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Effect of surfactant deficiency and surfactant replacement on airway patency in the piglet lung
Respiratory Physiology & Neurobiology 150 (2006) 173–181
Effect of surfactant deficiency and surfactant replacement on airway patency in the piglet lung Kevin M. Ellyett a , Patricia A. Cragg a,∗ , Roland S. Broadbent b b
a Department of Physiology, University of Otago Medical School, P.O. Box 913, Dunedin, New Zealand Department of Women’s and Children’s Health, University of Otago Medical School, P.O. Box 913 Dunedin, New Zealand
Received 21 December 2004; received in revised form 1 April 2005; accepted 1 April 2005
Abstract We investigated the effect of surfactant deficiency on airway patency and the effectiveness of surfactant replacement as either an instilled liquid bolus, a non-hygroscopic aerosol or a hygroscopic aerosol. Small airway patency was assessed in isolated piglet lungs by passing a continuous flow of gas though a cannulated airway. Occlusion was assessed by measuring increases in pressure in the cannula that resulted from airway obstruction. In surfactant-deficient conditions the amount of airway closure increased approximately three-fold. However, administration of exogenous surfactant as an instilled liquid bolus, non-hygroscopic aerosol or a hygroscopic aerosol decreased airway closure such that it was statistically similar to that recorded prior to induction of surfactant deficiency, although the instilled and hygroscopic aerosol surfactant both appeared superior to the non-hygroscopic aerosol. These experiments showed that pulmonary surfactant does have a role in maintaining airway patency and that airway closure induced by surfactant deficiency could be reduced by administration of surfactant in any of the aforementioned forms. © 2005 Elsevier B.V. All rights reserved. Keywords: Airway patency; Surfactant deficiency; Hygroscopic surfactant replacement; Piglet
1. Introduction Surfactant deficiency is one of the main causes of neonatal respiratory distress syndrome (NRDS). Impairment of lung function by surfactant deficiency is primarily due to a reduction in compliance of the alveoli. The effect of surfactant deficiency on the stability of airways has received relatively little attention although studies on isolated lungs (Macklem et al., 1970; En∗
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1569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2005.04.004
horning et al., 1995, 1996) and model airways (Kamm and Schroter, 1989; Liu et al., 1991) have shown that lung surfactant assists in maintaining distal airway patency. The forces acting on the airways, which affect airway mechanical stability, are adversely altered in surfactant-deficient conditions (Halpern and Grotberg, 1993; Wang et al., 1995; Hohlfeld et al., 1997). In addition the probability of liquid plug formation, which can occlude airways, is increased in surfactant-deficient conditions as shown in model airways by Kamm and Schroter (1989). These effects are further complicated
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by the fact that in order to re-open airways occluded by either mechanical collapse or liquid plug formation, greater pressures are required in surfactant-deficient conditions. We aimed to assess the degree to which airway closure was affected by surfactant deficiency and to what extent airway stability could be improved by the use of surfactant administration during mechanical ventilation in an experimental model that was similar in size to a premature human neonate. Surfactant is almost universally administered as an instilled liquid dose of surfactant in suspension delivered via an endotracheal tube (Rodriguez and Martin, 1999). This method, while effective, introduces excess fluid that may further impair function in a lung potentially already stressed with pulmonary oedema (Zola et al., 1993; Horbar et al., 1993). We wanted to test the effectiveness on airway patency of surfactant administered not only as a liquid but also as an aerosol. To have an effect on distal airway patency, exogenous surfactant must first traverse, with minimal retention, the upper conducting regions of the lung and be deposited in the distal regions of the lung. Conventional aerosol production and administration techniques (suitable for administration via a ventilator circuit) produce such poor deposition rates in the distal lung that aerosolized surfactant cannot be used as an economical means of surfactant administration (Gonda, 1990). On the other hand, hygroscopic (i.e. water absorbing) aerosols by evading deposition on upper airways have been shown to be effective in increasing deposition rates in distal regions of the lung (Arborelius, 1982; Hickey and Martonen, 1993). We wanted to apply this principle to surfactant aerosol administration. Thus, our aims were to determine the degree to which surfactant deficiency increases airway closure and whether surfactant delivered in aerosol form (as either a hygroscopic aerosol preparation or nonhygroscopic preparation) or as an instilled preparation was capable of improving distal airway patency. It was anticipated that instilled surfactant would be capable of restoring airway patency similar to the findings of Enhorning et al. (1995, 1996) who also used a form of instilled surfactant and a non-hygroscopic aerosol. The degree to which the aerosols would restore airway patency was unknown but it was predicted that the
hygroscopic aerosol would be more effective than the non-hygroscopic aerosol. 2. Methods We modified the method used, in the isolated rat lung, by Enhorning et al. (1995, 1996) to measure airway patency. However, we used neonatal piglet lungs, which have similar volumetric characteristics to those of premature human neonates (Rosen et al., 1993). The Enhorning method assesses the patency of airways by passing a continuous flow of gas through an airway while measuring pressure within the airway. Thus, when the airway spontaneously occludes, the pressure within it will increase until a point where the pressure is capable of re-opening the airway. Although airway closure can occur in any airway generation it is most likely that the smallest airways will be those that will close. Furthermore, although this method is not capable of determining whether collapse is due to formation of liquid plugs or mechanical collapse of terminal airways, it has the ability to measure the degree of airway closure and to indicate the effect of surfactant therapy on airway closure. 2.1. Lung preparation White Landrace piglets (n = 15) aged 5–7 days weighing 1.5–1.8 kg were initially anaesthetized in a chamber of 4% halothane in oxygen. Once sedated the animal was then transferred to a warming pad for surgery. Anaesthesia was maintained with 2.5% halothane via a mask at 2 l min−1 . A rectal probe was inserted and the heating pad adjusted so that rectal temperature was maintained at 39 ± 0.5 ◦ C. The trachea and carotid artery were cannulated and the piglet was killed by exsanguination via the carotid artery cannula. The lungs and heart were removed en bloc from the chest cavity. After the heart was freed from the lungs and the trachea clamped, the external surface of the lungs was carefully washed in warm (39 ◦ C) 0.9% NaCl solution. The lungs were suspended by attaching the tracheal cannula to an open port in the top of the experimental chamber (Fig. 1). The chamber temperature was maintained at 39 ◦ C with 100% humidity. The chamber was sealed and a negative pressure, initially of −25 cmH2 O, was applied to the chamber to inflate the lungs. This
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Fig. 1. Chamber for measurement of airway closure (the cannula for measuring pressure in an airway is shown for only one lung).
method of lung inflation was used (in preference to a fixed volume) as it limited the potential variation in lung size affecting the results. When the lungs had been fully inflated, the chamber pressure was then reduced to between −5 and −7 cmH2 O. 2.2. Airway cannulation A very fine cannula (PE 10, internal diameter 0.28 mm, external diameter 0.61 mm), referred to as the small-airway cannula, was then introduced via the tracheal cannula. This cannula had 3 mm of the final part to be inserted marked black to indicate how far it was to be later extracted (see below). The cannula was rotated as it was moved down through the airways, with minimal force applied, until it was close to the pleural surface of lower region of either the left or right main lobes. A moderate amount of force was then applied to pierce the pleura. The negative pressure in the chamber was then released and the collapsed lungs, with the small-airway cannula protruding, were removed from the chamber. Tension was then applied to the part of the cannula that protruded from the lung to draw the cannula out further through the pleura. Extraction of the cannula continued until the black marking could just be seen through the pleura
thus leaving only 3 mm of the cannula in the lung, such that the cannula was positioned approximately in airway generation 14–16 of the lung. The cannula was then cut so a length of approximately 5 mm protruded from the lung. Another short piece of polyethylene tube (PE 50, internal diameter 0.61 mm, external diameter 0.97 mm), approximately 1–2 mm longer than the length of the protruding small-airway cannula, was slid over the end of the cannula. This piece of tube had a flared flat end that provided a large surface area for adhesive glue which permitted the pressure cannula to be secured to the pulmonary pleura. This process was repeated for the second lung, so that each lung had one cannula inserted. This was necessary because during the various ventilation procedures one of the two cannulae were dislodged at some point during each experiment. The lungs were returned to the chamber and reinflated. The lungs were then ventilated via the trachea for 30 min with a ventilation frequency of 40 min−1 and a peak inspiratory pressure of 15 cmH2 O with a mechanical ventilator (BABYbird Mark 7 continuousflow infant ventilator). Ventilation was performed so that the ventilation history of the lung was the same for pre-lavage, post-lavage and post-treatment conditions. These ventilation regimens were the same as those used
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for our whole animal studies (Ellyett, 1999). After this 30-min ventilation period airway closure was assessed. 2.3. Assessment of airway closure Ventilation was stopped during airway closure assessment. The negative pressure in the chamber was reduced to zero to allow for the chamber to be opened. The small-airway cannulae were connected to fine tubes that were exteriorised (Fig. 1). The lungs were then returned to the chamber and re-inflated. The exteriorised small-airway cannulae were each attached to a ‘T’-junction. One side of the ‘T’-junction was supplied with a continuous flow of room air at 0.33 ml min−1 from an infusion pump. This flow was similar to that used by Enhorning et al. (1995, 1996) and generated an open system pressure of <0.25 cmH2 0. The other side of the ‘T’-junction was connected to a differential pressure transducer (Grass Model PT5A) and the pressure recorded at a sampling frequency of 200 Hz using a computer (MacLab ADInstruments and Macintosh LC). Once the infusion pump and pressure transducer were connected, the lung was re-inflated and the volume maintained by using a chamber pressure of −5 to −7 cmH2 O pressure. The pressure in the small-airway cannula was then recorded for a 4-min period. The initial control measurement was made at this point. (The airway cannulae were not connected during ventilation or lavage procedures as the movement of the lung could lever the cannulae off.) 2.4. Lung lavage After the control measurements, the lung was deflated and the connections to the small-airway cannulae were detached. The lungs were rendered surfactantdeficient by four double lavages with warm (39 ◦ C) 0.9% NaCl (50 ml kg−1 , original body mass). To perform the lung lavage, a tube with a reservoir attached (containing the lavage solution) was connected to the tracheal cannula. The reservoir was then raised above the level of the lung such that the filling pressure increased to, but did not exceed, 30 cmH2 O. Once the lavage fluid had entered the lung the reservoir was lowered below the level of the lung so that a maximum negative pressure of −30 cmH2 O was applied until the lavage fluid had been removed. This procedure was repeated thus performing a double lavage.
During removal of the lavage it was possible to completely collapse the lungs and, after disconnection from the lavage apparatus but prior to re-instatement of mechanical ventilation, the lungs were inverted to allow for further drainage. As a result, liquid retention was minimised. After each double lavage, the lung was reinflated and ventilated for 15 min (these double lavages interspersed with 15 min of ventilation was the protocol used for our whole animal studies (Ellyett, 1999)). After the final double lavage the lungs were re-inflated in the chamber and ventilated for 30 min using the aforementioned ventilation parameters. After this 30-min ventilation period airway closure was re-assessed for 4 min. Surfactant replacement was then given either as an instilled suspension or as one of two aerosols (n = 5 for each group). 2.5. Surfactant administration Surfactant was prepared from lung lavage collected from adult pig lungs obtained from a local abattoir. This lavage fluid underwent centrifugation (to remove cellular debris) followed by lipid extraction, which was performed to isolate the lipid soluble components of the surfactant. This produced a modified natural surfactant containing 88% phospholipid, 9.9% neutral lipids and 2.1% protein (Ellyett, 1999). The composition of this surfactant was similar to commercially available modified natural surfactant preparations (Halliday, 1996). Pulmonary surfactant was suspended in either 0.9 or 0.45% NaCl solution to produce a suspension containing 25 mg ml−1 surfactant. The surfactant preparations were administered via the trachea during 30 min of positive-pressure ventilation with the same frequency and pressures as used previously. The instilled group received surfactant boluses of 0.5 ml kg−1 (25 mg ml−1 surfactant in 0.9% NaCl solution) repeated at 1-min intervals until a total of 100 mg kg−1 had been administered, which took between 15 and 20 min. This has been shown to be an optimal dose for the administration of modified natural surfactant (Anon., 1993). Surfactant aerosol (100 mg kg−1 of a 25 mg ml−1 surfactant in 0.45% NaCl solution) was delivered as either a hygroscopic or non-hygroscopic aerosol preparation administered over a 30-min period although most of the aerosol supply was exhausted in the first 20 min. The aerosolized surfactant was produced from a surfactant suspension in 0.45% NaCl to
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ameliorate any effect of hypertonic irritation of airways in the hygroscopic group and to attempt to prevent an increase in aerosol particle size in the non-hygroscopic group. The non-hygroscopic aerosol was produced using an ultrasonic nebuliser (Devilbis Ultraneb 2000). The hygroscopic form was produced by passing the aerosol through a glass “drying” chamber, in which the aerosol was heated to 70–80 ◦ C, evaporating the water contained within the aerosol particles. This raises the water content in the air to a point where it is supersaturated when cooled to 39 ◦ C such that water will start to be absorbed by the aerosol and hence, increase aerosol particle size. Heating had no effect on the biophysical properties of the pulmonary surfactant (Ellyett, 1999). On entry to the lung the aerosol had cooled to 39 ◦ C. This method was similar to that described by us in a previous study (Ellyett et al., 1996). These aerosol preparations have been used by the experimenters previously and the hygroscopic and non-hygroscopic aerosols in the piglet were shown to have favourable distribution patterns relative to the lavaged model (Ellyett, 1999). At the completion of the surfactant administration period, ventilation was stopped and airway closure of the three treatment groups was assessed over a 4-min period. 2.6. Data analysis The mean pressures over each 4-min period were calculated for the control, post-lavage and posttreatment conditions. For each of the post-lavage and post-treatment conditions the data were normalised to the control measurement to give a ratio of airway closure (RAC). Therefore, by definition the control RAC was 1.0 and all other conditions for the individual lung were described as a ratio compared to this value. Statistical analysis was performed on the RAC and comprised a one-way analysis of variance (ANOVA). If a difference was observed significance was calculated with a Bonferroni analysis.
3. Results An example of the record of airway closure is presented in Fig. 2. This shows that even for control conditions the resistance to flow though the cannu-
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Fig. 2. Representative airway pressure trace for pre-lavage, postlavage and post-treatment conditions and mean pressures for respective conditions.
lated airway created a measurable pressure. After lavage, airway closures occurred almost continuously compared with pre-lavage conditions as shown by the increase in baseline and intermittent increases in pressure. Subsequent surfactant treatments practically eliminated airway closures although in most cases (and in the one presented) the mean airway pressure was increased from that of the original. The mean pressure for pre-lavage conditions ranged from 0.8 to 19.9 cmH2 O whereas post-lavage pressures ranged from 2.4 to 40.6 cmH2 O. The large range in pressures reflects the fact that the airway generation in which the cannula was positioned was not consistent. This principle whereby airways of varying calibre require differing opening pressures has previously been described by Gaver et al. (1990). However, the relative changes in pressure (with respect to each cannulation) were consistent. Pooled data, from all experiments (n = 15), shows that the RAC from the baseline of 1.0 increased to 2.9 ± 0.66 (mean ± S.D.) (P < 0.01) after lavage. Administration of surfactant reduced the amount of closure in all three groups irrespective of the degree to which airway closure had been increased. The degree to which airway closure was reduced varied between treatment groups. Surfactant treatment reduced (P < 0.01) RAC from 2.9 ± 0.6, 2.8 ± 0.7 and 2.8 ± 0.8 post-lavage to 0.8 ± 0.6, 1.8 ± 0.6 and 0.9 ± 0.5 for the instilled, non-hygroscopic aerosol and the hygroscopic aerosol group, respectively (Fig. 3). These results were also compared to pre-lavage conditions and there was no significant difference between pre-lavage
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Fig. 3. Ratio (mean ± S.D.) of airway closure pre-lavage, postlavage and post-surfactant treatment. Post-lavage ratios significantly different (P < 0.01) to pre-lavage; post-surfactant treatment not significantly different (P > 0.1) to pre-lavage; no significant difference between three types of surfactant treatment (P > 0.1).
and post-treatment conditions (P > 0.1). Although the hygroscopic aerosol and the instilled surfactant appeared to restore airway patency completely while the non-hygroscopic aerosol did not do so to the same degree (Fig. 3), there was no statistical difference between the three methods of administering surfactant (P > 0.1).
4. Discussion 4.1. Methodology Enhorning et al. (1995, 1996) assessed airway closure in the rat as the time the pressure in the cannula was greater than zero over a 4-min period; in the piglet we saw no isolated discrete airway closures and the pressure in the cannula remained above 0 cmH2 O for most of the experiment. However, there were continuous and discontinuous peaks throughout the 4-min period for all test conditions and the peaks increased in amplitude post-lavage and subsided post-surfactant administration (Fig. 2). The changes in peak amplitude were highly variable and illustrate the fact that increased pressures were required to re-open the airway. An explanation for these differences in pressure events could be based on the animal species and/or the modifications to the Enhorning method we had to introduce. Although our method was technically similar, there were four major differences re-
quired to apply the method to the ventilated piglet lung. One major difference was the chamber in which the piglet lungs were held was air-filled, not salinefilled as in Enhorning’s rat method. This change was made for a number of reasons. The necessity to ventilate the lungs required a medium surrounding the lungs that would permit the lungs to inflate and deflate. The length of the piglet lung (>10 cm) was such that if suspended in saline there would be a significant pressure gradient from the base to the apex—a problem that would not be encountered with the shorter rat lung. The buoyancy effect of the lungs was also of concern. The buoyancy of the piglet lung would put significant tension on the trachea and apex of the lung and, in addition to this, the buoyancy would tend to cause the apices of the lung to bend upwards deforming these regions and potentially altering the ventilation of the lungs. On the other hand, the buoyancy effect could have increased airway calibre in the rat because airway parenchymal tethering would be increased, and this may have been responsible for the difference in pressure events between the two species. In addition, as the lungs were orientated vertically and the cannula were at the base of the lung, there was a greater likelihood that gravity would move liquid towards the airway and hence, increase the probability of it occluding. The second major difference was the method used to induce surfactant deficiency. Enhorning et al. (1995, 1996) performed the saline lavage by passing a volume of 5–10 ml of saline through the isolated airway (i.e. flushing it) with the lung held at a static volume and thus only the airway that was cannulated was rendered surfactant-deficient. Our method induced surfactant deficiency by repeated lung lavage as this had proven to be the suitable method for the in vivo piglet lung (Ellyett, 1999). This produces a lung injury which is reproducable and in which aerosol surfactant distribution has proven to be uniform. Thirdly, the method of surfactant delivery was also dissimilar. Enhorning et al. delivered the surfactant by either flushing 1 ml of a surfactant suspension though the cannula or passing a surfactant aerosol through the cannula (i.e. from the distal airway towards the trachea not in the direction in which surfactant is usually administered). The final major difference was that we ventilated the lung prior to each assessment of airway closure.
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4.2. Airway closure Despite the differences in the methodology, both Enhorning et al.’s and our experiments showed that airway patency was severely affected by surfactant deficiency and airway patency could be restored with surfactant replacement therapy. Our data support the findings of others and confirm that the pulmonary surfactant system aids in maintaining small airway patency (Macklem et al., 1970; Enhorning et al., 1995, 1996). There are two potential mechanisms for airway closure: first structural collapse of the airway (compliant collapse), and secondly airway closure due to the formation of liquid plugs (liquid film instability). Surfactant reduces the occurrence of both these phenomena by reducing the surface tension of the liquid lining of both the alveoli and airways. The structural integrity of the airway wall, the surface tension of the liquid lining of the airway, radial traction on the airway from surrounding alveoli and the transmural pressure all influence mechanical stability of airways (Halpern and Grotberg, 1993; Wang et al., 1995; Hohlfeld et al., 1997). The factors that affect the liquid film stability are the thickness of the liquid film, relative to the airway diameter, and the surface tension of the film. Patency and stability of peripheral airways is a major determinant of airway resistance and functional residual capacity (FRC) (Kamm and Schroter, 1989; Liu et al., 1991; Enhorning et al., 1995, 1996). The mechanical forces which usually hold airways open vary in their contribution to airway closure depending on the location of the airway. In noncartilaginous airways the integrity of the airway lumen is maintained mainly by radial traction coming from the lung parenchyma and the tethering effect of the fibrous constituents of the parenchyma communicating directly with the lung periphery (Yap et al., 1994; Perun and Gaver, 1995). These forces are directly proportional to the transpulmonary pressure and thus, when the lung reduces its volume below FRC and transpulmonary pressure increases above zero, distal airways will tend to collapse. However, this collapse usually does not occur because, when the lung volume drops, radial traction reduction is disproportional to airway instability and therefore airway patency is maintained. Liquid film collapse can also lead to airway closure. Under normal conditions collapse of distal air-
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ways and their subsequent re-opening will result in the brief formation of liquid plugs which rapidly disperse with normal ventilatory pressures (Naureckas et al., 1994). However, the tendency for airways to collapse, and a subsequent difficulty for them to reopen, occurs in conditions where the surface tension and lung fluid volume have increased. The dysfunction of surfactant induced by albumin has been demonstrated to reduce airway patency due to the liquid forming plugs (Enhorning and Holm, 1993). The formation and/or longevity of liquid plugs can also be reduced by the reduction in surface tension (Otis et al., 1993). Therefore, the lowering of surface tension can reduce airway closure and hence, gas trapping. Our airway closure study was unable to distinguish between airway collapse and/or formation of liquid plugs. An increase in lung water content, as a result of the lavage and surfactant administration, would be expected. As the lavage procedures were identical in each group the only difference in water content would be due to the surfactant administration technique and therefore, it was predicted that the instilled group would retain the largest amount of water. Due to this increased liquid content in the lung, one would expect that the liquid film volume would also have increased and thus, formation of liquid plugs would be more likely. However, this was not observed in the instilled group as, in spite of the increase in lung water, airway closure was restored to normal. It is proposed that airway closure was reduced by all three forms of surfactant by either prevention of airway collapse and/or the formation of liquid plugs (Halpern and Grotberg, 1993; Otis et al., 1993; Hohlfeld et al., 1997; Halpern et al., 1998). Our results indicate that the hygroscopic aerosol surfactant was capable of reducing airway closure to the same extent as the instilled surfactant. The nonhygroscopic aerosol surfactant did not reduce the airway closure to the same extent as the hygroscopic aerosol but this difference was not statistically significant. Thus, irrespective of the mechanism of airway closure, all three surfactant replacement therapies were capable of restoring airway patency. 4.3. Conclusions Although pulmonary surfactant has been implicated in the maintenance of airway patency, since 1970
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(Macklem et al., 1970) the degree to which surfactant affects airways has remained relatively unresolved. Disruption of the normal function of the surfactant system has been implicated in obstructive conditions such as asthma, bronchiolitis, chronic obstructive pulmonary disease and lung transplantation (Griese, 1999). Indeed impairment of the surfactant system has been implicated in allergen-induced airway inflammation (Jarjour and Enhorning, 1999). These findings suggest that surfactant therapy has the potential to be extended to conditions in which distal airway closure is evident, or likely to occur, due to surfactant dysfunction. In summary, we have shown the importance of surfactant in the maintenance of the patency of small airways and that exogenous surfactant both as an aerosol and as an instilled suspension has the ability to reduce airway collapse in surfactant-deficient lungs. The aerosol method of surfactant administration should be considered when treating surfactantdeficient conditions. In addition to surfactant being used as a treatment for primarily surfactant-deficient conditions such as NRDS, surfactant therapy should be considered in certain pathological conditions in which surfactant dysfunction may further compromise the patency of peripheral airways.
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Effect of surfactant deficiency and surfactant replacement on airway patency in the piglet lung Kevin M. Ellyett a , Patricia A. Cragg a,∗ , Roland S. Broadbent b b
a Department of Physiology, University of Otago Medical School, P.O. Box 913, Dunedin, New Zealand Department of Women’s and Children’s Health, University of Otago Medical School, P.O. Box 913 Dunedin, New Zealand
Received 21 December 2004; received in revised form 1 April 2005; accepted 1 April 2005
Abstract We investigated the effect of surfactant deficiency on airway patency and the effectiveness of surfactant replacement as either an instilled liquid bolus, a non-hygroscopic aerosol or a hygroscopic aerosol. Small airway patency was assessed in isolated piglet lungs by passing a continuous flow of gas though a cannulated airway. Occlusion was assessed by measuring increases in pressure in the cannula that resulted from airway obstruction. In surfactant-deficient conditions the amount of airway closure increased approximately three-fold. However, administration of exogenous surfactant as an instilled liquid bolus, non-hygroscopic aerosol or a hygroscopic aerosol decreased airway closure such that it was statistically similar to that recorded prior to induction of surfactant deficiency, although the instilled and hygroscopic aerosol surfactant both appeared superior to the non-hygroscopic aerosol. These experiments showed that pulmonary surfactant does have a role in maintaining airway patency and that airway closure induced by surfactant deficiency could be reduced by administration of surfactant in any of the aforementioned forms. © 2005 Elsevier B.V. All rights reserved. Keywords: Airway patency; Surfactant deficiency; Hygroscopic surfactant replacement; Piglet
1. Introduction Surfactant deficiency is one of the main causes of neonatal respiratory distress syndrome (NRDS). Impairment of lung function by surfactant deficiency is primarily due to a reduction in compliance of the alveoli. The effect of surfactant deficiency on the stability of airways has received relatively little attention although studies on isolated lungs (Macklem et al., 1970; En∗
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horning et al., 1995, 1996) and model airways (Kamm and Schroter, 1989; Liu et al., 1991) have shown that lung surfactant assists in maintaining distal airway patency. The forces acting on the airways, which affect airway mechanical stability, are adversely altered in surfactant-deficient conditions (Halpern and Grotberg, 1993; Wang et al., 1995; Hohlfeld et al., 1997). In addition the probability of liquid plug formation, which can occlude airways, is increased in surfactant-deficient conditions as shown in model airways by Kamm and Schroter (1989). These effects are further complicated
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by the fact that in order to re-open airways occluded by either mechanical collapse or liquid plug formation, greater pressures are required in surfactant-deficient conditions. We aimed to assess the degree to which airway closure was affected by surfactant deficiency and to what extent airway stability could be improved by the use of surfactant administration during mechanical ventilation in an experimental model that was similar in size to a premature human neonate. Surfactant is almost universally administered as an instilled liquid dose of surfactant in suspension delivered via an endotracheal tube (Rodriguez and Martin, 1999). This method, while effective, introduces excess fluid that may further impair function in a lung potentially already stressed with pulmonary oedema (Zola et al., 1993; Horbar et al., 1993). We wanted to test the effectiveness on airway patency of surfactant administered not only as a liquid but also as an aerosol. To have an effect on distal airway patency, exogenous surfactant must first traverse, with minimal retention, the upper conducting regions of the lung and be deposited in the distal regions of the lung. Conventional aerosol production and administration techniques (suitable for administration via a ventilator circuit) produce such poor deposition rates in the distal lung that aerosolized surfactant cannot be used as an economical means of surfactant administration (Gonda, 1990). On the other hand, hygroscopic (i.e. water absorbing) aerosols by evading deposition on upper airways have been shown to be effective in increasing deposition rates in distal regions of the lung (Arborelius, 1982; Hickey and Martonen, 1993). We wanted to apply this principle to surfactant aerosol administration. Thus, our aims were to determine the degree to which surfactant deficiency increases airway closure and whether surfactant delivered in aerosol form (as either a hygroscopic aerosol preparation or nonhygroscopic preparation) or as an instilled preparation was capable of improving distal airway patency. It was anticipated that instilled surfactant would be capable of restoring airway patency similar to the findings of Enhorning et al. (1995, 1996) who also used a form of instilled surfactant and a non-hygroscopic aerosol. The degree to which the aerosols would restore airway patency was unknown but it was predicted that the
hygroscopic aerosol would be more effective than the non-hygroscopic aerosol. 2. Methods We modified the method used, in the isolated rat lung, by Enhorning et al. (1995, 1996) to measure airway patency. However, we used neonatal piglet lungs, which have similar volumetric characteristics to those of premature human neonates (Rosen et al., 1993). The Enhorning method assesses the patency of airways by passing a continuous flow of gas through an airway while measuring pressure within the airway. Thus, when the airway spontaneously occludes, the pressure within it will increase until a point where the pressure is capable of re-opening the airway. Although airway closure can occur in any airway generation it is most likely that the smallest airways will be those that will close. Furthermore, although this method is not capable of determining whether collapse is due to formation of liquid plugs or mechanical collapse of terminal airways, it has the ability to measure the degree of airway closure and to indicate the effect of surfactant therapy on airway closure. 2.1. Lung preparation White Landrace piglets (n = 15) aged 5–7 days weighing 1.5–1.8 kg were initially anaesthetized in a chamber of 4% halothane in oxygen. Once sedated the animal was then transferred to a warming pad for surgery. Anaesthesia was maintained with 2.5% halothane via a mask at 2 l min−1 . A rectal probe was inserted and the heating pad adjusted so that rectal temperature was maintained at 39 ± 0.5 ◦ C. The trachea and carotid artery were cannulated and the piglet was killed by exsanguination via the carotid artery cannula. The lungs and heart were removed en bloc from the chest cavity. After the heart was freed from the lungs and the trachea clamped, the external surface of the lungs was carefully washed in warm (39 ◦ C) 0.9% NaCl solution. The lungs were suspended by attaching the tracheal cannula to an open port in the top of the experimental chamber (Fig. 1). The chamber temperature was maintained at 39 ◦ C with 100% humidity. The chamber was sealed and a negative pressure, initially of −25 cmH2 O, was applied to the chamber to inflate the lungs. This
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Fig. 1. Chamber for measurement of airway closure (the cannula for measuring pressure in an airway is shown for only one lung).
method of lung inflation was used (in preference to a fixed volume) as it limited the potential variation in lung size affecting the results. When the lungs had been fully inflated, the chamber pressure was then reduced to between −5 and −7 cmH2 O. 2.2. Airway cannulation A very fine cannula (PE 10, internal diameter 0.28 mm, external diameter 0.61 mm), referred to as the small-airway cannula, was then introduced via the tracheal cannula. This cannula had 3 mm of the final part to be inserted marked black to indicate how far it was to be later extracted (see below). The cannula was rotated as it was moved down through the airways, with minimal force applied, until it was close to the pleural surface of lower region of either the left or right main lobes. A moderate amount of force was then applied to pierce the pleura. The negative pressure in the chamber was then released and the collapsed lungs, with the small-airway cannula protruding, were removed from the chamber. Tension was then applied to the part of the cannula that protruded from the lung to draw the cannula out further through the pleura. Extraction of the cannula continued until the black marking could just be seen through the pleura
thus leaving only 3 mm of the cannula in the lung, such that the cannula was positioned approximately in airway generation 14–16 of the lung. The cannula was then cut so a length of approximately 5 mm protruded from the lung. Another short piece of polyethylene tube (PE 50, internal diameter 0.61 mm, external diameter 0.97 mm), approximately 1–2 mm longer than the length of the protruding small-airway cannula, was slid over the end of the cannula. This piece of tube had a flared flat end that provided a large surface area for adhesive glue which permitted the pressure cannula to be secured to the pulmonary pleura. This process was repeated for the second lung, so that each lung had one cannula inserted. This was necessary because during the various ventilation procedures one of the two cannulae were dislodged at some point during each experiment. The lungs were returned to the chamber and reinflated. The lungs were then ventilated via the trachea for 30 min with a ventilation frequency of 40 min−1 and a peak inspiratory pressure of 15 cmH2 O with a mechanical ventilator (BABYbird Mark 7 continuousflow infant ventilator). Ventilation was performed so that the ventilation history of the lung was the same for pre-lavage, post-lavage and post-treatment conditions. These ventilation regimens were the same as those used
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for our whole animal studies (Ellyett, 1999). After this 30-min ventilation period airway closure was assessed. 2.3. Assessment of airway closure Ventilation was stopped during airway closure assessment. The negative pressure in the chamber was reduced to zero to allow for the chamber to be opened. The small-airway cannulae were connected to fine tubes that were exteriorised (Fig. 1). The lungs were then returned to the chamber and re-inflated. The exteriorised small-airway cannulae were each attached to a ‘T’-junction. One side of the ‘T’-junction was supplied with a continuous flow of room air at 0.33 ml min−1 from an infusion pump. This flow was similar to that used by Enhorning et al. (1995, 1996) and generated an open system pressure of <0.25 cmH2 0. The other side of the ‘T’-junction was connected to a differential pressure transducer (Grass Model PT5A) and the pressure recorded at a sampling frequency of 200 Hz using a computer (MacLab ADInstruments and Macintosh LC). Once the infusion pump and pressure transducer were connected, the lung was re-inflated and the volume maintained by using a chamber pressure of −5 to −7 cmH2 O pressure. The pressure in the small-airway cannula was then recorded for a 4-min period. The initial control measurement was made at this point. (The airway cannulae were not connected during ventilation or lavage procedures as the movement of the lung could lever the cannulae off.) 2.4. Lung lavage After the control measurements, the lung was deflated and the connections to the small-airway cannulae were detached. The lungs were rendered surfactantdeficient by four double lavages with warm (39 ◦ C) 0.9% NaCl (50 ml kg−1 , original body mass). To perform the lung lavage, a tube with a reservoir attached (containing the lavage solution) was connected to the tracheal cannula. The reservoir was then raised above the level of the lung such that the filling pressure increased to, but did not exceed, 30 cmH2 O. Once the lavage fluid had entered the lung the reservoir was lowered below the level of the lung so that a maximum negative pressure of −30 cmH2 O was applied until the lavage fluid had been removed. This procedure was repeated thus performing a double lavage.
During removal of the lavage it was possible to completely collapse the lungs and, after disconnection from the lavage apparatus but prior to re-instatement of mechanical ventilation, the lungs were inverted to allow for further drainage. As a result, liquid retention was minimised. After each double lavage, the lung was reinflated and ventilated for 15 min (these double lavages interspersed with 15 min of ventilation was the protocol used for our whole animal studies (Ellyett, 1999)). After the final double lavage the lungs were re-inflated in the chamber and ventilated for 30 min using the aforementioned ventilation parameters. After this 30-min ventilation period airway closure was re-assessed for 4 min. Surfactant replacement was then given either as an instilled suspension or as one of two aerosols (n = 5 for each group). 2.5. Surfactant administration Surfactant was prepared from lung lavage collected from adult pig lungs obtained from a local abattoir. This lavage fluid underwent centrifugation (to remove cellular debris) followed by lipid extraction, which was performed to isolate the lipid soluble components of the surfactant. This produced a modified natural surfactant containing 88% phospholipid, 9.9% neutral lipids and 2.1% protein (Ellyett, 1999). The composition of this surfactant was similar to commercially available modified natural surfactant preparations (Halliday, 1996). Pulmonary surfactant was suspended in either 0.9 or 0.45% NaCl solution to produce a suspension containing 25 mg ml−1 surfactant. The surfactant preparations were administered via the trachea during 30 min of positive-pressure ventilation with the same frequency and pressures as used previously. The instilled group received surfactant boluses of 0.5 ml kg−1 (25 mg ml−1 surfactant in 0.9% NaCl solution) repeated at 1-min intervals until a total of 100 mg kg−1 had been administered, which took between 15 and 20 min. This has been shown to be an optimal dose for the administration of modified natural surfactant (Anon., 1993). Surfactant aerosol (100 mg kg−1 of a 25 mg ml−1 surfactant in 0.45% NaCl solution) was delivered as either a hygroscopic or non-hygroscopic aerosol preparation administered over a 30-min period although most of the aerosol supply was exhausted in the first 20 min. The aerosolized surfactant was produced from a surfactant suspension in 0.45% NaCl to
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ameliorate any effect of hypertonic irritation of airways in the hygroscopic group and to attempt to prevent an increase in aerosol particle size in the non-hygroscopic group. The non-hygroscopic aerosol was produced using an ultrasonic nebuliser (Devilbis Ultraneb 2000). The hygroscopic form was produced by passing the aerosol through a glass “drying” chamber, in which the aerosol was heated to 70–80 ◦ C, evaporating the water contained within the aerosol particles. This raises the water content in the air to a point where it is supersaturated when cooled to 39 ◦ C such that water will start to be absorbed by the aerosol and hence, increase aerosol particle size. Heating had no effect on the biophysical properties of the pulmonary surfactant (Ellyett, 1999). On entry to the lung the aerosol had cooled to 39 ◦ C. This method was similar to that described by us in a previous study (Ellyett et al., 1996). These aerosol preparations have been used by the experimenters previously and the hygroscopic and non-hygroscopic aerosols in the piglet were shown to have favourable distribution patterns relative to the lavaged model (Ellyett, 1999). At the completion of the surfactant administration period, ventilation was stopped and airway closure of the three treatment groups was assessed over a 4-min period. 2.6. Data analysis The mean pressures over each 4-min period were calculated for the control, post-lavage and posttreatment conditions. For each of the post-lavage and post-treatment conditions the data were normalised to the control measurement to give a ratio of airway closure (RAC). Therefore, by definition the control RAC was 1.0 and all other conditions for the individual lung were described as a ratio compared to this value. Statistical analysis was performed on the RAC and comprised a one-way analysis of variance (ANOVA). If a difference was observed significance was calculated with a Bonferroni analysis.
3. Results An example of the record of airway closure is presented in Fig. 2. This shows that even for control conditions the resistance to flow though the cannu-
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Fig. 2. Representative airway pressure trace for pre-lavage, postlavage and post-treatment conditions and mean pressures for respective conditions.
lated airway created a measurable pressure. After lavage, airway closures occurred almost continuously compared with pre-lavage conditions as shown by the increase in baseline and intermittent increases in pressure. Subsequent surfactant treatments practically eliminated airway closures although in most cases (and in the one presented) the mean airway pressure was increased from that of the original. The mean pressure for pre-lavage conditions ranged from 0.8 to 19.9 cmH2 O whereas post-lavage pressures ranged from 2.4 to 40.6 cmH2 O. The large range in pressures reflects the fact that the airway generation in which the cannula was positioned was not consistent. This principle whereby airways of varying calibre require differing opening pressures has previously been described by Gaver et al. (1990). However, the relative changes in pressure (with respect to each cannulation) were consistent. Pooled data, from all experiments (n = 15), shows that the RAC from the baseline of 1.0 increased to 2.9 ± 0.66 (mean ± S.D.) (P < 0.01) after lavage. Administration of surfactant reduced the amount of closure in all three groups irrespective of the degree to which airway closure had been increased. The degree to which airway closure was reduced varied between treatment groups. Surfactant treatment reduced (P < 0.01) RAC from 2.9 ± 0.6, 2.8 ± 0.7 and 2.8 ± 0.8 post-lavage to 0.8 ± 0.6, 1.8 ± 0.6 and 0.9 ± 0.5 for the instilled, non-hygroscopic aerosol and the hygroscopic aerosol group, respectively (Fig. 3). These results were also compared to pre-lavage conditions and there was no significant difference between pre-lavage
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Fig. 3. Ratio (mean ± S.D.) of airway closure pre-lavage, postlavage and post-surfactant treatment. Post-lavage ratios significantly different (P < 0.01) to pre-lavage; post-surfactant treatment not significantly different (P > 0.1) to pre-lavage; no significant difference between three types of surfactant treatment (P > 0.1).
and post-treatment conditions (P > 0.1). Although the hygroscopic aerosol and the instilled surfactant appeared to restore airway patency completely while the non-hygroscopic aerosol did not do so to the same degree (Fig. 3), there was no statistical difference between the three methods of administering surfactant (P > 0.1).
4. Discussion 4.1. Methodology Enhorning et al. (1995, 1996) assessed airway closure in the rat as the time the pressure in the cannula was greater than zero over a 4-min period; in the piglet we saw no isolated discrete airway closures and the pressure in the cannula remained above 0 cmH2 O for most of the experiment. However, there were continuous and discontinuous peaks throughout the 4-min period for all test conditions and the peaks increased in amplitude post-lavage and subsided post-surfactant administration (Fig. 2). The changes in peak amplitude were highly variable and illustrate the fact that increased pressures were required to re-open the airway. An explanation for these differences in pressure events could be based on the animal species and/or the modifications to the Enhorning method we had to introduce. Although our method was technically similar, there were four major differences re-
quired to apply the method to the ventilated piglet lung. One major difference was the chamber in which the piglet lungs were held was air-filled, not salinefilled as in Enhorning’s rat method. This change was made for a number of reasons. The necessity to ventilate the lungs required a medium surrounding the lungs that would permit the lungs to inflate and deflate. The length of the piglet lung (>10 cm) was such that if suspended in saline there would be a significant pressure gradient from the base to the apex—a problem that would not be encountered with the shorter rat lung. The buoyancy effect of the lungs was also of concern. The buoyancy of the piglet lung would put significant tension on the trachea and apex of the lung and, in addition to this, the buoyancy would tend to cause the apices of the lung to bend upwards deforming these regions and potentially altering the ventilation of the lungs. On the other hand, the buoyancy effect could have increased airway calibre in the rat because airway parenchymal tethering would be increased, and this may have been responsible for the difference in pressure events between the two species. In addition, as the lungs were orientated vertically and the cannula were at the base of the lung, there was a greater likelihood that gravity would move liquid towards the airway and hence, increase the probability of it occluding. The second major difference was the method used to induce surfactant deficiency. Enhorning et al. (1995, 1996) performed the saline lavage by passing a volume of 5–10 ml of saline through the isolated airway (i.e. flushing it) with the lung held at a static volume and thus only the airway that was cannulated was rendered surfactant-deficient. Our method induced surfactant deficiency by repeated lung lavage as this had proven to be the suitable method for the in vivo piglet lung (Ellyett, 1999). This produces a lung injury which is reproducable and in which aerosol surfactant distribution has proven to be uniform. Thirdly, the method of surfactant delivery was also dissimilar. Enhorning et al. delivered the surfactant by either flushing 1 ml of a surfactant suspension though the cannula or passing a surfactant aerosol through the cannula (i.e. from the distal airway towards the trachea not in the direction in which surfactant is usually administered). The final major difference was that we ventilated the lung prior to each assessment of airway closure.
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4.2. Airway closure Despite the differences in the methodology, both Enhorning et al.’s and our experiments showed that airway patency was severely affected by surfactant deficiency and airway patency could be restored with surfactant replacement therapy. Our data support the findings of others and confirm that the pulmonary surfactant system aids in maintaining small airway patency (Macklem et al., 1970; Enhorning et al., 1995, 1996). There are two potential mechanisms for airway closure: first structural collapse of the airway (compliant collapse), and secondly airway closure due to the formation of liquid plugs (liquid film instability). Surfactant reduces the occurrence of both these phenomena by reducing the surface tension of the liquid lining of both the alveoli and airways. The structural integrity of the airway wall, the surface tension of the liquid lining of the airway, radial traction on the airway from surrounding alveoli and the transmural pressure all influence mechanical stability of airways (Halpern and Grotberg, 1993; Wang et al., 1995; Hohlfeld et al., 1997). The factors that affect the liquid film stability are the thickness of the liquid film, relative to the airway diameter, and the surface tension of the film. Patency and stability of peripheral airways is a major determinant of airway resistance and functional residual capacity (FRC) (Kamm and Schroter, 1989; Liu et al., 1991; Enhorning et al., 1995, 1996). The mechanical forces which usually hold airways open vary in their contribution to airway closure depending on the location of the airway. In noncartilaginous airways the integrity of the airway lumen is maintained mainly by radial traction coming from the lung parenchyma and the tethering effect of the fibrous constituents of the parenchyma communicating directly with the lung periphery (Yap et al., 1994; Perun and Gaver, 1995). These forces are directly proportional to the transpulmonary pressure and thus, when the lung reduces its volume below FRC and transpulmonary pressure increases above zero, distal airways will tend to collapse. However, this collapse usually does not occur because, when the lung volume drops, radial traction reduction is disproportional to airway instability and therefore airway patency is maintained. Liquid film collapse can also lead to airway closure. Under normal conditions collapse of distal air-
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ways and their subsequent re-opening will result in the brief formation of liquid plugs which rapidly disperse with normal ventilatory pressures (Naureckas et al., 1994). However, the tendency for airways to collapse, and a subsequent difficulty for them to reopen, occurs in conditions where the surface tension and lung fluid volume have increased. The dysfunction of surfactant induced by albumin has been demonstrated to reduce airway patency due to the liquid forming plugs (Enhorning and Holm, 1993). The formation and/or longevity of liquid plugs can also be reduced by the reduction in surface tension (Otis et al., 1993). Therefore, the lowering of surface tension can reduce airway closure and hence, gas trapping. Our airway closure study was unable to distinguish between airway collapse and/or formation of liquid plugs. An increase in lung water content, as a result of the lavage and surfactant administration, would be expected. As the lavage procedures were identical in each group the only difference in water content would be due to the surfactant administration technique and therefore, it was predicted that the instilled group would retain the largest amount of water. Due to this increased liquid content in the lung, one would expect that the liquid film volume would also have increased and thus, formation of liquid plugs would be more likely. However, this was not observed in the instilled group as, in spite of the increase in lung water, airway closure was restored to normal. It is proposed that airway closure was reduced by all three forms of surfactant by either prevention of airway collapse and/or the formation of liquid plugs (Halpern and Grotberg, 1993; Otis et al., 1993; Hohlfeld et al., 1997; Halpern et al., 1998). Our results indicate that the hygroscopic aerosol surfactant was capable of reducing airway closure to the same extent as the instilled surfactant. The nonhygroscopic aerosol surfactant did not reduce the airway closure to the same extent as the hygroscopic aerosol but this difference was not statistically significant. Thus, irrespective of the mechanism of airway closure, all three surfactant replacement therapies were capable of restoring airway patency. 4.3. Conclusions Although pulmonary surfactant has been implicated in the maintenance of airway patency, since 1970
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(Macklem et al., 1970) the degree to which surfactant affects airways has remained relatively unresolved. Disruption of the normal function of the surfactant system has been implicated in obstructive conditions such as asthma, bronchiolitis, chronic obstructive pulmonary disease and lung transplantation (Griese, 1999). Indeed impairment of the surfactant system has been implicated in allergen-induced airway inflammation (Jarjour and Enhorning, 1999). These findings suggest that surfactant therapy has the potential to be extended to conditions in which distal airway closure is evident, or likely to occur, due to surfactant dysfunction. In summary, we have shown the importance of surfactant in the maintenance of the patency of small airways and that exogenous surfactant both as an aerosol and as an instilled suspension has the ability to reduce airway collapse in surfactant-deficient lungs. The aerosol method of surfactant administration should be considered when treating surfactantdeficient conditions. In addition to surfactant being used as a treatment for primarily surfactant-deficient conditions such as NRDS, surfactant therapy should be considered in certain pathological conditions in which surfactant dysfunction may further compromise the patency of peripheral airways.
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