Respiratory Physiology & Neurobiology 210 (2015) 7–13
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
The effect of exogenous surfactant on alveolar interdependence Caterina Salito a,∗ , Andrea Aliverti a , Enrico Mazzuca a , Ilaria Rivolta b , Giuseppe Miserocchi b a b
Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano, Italy Dipartimento di Scienze della Salute, Università di Milano Bicocca, Milano, Italy
a r t i c l e
i n f o
Article history: Accepted 12 January 2015 Available online 17 January 2015 Keywords: Surfactant Alveolar mechanics In-vivo microscopy imaging
a b s t r a c t To investigate the nature of alveolar mechanical interdependence, we purposefully disturbed the equilibrium condition by administering exogenous surfactant in physiological non-surfactant deprived conditions. Changes in alveolar morphology induced by intra-tracheal delivery of CUROSURF were evaluated after opening a pleural window allowing in-vivo microscopic imaging of sub-pleural alveoli in 6 male anesthetized, tracheotomized and mechanically ventilated rabbits. Surfactant instillation increased the surface area of alveoli smaller than 20,000 m2 up to ∼50% at 15 min after instillation, reflecting a lowering of surface tension due to local surfactant enrichment. Conversely, for alveoli greater than 20,000 m2 , surface area decreased by ∼5%. Opposite changes in alveolar surface are interpreted as reflecting a new inter-alveolar mechanical equilibrium modified by local surfactant distribution and by a decrease in lung distending pressure. We propose that smaller alveoli, representing the majority of alveolar population, might mostly contribute to improve the oxygenation index following surfactant replacement therapy in case of surfactant deficiency. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recent studies from our group (Mazzuca et al., 2014; Salito et al., 2014) provided evidence of the existence of considerable geometrical heterogeneity in alveolar geometry and size that impact on the inter-alveolar differences in mechanical behavior that, in turn reflects the balance between elastic and surface forces (Gil et al., 1979; Tabuchi et al., 2008). The inter-alveolar differences in mechanical properties ought to be framed within a model of mechanically inter-dependent units (Bates and Suki, 2008). Although local elastic distending pressure can be evaluated under static conditions, alveolar surface tension may vary due the thickness of the surfactant layer that depends upon alveolar surface and inter-alveolar surface tension gradients sustaining the Marangoni flow. Previous data suggest that local changes in surface forces appear more important than changes in elastic forces during alveolar expansion in the volume range 40–60% TLC (Tabuchi et al., 2008; Wilson, 1982). Aiming to investigate the nature of the alveolar interdependence when potential inter-alveolar mechanical differences are present,
∗ Corresponding author. Tel.: +39 02 2399 9026x9001; fax: +39 02 2399 9000. E-mail address: [email protected] (C. Salito). http://dx.doi.org/10.1016/j.resp.2015.01.009 1569-9048/© 2015 Elsevier B.V. All rights reserved.
we purposefully disturbed the equilibrium condition by administering exogenous surfactant that causes a sudden decrease in surface tension in alveoli being reached by surfactant. 2. Methods 2.1. Animal preparation A general consensus for the experimental procedures used in our research activity was obtained from the Local Ethical Committee. Experiments were performed on 6 adult (New Zealand White) male rabbits (weight range 1–1.5 kg) anesthetized with a bolus of 2.5 ml/kg of a saline solution containing 0.25 g/ml of urethane injected into an ear vein. To maintain a constant depth of anesthesia throughout the procedure subsequent doses of 0.5 ml of anesthetic were administered every 20 min: this anesthesia strategy proved effective to maintain the absence of the corneal reflex between successive administrations. Tracheostomy was performed, and a plastic cannula (length 50 mm, inner diameter 2.5 mm) was inserted into the distal trachea. Before connecting the animal to the ventilator, paralysis was accomplished by pancuronium bromide (1 mg/kg body weight initial dose, supplemented by 0.33 mg/kg every 40 min). Mechanical ventilation was provided with a tidal volume of ∼20 ml and a frequency of 16 breaths/min
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C. Salito et al. / Respiratory Physiology & Neurobiology 210 (2015) 7–13
that we previously used in rabbits to guarantee an end-tidal PCO2 of ∼36 mmHg (Miserocchi et al., 2001). Tracheal and oesophageal (through oesophageal balloon) pressures were measured by disposable pressure transducers (Edwards Baxter, Irvine, CA, USA). All signals were digitized by an analogue-to-digital board (NI BNC-2090, National Instruments, Austin, TX, USA) and dedicated software for real-time signal acquisition and visualization was implemented in LabView (version 8.2, National Instruments) and for the signal processing in Matlab (The Mathworks, Inc., Natick, MA, USA). An “intact pleural window” was prepared in the sixth intercostal space to allow a view of the lower lobe where the cardiac artifacts are relatively small. The skin and superficial muscles on the right side of the chest were resected and when reaching the layer of the intercostal muscles, a surface area of about 0.5 cm2 was freed from muscles down to the endothoracic fascia. Under stereomicroscopic view and with fine forceps, we then carefully stripped the endothoracic fascia, exposing the parietal pleura (about 10 m thick) over an average surface of about 2.5 mm2 , through which the lung surface with its alveolar texture could be clearly detected. This approach allowed us to neatly visualize the subpleural alveoli freely moving by preserving intact pleural space mechanics. 2.2. Experimental protocol The animal was placed under the field of a fixed microscope (Nikon SMZ-2T) in left lateral decubitus; therefore, we imaged the change in size of alveoli of the caudal part of the right lower lobe where no interlobar fissures are present. Given the animal position in left lateral decubitus, the alveoli studied were in the less dependent portion of the pleural cavity. Images of the lung surface through the intact parietal pleura were acquired with a video camera (CoolSnap EZ, Photometrics), connected to the microscope, interfaced through a IEEE-1394 data-transfer interface card with a personal computer equipped with an image-processing software (MetaMorph® System, Molecular Devices). Total microscopic magnification was 60×. A LED ring-light illuminator was anchored to microscopic optics to provide a uniform lighting of the alveolar field. 2.3. Perturbation of alveolar mechanical equilibrium by surfactant instillation Alveolar image acquisition (10 images/s) was triggered during the last part of the expiratory phase, but only images corresponding to Functional Residual Capacity (FRC), at zero flow, were considered for the analysis. After baseline data collection, a single bolus of 2.5 ml of surfactant (CUROSURF, Chiesi Farmaceutici, Parma, Italy), corresponding to about 50 mg/kg of phospholipids, was delivered into the trachea down to the carina, with the animal in lateral decubitus. Mechanical ventilation was continued and images were acquired in baseline condition at end expiration for about 20 min. The values corresponding to the last 5 min were considered as baseline before surfactant instillation. Thereafter, surfactant instillation was performed (considered as time 0) and images were acquired at 5, 10, 15 and 20 min for the same group of alveoli to obtain their surface area vs time relationships. At the end of the experiment, the animals were euthanized with an anesthetic over-dose. 2.4. Image analysis The transparency of the parietal pleura allowed a clear identification of the alveolar texture. Alveoli were manually segmented by following their borders well identified by the grey level gradient between air and tissue phase. Alveolar segmentation allowed to derive the alveolar 2D surface area.
2.5. Statistical analysis Quartile coefficient of variation (QVC), equal to the ratio between interquartile range and the sum of the 75th and the 25th percentiles, was calculated for alveolar size distribution. Kruskal–Wallis One Way Analysis of Variance on Ranks was performed to compare the changes in alveolar surface area for alveoli, respectively, smaller and greater than 20,000 m2 . Post-hoc analysis for multiple comparisons of the changes in alveolar area after surfactant instillation was based on Holm-Sidak method. Statistical analysis was performed by SigmaStat software v11.0 (San Jose, CA, USA). All data are reported as mean values ± standard error. P-values lower than 0.05 were considered to indicate significant differences. 3. Results Fig. 1A shows that in baseline condition the overall distribution (N = 60) of alveolar surface areas (A0 ) had a median value of 11,444.5 m2 ; about 80% of the alveoli had an alveolar surface area below about 22,000 m2 , with a mean alveolar diameter of about 75 m. The distribution was not normal with a QVC equal to 0.40. Fig. 1B–E shows the distribution of alveolar surface area at 5 (A5 ), 10 (A10 ), 15 (A15 ) and 20 (A20 ) min after surfactant instillation. Fig. 2A shows that under control conditions there were no significant changes in alveolar areas over 20 min before surfactant instillation. Fig. 2B–D reports the statistical coefficients over time (median, skewness, kurtosis and QVC) for the frequency distributions shown in Fig. 1. Median value increased up to 15 min then decreased at 20 min. Skewness remained constant while kurtosis increased sharply at 5 min after surfactant instillation. QVC remained essentially unchanged. 3.1. The effect of surfactant instillation Fig. 3A shows representative results of surfactant administration on the relative change in size for a group of alveoli (identified in Fig. 3B and referring to 15 min after instillation) as a function of time. One can appreciate a variability in the changes of alveolar surface area: some alveoli showed an increase in size of different entity (numbers 4 and 7), some did not change (numbers 6 and 8), while others showed a decrease (numbers 1, 3 and 5). Fig. 3C allows to appreciate that an inverse significant relationship could be described by plotting A/A0 versus A0 : for alveolar areas smaller than 20,000 m2 an increase of A/A0 was observed following surfactant instillation, the opposite was true for alveolar areas greater than about 20,000 m2 . Note that remarkable changes of opposite sign were observed for adjacent alveoli, namely only separated by septal tissue with no intervening alveoli (e.g. alveolar couples 4-3, 4-5, 7-8). In Fig. 4 all values of A/A0 at 5, 10 and 15 min following surfactant instillation are shown for alveoli of different size. Clearly most of the increase in A/A0 occurs for alveoli having A0 lower than 20,000 m2 , for larger alveoli either no change or decrease of A/A0 was observed. ANOVA on ranks for alveoli, respectively, smaller and greater than 20,000 m2 yielded a significant difference at P = 0.020. In Fig. 5 are plotted as function of time the average values of the increase of A/A0 for alveoli with A0 lower than 20,000 m2 ; we fitted the data with an exponential function providing a time constant of about 12 min for increase in alveolar surface area after surfactant instillation. Esophageal pressure, reflecting the lung distending pressure (Pd ), decreased at 5 min after surfactant administration from 2.5 ± 0.2 to 1.5 ± 0.2 cmH2 O and remained essentially steady up to the 20 min of observation (P = 0.10).
C. Salito et al. / Respiratory Physiology & Neurobiology 210 (2015) 7–13
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Fig. 2. (A) Relative changes in alveolar surface area for all the alveoli considered up to 20 min before surfactant instillation. (B)–(D) Median value, skewness and kurtosis, and QVC, respectively, for the frequency distributions shown in Fig. 1.
Fig. 6 shows for a group of alveoli from one animal, that the decrease in the action of surfactant occurred with different onset time and with different kinetics. One way ANOVA Pairwise Multiple Comparison Procedures (Holm-Sidak method) for these alveoli yielded a significant difference between times 5, 10, 15 and 20 compared to control (time 0) (P < 0.001). Fig. 7 presents the average percentage decrease in surfactant effect by pooling all available data for alveoli
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
The effect of exogenous surfactant on alveolar interdependence Caterina Salito a,∗ , Andrea Aliverti a , Enrico Mazzuca a , Ilaria Rivolta b , Giuseppe Miserocchi b a b
Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano, Italy Dipartimento di Scienze della Salute, Università di Milano Bicocca, Milano, Italy
a r t i c l e
i n f o
Article history: Accepted 12 January 2015 Available online 17 January 2015 Keywords: Surfactant Alveolar mechanics In-vivo microscopy imaging
a b s t r a c t To investigate the nature of alveolar mechanical interdependence, we purposefully disturbed the equilibrium condition by administering exogenous surfactant in physiological non-surfactant deprived conditions. Changes in alveolar morphology induced by intra-tracheal delivery of CUROSURF were evaluated after opening a pleural window allowing in-vivo microscopic imaging of sub-pleural alveoli in 6 male anesthetized, tracheotomized and mechanically ventilated rabbits. Surfactant instillation increased the surface area of alveoli smaller than 20,000 m2 up to ∼50% at 15 min after instillation, reflecting a lowering of surface tension due to local surfactant enrichment. Conversely, for alveoli greater than 20,000 m2 , surface area decreased by ∼5%. Opposite changes in alveolar surface are interpreted as reflecting a new inter-alveolar mechanical equilibrium modified by local surfactant distribution and by a decrease in lung distending pressure. We propose that smaller alveoli, representing the majority of alveolar population, might mostly contribute to improve the oxygenation index following surfactant replacement therapy in case of surfactant deficiency. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Recent studies from our group (Mazzuca et al., 2014; Salito et al., 2014) provided evidence of the existence of considerable geometrical heterogeneity in alveolar geometry and size that impact on the inter-alveolar differences in mechanical behavior that, in turn reflects the balance between elastic and surface forces (Gil et al., 1979; Tabuchi et al., 2008). The inter-alveolar differences in mechanical properties ought to be framed within a model of mechanically inter-dependent units (Bates and Suki, 2008). Although local elastic distending pressure can be evaluated under static conditions, alveolar surface tension may vary due the thickness of the surfactant layer that depends upon alveolar surface and inter-alveolar surface tension gradients sustaining the Marangoni flow. Previous data suggest that local changes in surface forces appear more important than changes in elastic forces during alveolar expansion in the volume range 40–60% TLC (Tabuchi et al., 2008; Wilson, 1982). Aiming to investigate the nature of the alveolar interdependence when potential inter-alveolar mechanical differences are present,
∗ Corresponding author. Tel.: +39 02 2399 9026x9001; fax: +39 02 2399 9000. E-mail address: [email protected] (C. Salito). http://dx.doi.org/10.1016/j.resp.2015.01.009 1569-9048/© 2015 Elsevier B.V. All rights reserved.
we purposefully disturbed the equilibrium condition by administering exogenous surfactant that causes a sudden decrease in surface tension in alveoli being reached by surfactant. 2. Methods 2.1. Animal preparation A general consensus for the experimental procedures used in our research activity was obtained from the Local Ethical Committee. Experiments were performed on 6 adult (New Zealand White) male rabbits (weight range 1–1.5 kg) anesthetized with a bolus of 2.5 ml/kg of a saline solution containing 0.25 g/ml of urethane injected into an ear vein. To maintain a constant depth of anesthesia throughout the procedure subsequent doses of 0.5 ml of anesthetic were administered every 20 min: this anesthesia strategy proved effective to maintain the absence of the corneal reflex between successive administrations. Tracheostomy was performed, and a plastic cannula (length 50 mm, inner diameter 2.5 mm) was inserted into the distal trachea. Before connecting the animal to the ventilator, paralysis was accomplished by pancuronium bromide (1 mg/kg body weight initial dose, supplemented by 0.33 mg/kg every 40 min). Mechanical ventilation was provided with a tidal volume of ∼20 ml and a frequency of 16 breaths/min
8
C. Salito et al. / Respiratory Physiology & Neurobiology 210 (2015) 7–13
that we previously used in rabbits to guarantee an end-tidal PCO2 of ∼36 mmHg (Miserocchi et al., 2001). Tracheal and oesophageal (through oesophageal balloon) pressures were measured by disposable pressure transducers (Edwards Baxter, Irvine, CA, USA). All signals were digitized by an analogue-to-digital board (NI BNC-2090, National Instruments, Austin, TX, USA) and dedicated software for real-time signal acquisition and visualization was implemented in LabView (version 8.2, National Instruments) and for the signal processing in Matlab (The Mathworks, Inc., Natick, MA, USA). An “intact pleural window” was prepared in the sixth intercostal space to allow a view of the lower lobe where the cardiac artifacts are relatively small. The skin and superficial muscles on the right side of the chest were resected and when reaching the layer of the intercostal muscles, a surface area of about 0.5 cm2 was freed from muscles down to the endothoracic fascia. Under stereomicroscopic view and with fine forceps, we then carefully stripped the endothoracic fascia, exposing the parietal pleura (about 10 m thick) over an average surface of about 2.5 mm2 , through which the lung surface with its alveolar texture could be clearly detected. This approach allowed us to neatly visualize the subpleural alveoli freely moving by preserving intact pleural space mechanics. 2.2. Experimental protocol The animal was placed under the field of a fixed microscope (Nikon SMZ-2T) in left lateral decubitus; therefore, we imaged the change in size of alveoli of the caudal part of the right lower lobe where no interlobar fissures are present. Given the animal position in left lateral decubitus, the alveoli studied were in the less dependent portion of the pleural cavity. Images of the lung surface through the intact parietal pleura were acquired with a video camera (CoolSnap EZ, Photometrics), connected to the microscope, interfaced through a IEEE-1394 data-transfer interface card with a personal computer equipped with an image-processing software (MetaMorph® System, Molecular Devices). Total microscopic magnification was 60×. A LED ring-light illuminator was anchored to microscopic optics to provide a uniform lighting of the alveolar field. 2.3. Perturbation of alveolar mechanical equilibrium by surfactant instillation Alveolar image acquisition (10 images/s) was triggered during the last part of the expiratory phase, but only images corresponding to Functional Residual Capacity (FRC), at zero flow, were considered for the analysis. After baseline data collection, a single bolus of 2.5 ml of surfactant (CUROSURF, Chiesi Farmaceutici, Parma, Italy), corresponding to about 50 mg/kg of phospholipids, was delivered into the trachea down to the carina, with the animal in lateral decubitus. Mechanical ventilation was continued and images were acquired in baseline condition at end expiration for about 20 min. The values corresponding to the last 5 min were considered as baseline before surfactant instillation. Thereafter, surfactant instillation was performed (considered as time 0) and images were acquired at 5, 10, 15 and 20 min for the same group of alveoli to obtain their surface area vs time relationships. At the end of the experiment, the animals were euthanized with an anesthetic over-dose. 2.4. Image analysis The transparency of the parietal pleura allowed a clear identification of the alveolar texture. Alveoli were manually segmented by following their borders well identified by the grey level gradient between air and tissue phase. Alveolar segmentation allowed to derive the alveolar 2D surface area.
2.5. Statistical analysis Quartile coefficient of variation (QVC), equal to the ratio between interquartile range and the sum of the 75th and the 25th percentiles, was calculated for alveolar size distribution. Kruskal–Wallis One Way Analysis of Variance on Ranks was performed to compare the changes in alveolar surface area for alveoli, respectively, smaller and greater than 20,000 m2 . Post-hoc analysis for multiple comparisons of the changes in alveolar area after surfactant instillation was based on Holm-Sidak method. Statistical analysis was performed by SigmaStat software v11.0 (San Jose, CA, USA). All data are reported as mean values ± standard error. P-values lower than 0.05 were considered to indicate significant differences. 3. Results Fig. 1A shows that in baseline condition the overall distribution (N = 60) of alveolar surface areas (A0 ) had a median value of 11,444.5 m2 ; about 80% of the alveoli had an alveolar surface area below about 22,000 m2 , with a mean alveolar diameter of about 75 m. The distribution was not normal with a QVC equal to 0.40. Fig. 1B–E shows the distribution of alveolar surface area at 5 (A5 ), 10 (A10 ), 15 (A15 ) and 20 (A20 ) min after surfactant instillation. Fig. 2A shows that under control conditions there were no significant changes in alveolar areas over 20 min before surfactant instillation. Fig. 2B–D reports the statistical coefficients over time (median, skewness, kurtosis and QVC) for the frequency distributions shown in Fig. 1. Median value increased up to 15 min then decreased at 20 min. Skewness remained constant while kurtosis increased sharply at 5 min after surfactant instillation. QVC remained essentially unchanged. 3.1. The effect of surfactant instillation Fig. 3A shows representative results of surfactant administration on the relative change in size for a group of alveoli (identified in Fig. 3B and referring to 15 min after instillation) as a function of time. One can appreciate a variability in the changes of alveolar surface area: some alveoli showed an increase in size of different entity (numbers 4 and 7), some did not change (numbers 6 and 8), while others showed a decrease (numbers 1, 3 and 5). Fig. 3C allows to appreciate that an inverse significant relationship could be described by plotting A/A0 versus A0 : for alveolar areas smaller than 20,000 m2 an increase of A/A0 was observed following surfactant instillation, the opposite was true for alveolar areas greater than about 20,000 m2 . Note that remarkable changes of opposite sign were observed for adjacent alveoli, namely only separated by septal tissue with no intervening alveoli (e.g. alveolar couples 4-3, 4-5, 7-8). In Fig. 4 all values of A/A0 at 5, 10 and 15 min following surfactant instillation are shown for alveoli of different size. Clearly most of the increase in A/A0 occurs for alveoli having A0 lower than 20,000 m2 , for larger alveoli either no change or decrease of A/A0 was observed. ANOVA on ranks for alveoli, respectively, smaller and greater than 20,000 m2 yielded a significant difference at P = 0.020. In Fig. 5 are plotted as function of time the average values of the increase of A/A0 for alveoli with A0 lower than 20,000 m2 ; we fitted the data with an exponential function providing a time constant of about 12 min for increase in alveolar surface area after surfactant instillation. Esophageal pressure, reflecting the lung distending pressure (Pd ), decreased at 5 min after surfactant administration from 2.5 ± 0.2 to 1.5 ± 0.2 cmH2 O and remained essentially steady up to the 20 min of observation (P = 0.10).
C. Salito et al. / Respiratory Physiology & Neurobiology 210 (2015) 7–13
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Fig. 2. (A) Relative changes in alveolar surface area for all the alveoli considered up to 20 min before surfactant instillation. (B)–(D) Median value, skewness and kurtosis, and QVC, respectively, for the frequency distributions shown in Fig. 1.
Fig. 6 shows for a group of alveoli from one animal, that the decrease in the action of surfactant occurred with different onset time and with different kinetics. One way ANOVA Pairwise Multiple Comparison Procedures (Holm-Sidak method) for these alveoli yielded a significant difference between times 5, 10, 15 and 20 compared to control (time 0) (P < 0.001). Fig. 7 presents the average percentage decrease in surfactant effect by pooling all available data for alveoli
