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1Istituto di Fisiologia Umana I, Università degli Studi di Milano, Milan; 2Servizio di Coagulazione & Unità di Ricerca Trombosi, IRCCS Ospedale San Raffaele, Milan, Italy; 3Critical Care Department, University of Athens, Athens, Greece; and 4Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada
Submitted 10 December 2004 ; accepted in final form 4 March 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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in serum and bronchoalveolar lavage fluid were assessed in eight closed and eight open chest, normal anesthetized rabbits undergoing prolonged (3–4 h) mechanical ventilation (MV) at low volume with physiological tidal volumes (10 ml/kg). Relative to initial MV on positive end-expiratory pressure (PEEP), MV at low volume increased lung quasi-static elastance (+267 and +281%), airway (+471 and +382%) and viscolelastic resistance (+480 and +294%), and decreased NOe (–42 and –25%) in closed and open chest rabbits, respectively. After restoration of PEEP, viscoelastic resistance returned to control, whereas airway resistance remained elevated (+120 and +31%) and NOe low (–25 and –20%) in both groups of rabbits. Elastance remained elevated (+23%) only in closed-chest animals, being associated with interstitial pulmonary edema, as reflected by increased lung wet-to-dry weight ratio with normal albumin concentration in bronchoalveolar lavage fluid. In contrast, in 16 additional closed- and open-chest rabbits, there were no changes of lung mechanics or NOe after prolonged MV on PEEP only. At the end of prolonged MV, TNF- was practically undetectable in serum, whereas its concentration in bronchoalveolar lavage fluid was low and similar in animals subjected or not subjected to ventilation at low volume (62 vs. 43 pg/ml). These results indicate that mechanical injury of peripheral airways due to their cyclic opening and closing during ventilation at low volume results in changes in lung mechanics and reduction in NOe and that these alterations are not mediated by a proinflammatory process, since this is expressed by TNF- levels. lung elastance; interrupter resistance; viscoelasticity; proinflammatory cytokines; exhaled vapor condensate
Recruitment of polymorphonuclear leukocytes in the alveolar walls during ventilation at low volume (12) fits a recently described type of ventilator-induced lung injury called biotrauma (14). Under this condition, parenchymal overdistension and abnormal shear forces could represent the mechanical stimuli leading to release of mediators that prime polymorphonuclear leukocytes, which may represent the major effector cells in the generation of tissue injury and upregulation of the inflammatory response (14, 39). Increased concentrations of proinflammatory cytokines, mainly tumor necrosis factor (TNF)- and IL-6, have been in fact observed in bronchoalveolar lavage (BAL) fluid of excised rat lungs after prolonged ventilation at ZEEP (6, 38). Because these cytokines have been shown to enhance the expression of inducible nitric oxide (NO) synthase (iNOS) in an in vitro preparation (2), NO concentration in expired air (NOe) could eventually increase also in normal rabbits ventilated at low lung volume and thus serve as a marker of parenchymal inflammation. Other studies have shown, however, that injurious ventilation in initially intact rats does not affect in vivo proinflammatory cytokine production (18, 30, 41). Human alveolar macrophages and epithelial cells subjected to prolonged cyclic stretching release IL-8, which is involved in the recruitment of polymorphonuclear leukocytes, but not proinflammatory cytokines, such as TNF- or IL-6 (29, 42). Moreover, anti-inflammatory mediators are also expressed during ventilation at low volume (38), thus making it difficult to recognize the effective orientation of cytokine balance (24). On this basis, increased concentration of NOe should not be expected to occur in normal rabbits during prolonged ventilation at low volume. In contrast, because most of the NO from the lungs is produced by small airway epithelium, a reduction in NOe levels could be a useful marker of the extent of the mechanical injury of the peripheral airways due to their cyclic opening and closing during tidal ventilation at low lung volumes (11, 25). Moreover, NO production could be reduced by prostaglandins E2 and F2a (21) released by alveolar macrophages, polymorphonuclear leukocytes, airway and alveolar epithelial cells activated by mechanical insults (23), as well as by the vasoactive intestinal peptide (13), which also may be involved in the inflammatory response of the lung (33).
The purpose of the present investigation in normal rabbits is therefore to assess the effects of 3–4 h of ventilation at low lung volume on NO production from the tracheobronchial tree, also in relation to possible inflammatory reaction, as monitored by TNF- levels in serum and BAL fluid. Although previous studies (11, 12) on the morphological and mechanical effects of ventilation at low lung volume were performed on open-chest rabbits only, the present experiments also included closed-chest rabbits to avoid confounding inflammatory responses elicited by the major surgical intervention required in open-chest animals.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Airflow (
) was measured with a heated Fleisch pneumotachograph no. 00 (HS Electronics, March-Hugstetten, Germany) connected to the tracheal cannula and a differential pressure transducer (Validyne MP45, ±2 cmH2O; Northridge, CA). The response of the pneumotachograph was linear over the experimental range of . Tracheal (Ptr), esophageal (Pes), and systemic blood pressure were measured with pressure transducers (model 1290A; Hewlett-Packard, Palo Alto, CA) connected to the side arm of the tracheal cannula, a latex balloon (2.5 cm long, 0.5-cm inner diameter) suitably placed in the lower esophagus, and the femoral artery, respectively. There was no appreciable shift in the signals or alteration in amplitude up to 20 Hz. NO was measured using a chemiluminescence analyzer (NOA 280i, Sievers, Boulder, CO) attached to a side port of the tracheal cannula through Teflon tubing and set to draw air at a rate of 200 ml/min. Zeroing and calibration of the NO analyzer was verified repeatedly during the experiment using the Zero Air Filter and certified gas mixture provided by the manufacturer. The signals from the transducers were amplified (model RS3800; Gould Electronics, Valley View, OH), sampled at 200 Hz by a 12-bit analog-to-digital converter (AT MIO16E-10; National Instruments, Austin, TX), and stored on a desk computer, together with the signal from the NO analyzer. Volume changes (
V) were obtained by numerical integration of the digitized airflow signal. Arterial blood PO2 (PaO2), PCO2 (PaCO2), and pH were measured by means of a blood gas analyzer (IL 1620; Instrumentation Laboratory, Milan, Italy) on samples drawn from the carotid artery at the beginning and end of each test session, whereas the pH of the deareated, exhaled airway vapor condensate (20) was measured using an Amersham Pharmacia C900 pH meter (Uppsala, Sweden).
After completion of the surgical procedure and instrumentation, the rabbits were ventilated with a specially designed, computer-controlled ventilator, delivering a NO-free [NO concentration <0.5 parts/billion (ppb)], water-saturated gas mixture from a high-pressure source (4 atm) at constant flow of different selected magnitudes and duration. A three-way stopcock allowed the connection of the expiratory valve of the ventilator either to the ambient (ZEEP) or to a drum in which the pressure could be made positive (PEEP) or negative [negative end-expiratory pressure (NEEP)] by means of a flow-through system. A detailed description of the ventilator can be found elsewhere (12).
For all animals, the baseline ventilator settings consisted of fixed VT (10 ml/kg), inspiratory duration (TI; 0.25 s), and cycle duration (1.8 s). An end-inspiratory pause of 0.35 s was applied to ensure a normal mean lung volume during the respiratory cycle. During NO measurements, both the TI and expiratory duration were set at 1 s and the end-inspiratory pause was removed. With the above settings, no intrinsic PEEP was present under any experimental condition, as evidenced by an end-expiratory pause (zero flow) and absence of Ptr changes with airway occlusion at end expiration. Although open-chest rabbits were always ventilated with air, closed-chest animals were intermittently ventilated with 70–80% oxygen during MV on NEEP to limit the profound, life-threatening hypoxia that occurred otherwise. However, measurements were always performed during air breathing.
Procedure and data analysis. Rabbits of groups A and B were equally divided into two subgroups, group Acontrol and Atest, and Bcontrol and Btest, respectively. Rabbits of group Atest and Btest were subjected to the following sequence of PEEP and NEEP or ZEEP: 1) 30 min of MV with PEEP (PEEP1); 2) 3–4 h of MV at NEEP or ZEEP; 3) 1.5 h of MV with PEEP. Rabbits of group Acontrol and Bcontrol were subjected for the same cumulative time to MV with PEEP only. In open-chest animals, the end-expiratory pressure was almost the same during the initial and final period of MV on PEEP, averaging 2.3 ± 0.1 cmH2O. In closed-chest animals, an end-expiratory pressure of 1.2 ± 0.1 cmH2O was applied to limit or prevent the expected fall in the end-expiratory lung volume with anesthesia and paralysis, whereas NEEP was –7.7 ± 0.1 cmH2O. On completion of in vivo measurements, the animals were killed with an overdose of anesthetic, the lungs were isolated, the main right bronchus was tied off, and the right lung was removed, weighed immediately, left overnight in an oven at 120°C, and weighed again to compute the wet-to-dry weight ratio. The left lung was lavaged four times using 3-ml aliquots of normal saline, fluid recovery ranging from 40 to 50%. The effluents were pooled and centrifuged (Harrier 18/80, Sanyo Gallenkamp PLC, Loughborough, UK) at 2,000 rpm for 10 min, and the supernatant was frozen and stored at –20°C. The animals were from a single cohort, and the experiments were done in random order.
To assess TNF levels in the BAL fluid before the prolonged MV at low or normal end-expiratory lung, in an additional group of eight closed-chest rabbits (group C), instrumented as described above, BAL fluid was obtained after 15 min of MV with PEEP, while blood samples were taken before and after induction of anesthesia and paralysis, on completion of the surgical maneuvers, and at PEEP1.
Mechanical characteristics were studied during PEEP1, at start (NEEP1 or ZEEP1) and end of the NEEP or ZEEP period (NEEP2 or ZEEP2), and 
15 min after MV on PEEP had been restored (PEEP2). Before all measurements on PEEP, the lungs were inflated three to four times to Ptr of 30 cmH2O. Two types of measurements were carried out: 1) while VT was kept at baseline values, test breaths were intermittently performed with different inspiratory and TI in the range of 0.25–3 s to assess mechanics at end inflation; and 2) while keeping inspiratory at baseline values, test breaths were intermittently performed with different VT to obtain quasi-static inflation volume-pressure curves. End-inspiratory occlusions lasting 5 s were made in all test breaths, which were performed in random order and repeated three to five times. During ventilation at NEEP or ZEEP, end-inspiratory occlusions were performed only for VT greater than or equal to baseline VT. In open-chest rabbits and during ventilation with PEEP, the expiratory valve was opened to the ambient to measure the difference between the end-expiratory and the resting lung volume (EELV). In closed-chest animals, EELV was obtained as the volume exhaled with a Ptr of –20 cmH2O. Mechanical parameters were assessed with the rapid airway occlusion method (3, 10). The end-inspiratory airway occlusions were followed by a rapid initial drop in pressure (P1) and by a slow decay (P2) to an apparent plateau value (Pst). This pressure, computed as the mean pressure recorded during the last 0.5 s of occlusion, was taken to represent the quasi-static pressure, whereas P1 and P2 divided by inspiratory yielded the interrupter resistance (Rint) and additional (R) resistance, respectively. Viscoelastic parameters of resistance (Rvisc) and 
visc [Rvisc/viscoelastic elastance (Est)] were computed by fitting the values of R and TI with the function (10)
 | (1) |
Tracheal NO concentration was continuously measured for 15–20 min at the transition from PEEP (PEEP1) to NEEP or ZEEP (NEEP1 or ZEEP1) and from NEEP or ZEEP (NEEP2 or ZEEP2) to PEEP (PEEP2), and, for 10-min periods, 60 and 90 min after restoration of MV on PEEP to check for any possible deterioration of the preparation. Moreover, during this period, administration of epinephrine, boluses of bicarbonate (1 M) solution, and/or short periods of hyperventilation and oxygen breathing were performed to keep the values of arterial pressure, PaO2, PaCO2, and pHa close to those with PEEP1. For a given condition, 60 breaths were ensemble averaged, and the mean concentration of NO during expiration was used as the NOe concentration. Moreover, during PEEP1, NEEP2 or ZEEP2, and PEEP2, part of the tubing beyond the expiratory valve of the ventilator was immersed in ice-cold water to obtain 1 ml of exhaled airway vapor condensate (20).
Analysis of TNF- was carried out in a blinded fashion on BAL fluid and serum collected under conditions PEEP1, NEEP2 or ZEEP2, and PEEP2 in groups A and B, and before and after induction of anesthesia and at the start and end of the 15-min period of MV on PEEP in group C, using a commercially available ELISA kit (BD Bioscience, Franklin Lakes, NJ), specific for rabbit. TNF- color development was measured at 405 nm (Titertek Multiskan MCC, Flow Laboratories, Milan, Italy), with background absorbancy of blank wells being subtracted from the standards and samples before determination of the concentration. The lower limit of detection was 10 pg/ml, in which case TNF- concentration was assumed to be nil. The albumin concentration of the BAL fluid supernatant and serum obtained shortly before lung lavage was determined with a clinical chemistry analyzer (Falcor 350, Menarini Diagnostics, Florence, Italy) at 630 and 700 nm using the BCG method (Menagent, Menarini Diagnostics) with bovine albumin as standard.
Statistics.
Results are presented as means ± SE, except for TNF- measurements. The least-square regression method was used to assess the parameters in Eq. 1 and the pressure-volume relationship of the lungs. Comparisons among experimental conditions were performed using one-way ANOVA; when significant differences were found, the Bonferroni test was performed to determine significant differences between different experimental conditions. Results from TNF- measurements are expressed as median and range, and the statistical analysis was performed using the Mann-Whitney test. The level for statistical significance was taken at P 
0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Mechanics. In closed-chest animals, the quasi-static volume-pressure curve of the chest wall on PEEP1 and PEEP2 did not differ (Fig. 1). Moreover, chest wall viscous resistance and viscoelastic properties were the same under all conditions (see Tables 3 and 4). Hence, any change in the mechanical properties of the respiratory system because of prolonged ventilation at low volume was the consequence of changes in lung mechanics.
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EELV was significantly larger in open- than in closed-chest animals (Table 2). With PEEP1 and PEEP2, EELV was the same in open-chest rabbits and in closed-chest rabbits ventilated on PEEP only, whereas in rabbits ventilated on NEEP, EELV decreased significantly (–4.2 ± 1.4 ml; P < 0.025) with PEEP2 (Table 2).
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On NEEP or ZEEP, the quasi-static inflation volume-pressure curve of the lung shifted downward both in closed- and open-chest animals (Fig. 2). Moreover, the volume-pressure curve of open-chest animals, which on PEEP was concave toward the pressure axis, became markedly S shaped, as in closed-chest animals. All these changes increased with NEEP2 or ZEEP2.
Elastance.
On the basis of the Vo and EELV values in Table 2, tidal ventilation with PEEP occurred in the range 25–50 and 33–65% Vo in closed- and open-chest animals, respectively, whereas during NEEP or ZEEP, tidal ventilation occurred in the range 0–26% Vo in both closed- and open-chest animals. The average values of Est obtained in the various groups of animals and conditions are reported in Table 3. With PEEP1, Est was significantly larger in closed- than in open-chest animals (209 ± 11 vs. 144 ± 5 cmH2O/l; P < 0.001). With PEEP2, Est increased significantly only in group Atest rabbits. Est increased markedly and progressively from NEEP1 or ZEEP1 to NEEP2 or ZEEP2 and more in closed- than in open-chest animals. Relative to PEEP1, Est increased, however, by a similar amount in group Atest and Btest rabbits both with NEEP1 or ZEEP1 (225 ± 18 vs. 203 ± 25%; P = 0.5) and with NEEP2 or ZEEP2 (267 ± 19 vs. 281 ± 28%; P > 0.5).
Rint. At the end-inspiratory volume of baseline ventilation, the pulmonary Rint was independent of flow, at least in the range of 10–100 ml/s, in all animals and conditions; hence, the values of Rint obtained in each rabbit and condition were averaged (Table 3). With PEEP1, Rint was similar in closed- and open-chest animals (11.3 ± 0.9 vs. 10.4 ± 0.5 cmH2O·s·l–1; P = 0.38). With PEEP2, Rint did not differ significantly from PEEP1 values in animals ventilated on PEEP only (group Acontrol and Bcontrol), whereas in animals ventilated on NEEP or ZEEP (group Atest and Btest) Rint increased significantly and more in closed- than in open-chest animals, both in absolute (14.6 ± 3.5 vs. 2.9 ± 0.6 cmH2O·s·l–1; P = 0.005) and relative terms (120 ± 32 vs. 31 ± 6%; P = 0.014). Rint increased progressively from NEEP1 or ZEEP1 to NEEP2 or ZEEP2 and more in closed- than in open-chest animals. Relative to PEEP1, Rint increased, however, by a similar amount in group Atest and Btest rabbits both with NEEP1 or ZEEP1 (292 ± 22 vs. 210 ± 31%; P = 0.11) and with NEEP2 or ZEEP2 (471 ± 46 vs. 382 ± 36%; P = 0.15).
Viscoelastic properties.
In all animals and conditions, a unique function in the form of Eq. 1 adequately described the experimental R-TI data of the respiratory system, lung, and chest wall (r > 0.92), allowing computation of the dependent Rvisc and visc values. Figure 3 depicts the group mean relationships of R to TI of the lung obtained under the various experimental conditions, whereas the group mean values of Rvisc and visc are reported in Table 4. With PEEP1, both the Rvisc and visc values did not differ significantly between closed- and open-chest animals (72 ± 6 vs. 65 ± 7 cmH2O·s·l–1; P > 0.5, and 1.30 ± 0.11 vs. 1.34 ± 0.13 s; P = 0.09). With PEEP2, neither Rvisc nor visc changed significantly relative to corresponding PEEP1 values. Rvisc increased progressively from NEEP1 or ZEEP1 to NEEP2 or ZEEP2 and more in closed- than in open-chest animals (258 ± 30 vs. 134 ± 21 cmH2O·s·l–1, P = 0.005, and 330 ± 52 vs. 191 ± 15 cmH2O·s·l–1; P = 0.022). Although in open-chest animals visc did not change with ZEEP, in closed-chest animals visc increased during NEEP ventilation (visc = 0.39 ± 0.09 s; P < 0.001).
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With prolonged MV on PEEP only, there was a tendency for NOe to decrease; however, during the 5–5.5 h of MV, the rate of decay of NOe concentration was not significant both in closed- (–0.55 ± 0.95 ppb/h; P > 0.5) and open-chest animals (–0.37 ± 0.47 ppb/h; P = 0.44). Similarly, none of the other parameters changed significantly with MV on PEEP only (Figs. 4 and 5).
On transition from PEEP to NEEP or ZEEP, NOe concentration increased both in closed- (2.8 ± 2.4 ppb) and open-chest animals (3.9 ± 0.2 ppb), the change being significant only in the latter group of rabbits (P < 0.001). On NEEP or ZEEP, NOe concentration decreased more in closed- (–12 ± 2.7 ppb; P < 0.001) than in open-chest animals (–5.7 ± 1.9 ppb; P < 0.025), whereas on transition to PEEP there was a small significant increase in NOe concentration in closed- and open-chest animals (2.4 ± 0.5 and 1.4 ± 0.1 ppb; P < 0.001). On the other hand, no further changes in NOe concentration occurred during the subsequent 1.5 h of MV on PEEP both in closed- and open-chest rabbits (Figs. 4 and 5). During this period, the average NOe concentration was similar in closed- and open-chest animals (19.5 ± 2.3 vs. 16.7 ± 0.9 ppb; P = 0.25) and markedly lower than that on PEEP1 (Figs. 4 and 5), amounting to 69 and 78% of PEEP1 values in group Atest and Btest rabbits.
As shown in Figs. 4 and 5, no significant changes in PaCO2 and pHa occurred on transition from PEEP to NEEP or ZEEP, whereas PaO2 decreased markedly both in closed- and open-chest animals (–40 ± 3 and –23 ± 5 Torr; P < 0.001). With prolonged MV at NEEP or ZEEP, PaCO2 increased by 10 Torr, pHa decreased by 0.14, and PaO2 dropped by an additional 15 Torr, whereas on transition from NEEP or ZEEP to PEEP (PEEP2) only PaO2 showed a significant increase of 15 Torr. With NEEP2 and ZEEP2, mean systemic arterial pressure decreased significantly both in closed- and open-chest animals and remained significantly lower than control values also on PEEP2 (Figs. 4 and 5). During the final 1.5 h of MV on PEEP, administration of epinephrine, boluses of bicarbonate (1 M) solution, and/or short periods of hyperventilation and oxygen breathing brought the values of all these parameters back to those with PEEP1 (Figs. 4 and 5).
Airway vapor condensate. The mean pH of exhaled airway vapor condensate sampled in closed- and open-chest animals on PEEP1, PEEP2, NEEP2, and ZEEP2 are shown in Table 5. No significant differences were found between pH values of closed- and open-chest rabbits under all conditions or among the pH values obtained under the various conditions in either closed- or open-chest rabbits. When pH values measured in all animals and conditions were pooled, the average pH of the exhaled airway vapor condensate was 6.89 ± 0.05 (range: 6.15–7.61).
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Under all conditions, TNF- concentration in BAL fluid was low but significant. Although the largest median value occurred in animals subjected to prolonged MV on NEEP (group Atest), TNF- levels did not differ significantly among all groups of animals (Table 6). Similarly, although the lowest median value of TNF- concentration in BAL fluid occurred in animals not subjected to prolonged MV (group C), this concentration did not differ significantly from that of group A and B rabbits.
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concentration was high and similar in all groups of closed- and open-chest animals (Table 6). In contrast, serum TNF- concentration was essentially nil during NEEP2, ZEEP2, and PEEP2, as well as in group C rabbits before induction of anesthesia. In these rabbits, serum TNF- levels during MV on PEEP1 were, however, high and similar to those found under the same condition in group A and B rabbits (Table 6). Figure 6 provides a composite picture of the time course of serum TNF- concentration in group Atest, Btest, and C rabbits. Serum TNF- levels grew rapidly during the surgical interventions, peaking at their end, and then declined, becoming nil after 3–4 h or likely earlier.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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concentration in BAL fluid and serum, and that both the fall of NOe and the adverse mechanical effects on the lung (Tables 3 and 4; Fig. 1) are greater in closed- than in open-chest animals. In addition, in closed-chest rabbits, there is a significant increase in lung elastance, probably due to interstitial edema, as reflected by increased wet-to-dry weight ratio but normal albumin concentration in BAL fluid. On the other hand, when lung volumes are kept within the physiological range with PEEP, prolonged MV does not result in significant changes in NOe concentration, lung mechanics, and TNF- levels in BAL fluid and serum.
Lung mechanics.
In line with previous results (11, 12), during MV with PEEP, there was no evidence of airway closure in open-chest animals, since the static inflation volume-pressure curve of the lung was concave to the pressure axis (16), as shown in Fig. 2. In closed-chest animals, however, the initial part of the inflation volume-pressure curve was slightly convex to the pressure axis, suggesting some progressive reopening of small airway (<1 mm in diameter; Ref. 19) during tidal ventilation. Indeed, small airway closure is likely to occur in the dependent zones of the lung in supine, anesthetized, intact rabbits at functional residual capacity, since under this condition pleural surface pressure in the lowermost part of the pleural space is nil (8). In contrast, at low end-expiratory volume, the inflation volume-pressure curve became markedly sigmoidal both in closed- and open-chest animals (Fig. 2), indicating that, under this condition, there was substantial cyclic airway opening and closing, which should be responsible for the histological alterations previously observed in open-chest animals (11, 12), as well as the increase in Rint on PEEP2 relative to PEEP1. The presence of the vertical gradient of transpulmonary pressure in the closed-chest rabbits probably explains the greater convexity of the initial part of the quasi-static inflation volume-pressure curve from low end-expiratory volumes in closed- than in open-chest rabbits (Fig. 2). Indeed, the ratio between Est with the lowest inflation volume (4 ml/kg) and baseline VT was significantly larger in group Atest than in Btest both at the beginning (2.22 ± 0.07 vs 1.18 ± 0.06; P < 0.001) and at the end of the NEEP and ZEEP period (2.83 ± 0.16 vs. 1.30 ± 0.06; P < 0.001), respectively. This should indicate that, in closed-chest animals, more airways were involved in cyclic opening and closing with greater mechanical alterations (Table 3) and, possibly, histological damage.
Both in closed- and open-chest rabbits, there was a significant increase of Est, Rint, and Rvisc relative to PEEP1, which was significantly greater after 3–4 h (NEEP2 and ZEEP2) than after 15 min (NEEP1 and ZEEP1) of ventilation at low lung volume (Tables 3 and 4). Similar results have been reported in previous studies on open-chest rabbits (11, 12, 36) in which the increase of Est and Rvisc was attributed to a higher surface tension and a decrease of ventilated tissue due to airway closure and dependent gas trapping, and the increase of Rint was attributed to reduction of ventilated tissue, uncoupling between peripheral airways and lung parenchyma, and, possibly, increased bronchomotor tone (11, 12). The first mechanism should contribute mainly to the increase in Rvisc, especially on ZEEP2, since most of Rvisc should reside in the air-liquid interface (1), whereas the second accounts for the proportional changes of Est and Rvisc with an essentially constant visc (Tables 3 and 4). Enhanced depletion or inactivation of lung surfactant and greater extent of airway collapse and cyclic opening and closing should explain the greater increase of Est, Rint, and Rvisc observed in closed-chest animals, whereas an increased inequality of regional lung expansion with decreasing lung volume may account for the augmented visc and loss of proportionality between Rvisc and Est relative changes (Tables 3 and 4). Greater inhomogeneity of ventilation distribution in closed-chest animals, poorly compensated by the PO2-dependent redistribution of pulmonary perfusion, is further supported by the lower values of PaO2 (Table 1; Figs. 4 and 5) despite intermittent oxygen administration.
Increased surface tension, differences in pulmonary vascular pressures, and release of vasoactive substances (see below) could explain the presence of pulmonary edema in closed-chest animals with NEEP, as reflected by the significant increase of wet-to-dry weight ratio (Table 1), the downward shift of the inflation volume-pressure curve (Figs. 1 and 2), and the reduction of EELV (Table 2). Edema was, however, limited to the interstitium, because both the albumin concentration in BAL fluid and the ratio between albumin concentration in BAL fluid and serum were normal. Moreover, on dissecting the lungs, no foam could be observed in peripheral units and airways.
After return to PEEP (PEEP2), Rvisc and Est of open-chest animals reversed to the initial (PEEP1) values (Tables 3 and 4). In closed-chest animals, however, Est, although partially restored, remained significantly larger (Table 3), likely because of interstitial edema and increased surface forces. In contrast, Rint was significantly increased both in closed- and open-chest animals (Table 3). The increase in Rint could not be related to changes in arterial blood gases or pH (Table 1) nor to changes in elastic recoil, which, relative to PEEP1, was either the same or moderately increased in open- or closed-chest rabbits, respectively (Table 3; Fig. 2). This increase is due to damage of peripheral airway (11), changes in the mechanical coupling between peripheral airways and lung parenchyma (12), interstitial edema, and possibly increased bronchomotor tone. These mechanisms played a greater role in closed-chest animals, because Rint increased more in closed- (120 ± 32%) than in open-chest rabbits (31 ± 6%). Based on greater wet-to-dry weight ratio (Table 1), small airway edema could have been more pronounced in closed-chest animals. Similarly, because of the greater number of airways involved in cyclic opening and closing and higher airway opening pressures in closed- than open-chest animals during low-volume ventilation (Fig. 2), larger inhomogeneous shear stress should have occurred in the former group of rabbits, thus enhancing the functional alterations.
NOe concentration.
In both closed- and open-chest animals ventilated for 3–4 h on PEEP, NOe concentration did not change significantly (Figs. 4 and 5). Furthermore, NOe values on PEEP1 were comparable with those of normal, anesthetized rabbits (15, 17). They were, however, significantly higher in closed- than open-chest rabbits. This could reflect the lower body temperature in open- than closed-chest animals (36.3 vs. 37.5°C), a difference that might have been larger at the level of airway epithelium. Moreover, the major surgery required to open the chest may also have played a role, although the concentration of the proinflammatory cytokine TNF- did not differ significantly between open- and closed-chest animals (Table 6). At ZEEP1 and NEEP1, there was a small increase in NOe (Figs. 4 and 5), likely related to the decrease in pulmonary blood flow (4, 7) with increasing pulmonary vascular resistance due to the reduction in lung volume.
Prolonged MV at low volume substantially lowered NOe both in closed- and open-chest animals, a reduction that persisted after restoration of the end-expiratory volume (Figs. 4 and 5). Because VT was the same under all conditions, the fall of NOe was the consequence of decreased elimination of NO from injured terminal and respiratory bronchioles, which in our preparation are the main source of NOe (27). Indeed, prolonged MV at low lung volume causes small airway injury with epithelial necrosis and sloughing in open-chest rabbits (11). Although peripheral airway injury has not been studied histologically in closed-chest rabbits, it seems likely that the greater reduction of NOe found in these animals (Figs. 4 and 5) was related to greater histological and mechanical damage.
In open-chest animals ventilated on ZEEP, necrosis and epithelial sloughing involved 12% of peripheral airways (11). Considering that those rabbits were ventilated with lower inflation flows than the present ones and that higher inflation flows cause greater functional and morphological damage (12), the percentage of injured airways might have been even larger in the present open-chest animals ventilated on ZEEP. Interestingly, in these animals, the fall in NOe at PEEP2 averaged 22% relative to PEEP1. However, the satisfactory correspondence between percentage of injured airways and relative decrease of NOe may be fortuitous, also because other factors can reduce NO production and elimination from airway epithelium. In isolated, blood-perfused rabbit lungs ventilated with a fixed pattern, decreasing PaO2 to 33 Torr caused a nearly 40% decrease in end-expiratory NO concentration (4). Therefore, hypoxia could explain the reduced NO elimination on NEEP and ZEEP (Figs. 4 and 5) as well as the larger decrease of NOe in closed- than in open-chest animals. Hypercapnia can also depress NO formation: in intact, anesthetized rabbits, an increase of PaCO2 from 30 to 75 Torr decreased NOe by 30% within a few minutes (35). On the other hand, the effect of hypoxia and hypercapnia are rapidly reversible (4, 35), although in the present animals NOe remained well below control values long after return to PEEP and initial PaO2, PaCO2, and pHa values (Figs. 4 and 5). It should be noted, however, that in the present experiments hypoxia was maintained for hours instead of minutes as in the isolated rabbit lung preparation.
Changes in the volume or composition of the fluid lining the lower airways may also affect NO formation. NOe is markedly decreased in isolated pig lungs with developing interstitial edema (7) and with administration of nebulized aqueous solutions in healthy subjects (22), whereas increased hydrogen ion concentration in the lining fluid leads to an increased NOe (20). In the present animals, the acid-base balance of this fluid was essentially unaffected, since the pH of the condensate was the same under all conditions (Table 5). On the other hand, in animals ventilated at low volume, interstitial edema (Table 1) could have contributed to the fall of NOe, which might have been also due to the depressant action exerted on NO production by prostaglandins E2 and F2a, VIP, and free radicals (13, 21, 26). Indeed, damage and shedding of small airway epithelium, by exposing sensory nerve endings, fibroblasts, and collagen, can eventually cause release of tachykinins and VIP, activation of bradykinin with release of prostaglandins E2 and F2a, and formation of free radicals (28, 40), whereas alveolar epithelial cells, macrophages, and polymorphonuclear leukocytes activated by mechanical insults could represent an additional source of VIP and prostaglandins E2 and F2a (13, 23). Moreover, because bradykinin causes bronchoconstriction of mainly peripheral airways and systemic vasodilation (28), it could have contributed to the persistent increase of Rint after NEEP or ZEEP ventilation (Table 3), as well as to the significant fall of mean systemic arterial pressure on NEEP2, ZEEP2, and PEEP2 (Figs. 4 and 5). The latter effect was not related to reduced heart performance, since additional experiments in open-chest rabbits showed that, although mean systemic arterial pressure decreased progressively during the 3- to 4-h period of ventilation on ZEEP, mean pulmonary artery pressure remained essentially unchanged.
Proinflammatory cytokines.
Serum TNF- levels were essentially undetectable before and after induction of anesthesia and paralysis, peaked immediately after surgery (PEEP1), and were back to initial values after 3–4 h of ventilation both at normal or decreased lung volume (Fig. 6), consistent with the known kinetics of this cytokine (43). The time course of serum TNF- could suggest that surgery was involved in this response; but peak levels were similar in closed- and open-chest rabbits, despite more extensive surgery in the latter animals. In contrast with serum TNF- levels, those in BAL fluid, although higher with PEEP2, did not differ significantly between PEEP1 and PEEP2, nor at PEEP2 between animals undergoing prolonged ventilation at low (group Atest and Btest) and physiological end-expiratory volume (group Acontrol and Bcontrol), independent of closed or open chest (Table 6). This indicates that 1) the greater decrease of NOe production in animals subjected to prolonged ventilation at low volume (Figs. 4 and 5) cannot be related to lower TNF- levels; 2) the mechanical alterations caused by this type of ventilation (Tables 2–4), as well as histological damage of peripheral airway (11, 12), are largely independent of cytokine production; and 3) relative to ventilation at physiological end-expiratory lung volume, prolonged ventilation at low volume does not induce any significant change in TNF- production. Indeed, in most of the present rabbits, both serum and BAL fluid TNF- levels were too low to produce injury (37), in line with recent results showing that in excised mouse lungs a positive cytokine response is elicited only with NEEP of –15 cmH2O (5). Nevertheless, the higher TNF- levels in rabbits ventilated at low volume (Table 6), possibly coupled with a secondary release of IL-8 (32), may account for the greater recruitment of polymorphonuclear leukocytes in the alveolar walls observed in a previous study (12). On the other hand, it has been suggested that ventilation at low volume can increase cytokine production (6, 38); but this seems to depend mainly on the VT used. Although in excised rat lungs TNF-, IL-6, and macrophage levels were higher during ventilation with ZEEP and large VT (15 ml/kg) than with PEEP of 3 cmH2O and low VT (7 ml/kg), they were, however, similar to corresponding values with PEEP and large VT (38). Similarly, inflammatory cytokines were higher in BAL fluid from lungs ventilated with low VT on ZEEP than atelectatic lungs but similar to those in BAL fluid from lungs ventilated with low VT and PEEP of 5 cmH2O (6).
In conclusion, the present results show that MV at low volume with physiological VT causes an increase in airway resistance in both open- and closed-chest normal rabbits. In closed-chest animals, this increase is more pronounced and associated with an increase of lung elastance, likely due to interstitial edema and surfactant depletion or inactivation. Ventilation at low volume decreases NOe without affecting pH of exhaled vapor condensate and concentration of the proinflammatory cytokine TNF- in BAL fluid and serum. The decreased NOe should mainly reflect necrosis and sloughing of the epithelium of the respiratory and membranous bronchioles due to the abnormal shear stress related to their cyclic opening and closing. On the other hand, the low TNF- levels suggest that damage of small airways with ventilation at low volume is due to direct mechanical injury rather than biotrauma elicited by increased release of proinflammatory cytokines.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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a), arterial pH (pHa), arterial PCO2 (PaCO2), and arterial PO2 (PaO2) in 8 closed-chest rabbits (Group Atest; hatched bars) during ventilation with PEEP of 1.2 cmH2O before (PEEP1) and after 3–4 h of ventilation on NEEP (PEEP2), and during the initial (NEEP1) and final period (NEEP2) of ventilation on NEEP, and in 8 closed-chest rabbits (Group Acontrol; open bars) during ventilation with PEEP before (PEEP1) and after 3–4 h of ventilation on PEEP (PEEP2). During the final 3–3.5 h on NEEP, the rabbits were ventilated with 70–80% oxygen. Bars show SE. ppb, Parts/billion. *Significantly different (P < 0.05) from those on PEEP1. Number in parentheses indicate the average time (minutes) elapsed from the initial measurements on PEEP.
