to the MVZP group (both increased significantly vs. the MVHP group). *P < 0.05 vs. Control, MVHP, MVZP; P < 0.05 vs. Control, MVHP; §P < 0.05 vs. Control; ¶P < 0.05 vs. MVHP.

Ratio of study group BAL cytokine concentrations relative to saline-treated controls. LPS ( ) pretreatment resulted in significantly increased levels of TNF for three of the ventilatory strategies (i.e., an approximately 5-fold increase for C, an approximately 30fold increase for MVHP, and an approximately 37-fold increase for MVZP) as compared to saline-treated (O) controls. LPS also increased levels of MIP-2 for all four ventilatory strategies, whereas no significant changes were seen with the other four cytokines assessed. As IL-6 and IFN were undetectable in saline-treated controls, an arbitrary value of 1 was assigned to allow comparison. *P < 0.05 vs. saline treated group

Ventilation strategy and c-fos expression Mechanical ventilation increased expression of c-fos which was enhanced by LPS Ventilation with high volumes or no PEEP further increased c-fos expression Northern blot analysis of lung homogenate c-fos mRNA for the various ventilation strategies. Densitometric values for c-fos were standardized to 28S ribosomal RNA. A similar trend to that observed for the BAL cytokine concentrations was seen in both saline- and LPS-treated animals. The presence or absence of LPS was not found to make a significant difference in c-fos mRNA levels. *P < 0.05 vs. Control; P < 0.05 vs. MVHP.

Ventilation strategy and TNFa expression Mechanical ventilation increased expression of TNFa which was enhanced by LPS Ventilation with high volumes or no PEEP further increased TNFa expression Northern blot analysis of lung homogenate TNF mRNA levels for the various ventilation strategies. Densitometric values for TNF were standardized to 28S ribosomal RNA. TNF mRNA in the saline-treated animals increased for three of the ventilatory strategies as compared to the controls (§P < 0.05 vs. Control). In LPStreated animals, TNF mRNA was significantly greater for MVHP and MVZP as compared to controls or HVZP (*P < 0.05 vs. Control HVZP). Within ventilatory strategies, LPS was found to increase TNF mRNA for three of the four ventilation strategies used (C, MVHP, MVZP; P < 0.05).

Ventilation Strategy and Lung Edema formation Ventilation with lower tidal volume reduces lung edema formation Rate of pulmonary edema formation, measured as excess extravascular lung water/h, was significantly lower with lower tidal volume ventilation [*P < 0.05 compared with the 3-ml/kg group, P < 0.05 compared with the 6-ml/kg group, P < 0.05 compared with the 12-ml/kg positive end-expiratory pressure (PEEP) 5-cmH2O group]. All acid-injured rats (solid bars) had a significantly more extravascular lung water than did ventilated control rats instilled with saline instead of acid (open bars; P < 0.001 by ANOVA correction for multiple comparisons). Excess lung water did not differ among the control groups (P > 0.05). Sample water did not differ among the control groups (P > 0.05). Sample sizes: 12 ml/kg PEEP 10 cmH2O, n = 4; 12 ml/kg PEEP 5cm H2O, n = 8; 6 ml/kg PEEP 10 cmH2O, n = 13; 3 ml/kg PEEP 10 cmH2O, n = 12. Data are means ± SD.

Light photomicrographs of rat lungs ventilated with 3 ml/kg PEEP 10 cmH2O (A); 6 ml/kg PEEP 10 cmH2O (B); 12 ml/kg PEEP 5 cmH2O (C); and 12 ml/kg PEEP 10 cmH2O (D) after lung acid injury. There was significantly more interstitial and alveolar edema after higher tidal volume ventilation. Lung injury scores based on edema, hyaline membranes, septal thickening, inflammatory cell infiltration, and small airway epithelial injury were significantly higher in rats ventilated with 12 ml/kg PEEP 10 cmH2O, although injury was patchy in all groups (original magnification, ×40; hematoxylin-eosin).

Summary thus far (what we have learned) High tidal volume ventilation induces lung injury Alkalosis induces lung injury Synergistic with other factors that induce lung injury Lung injury is reduced by use of CPAP or PEEP

Hypercapnea may protect the lung Wean, Wean, WEAN

Protective effects of hypercapnic acidosis on VLI Sinclair et al: Am J Respir Crit Care Med 2002 Rabbit model All ventilated with high VT ~ 25 ml/kg to induce lung injury for 4 hours Randomized to eucapnia (PaCO2 ~ 40 mm Hg or hypercapnia (PaCO2 80-100 mm Hg) Achieved by addition of CO2 to inspired gas (4-5% vs 12%) Respiratory rate kept constant (32 breaths/min) FiO2 kept constant 0.5

Effects of hypercapnic acidosis on oxygenation in ventilator induced lung injury Oxygenation was better preserved in hypercapnic acidosis Arterial partial pressure of oxygen as it varies over time between the two study groups. Solid squares = hypercapnic group open circles = eucapnic group. *p < 0.05 compared with time zero value within the same group. p < 0.05 compared with hypercapnic group at same time point.

Effects of hypercapnea on lung injury indices in VILI All four types of injury are reduced in the hypercapnic group, particularly PMN infiltration and hemorrhage. Black bars = hypercapnic group White bars = eucapnic group. p < 0.05.

Effects of hypercapnea on BAL indices in ventilator induced lung injury Hypercapnic acidosis was associated with a reduction in the inflammatory infiltrate in BAL This was predominately due to decrease in neutrophils BAL fluid total and differential cell counts. Eucapnic animals had significantly more nucleated cells per ml, most of which were neutrophils, whereas fewer cells, predominately macrophages, were found in the hypercapnic group. p < 0.05.

Protective effects of hypercapnia in pulmonary reperfusion injury Laffey et al: Am J Respir Crit Care Med 2000 Rabbit model FiCO2 altered to produce hypercapnea FiCO2 of 0.00 (control) vs. 0.06 (hypercapnea) Very high PaCO2 (50-60 torr vs.100 torr) FiO2 constant (0.75) VT 6 ml kg-1 , constant rate and PEEP Experimental protocol Warm ischemia – reperfusion of left lung Ligature of left hilum followed by reperfusion

Hypercapnea improves pulmonary mechanics following reperfusion injury Hypercapnic acidosis was associated with improved pulmonary mechanics Peak airway pressure (Paw). Paw was comparable in both groups at baseline; increased significantly in both groups following reperfusion; and was significantly lower in TH versus CON (*p < 0.05). (B) Static Inflation compliance. Compliance was comparable in both groups at baseline, and decreased significantly in both groups following IR. The final static lung compliance value was significantly greater in TH versus CON (*p < 0.05 versus baseline in both groups, and p < 0.05 TH versus CON). (C ) Dynamic expiratory compliance. Dynamic expiratory compliance was comparable in both groups at baseline and decreased significantly in both groups following reperfusion. The final value of dynamic lung compliance was significantly greater (and the decrement smaller) in TH versus CON (*p < 0.05). (D) Lung wet:dry ratio. The wet: dry ratio was significantly lower in TH versus CON (*p < 0.05).

Hypercapnea reduces inflammatory mediators following reperfusion injury Hypercapnic acidosis was associated with a reduction in inflammatory mediators following reperfusion injury (A) The protein concentration in the BAL fluid was significantly lower in TH versus CON groups (*p < 0.05). (B) BALF TNF- was significantly lower in the TH versus CON groups (*p < 0.05). (C ) The lung tissue 8-Isoprostane concentration was lower in TH versus CON (*p < 0.05). (D). Lung tissue myeloperoxidase levels were similar in both groups.

Hypercapnea reduces oxidant injury and Apotosis following reperfusion injury Hypercapnic acidosis was associated with a reduction in oxidant injury (nitration of tyrosine) and reduction in apoptosis of lung cells Control Hypercapnea

Protective effect of hypercapnia on endotoxin induced lung injury Laffey et al: Am J Resp Cir Care Med 2004 Rat Model (adult) Mechanical ventilation FiO2 0.30, VT 4.5 ml kg-1, 90 bpm, PEEP 2.5 Hypercapnia induced by added CO2 (FiCO2 0.06) Therapeutic vs. prophylactic strategies Intratracheal instillation of E coli LPS

Hypercapnia reduces lung inflammation following endotoxin installation Hypercapnic acidosis was associated with a reduction in lung inflammation A) Control (B) Therapeutic hypercapnic acidosis

Hypercapnia improves lung function following endotoxin installation Oxygenation (lower A-a gradient) is maintained by therapeutic hypercapnia Lung compliance is maintained by hypercapnia

Caveats and Concerns Therapeutic hypercapnia may not be as effective in surfactant deficiency Rai et al: Pediatr Res 2004 Surfactant depleted rabbits (saline lavage) Compared high and normal CO2 (FiCO2 0.12) Vt 12 mL/kg, PEEP 0 cm H2O, and a rate of 19 breaths/min vs. Vt 5 mL/kg, with PEEP 12.5 cm H2O and rate 52 Found little differences between treatment groups Compliance, cytokine levels, oxygenation

Caveats and Concerns What about the brain??? Potential adverse effects of high CO2 on incidence of IVH CO2 increases cerebral blood flow Van Hulst et al Clin Physiol Funct Imaging 2004 Effects of hypocapnia and hypercapnia on brain glucose and lactate (pigs) Hypercapnia has no effect Hypocapnia decreased brain glucose and increased lactate Kamper et al Acta Paediatr 2004 Similar survival and neurological outcome between ELBW infants treated with early CPAP and permissive hypercapnia and those treated conventionally

Summary (what we have learned) High tidal volume ventilation induces lung injury Alkalosis induces lung injury Synergistic with other factors that induce lung injury Lung injury is reduced by use of CPAP or PEEP Optimal PEEP strategies Hypercapnea protects the lung from injury Protects lung against injury from other causes Even when high tidal volumes are used