Respiratory Physiology & Neurobiology 162 (2008) 8592

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Respiratory Physiology & Neurobiology

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Breathing pattern, CO2 elimination and the absence of exhaled NO in freely diving Weddell seals
K.J. Falke a, , T. Busch b , O. Hoffmann c , G.C. Liggins d , J. Liggins d , R. Mohnhaupt a , J.D. Roberts Jr. e , K. Stanek e , W.M. Zapol e
a

Klinik f ur Anaesthesiologie und Operative Intensivmedizin, Charit e Campus Virchow Klinikum, Universitaetsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany Klinik und Poliklinik fuer Anaesthesiologie und Intensivtherapie, Universitaetsklinikum Leipzig, D-04103 Leipzig, Germany Klinik und Poliklinik fuer Neurologie, Charit e Campus Mitte, Universitaetsmedizin Berlin, D-10098 Berlin, Germany d Research Centre in Reproductive Medicine, Department of Obstetrics and Gynaecology, University of Auckland, Auckland 1142, New Zealand e Department of Anesthesia and Critical Care, Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street, Boston 02114-2696, MA, USA
b c

a r t i c l e

i n f o

a b s t r a c t
Weddell seals undergo lung collapse during dives below 50 m depth. In order to explore the physiological mechanisms contributing to restoring lung volume and gas exchange after surfacing, we studied ventilatory parameters in three Weddell seals between dives from an isolated ice hole on McMurdo Sound, Antarctica. Methods: Lung volumes and CO2 elimination were investigated using a pneumotachograph, infrared gas analysis, and nitrogen washout. Thoracic circumference was determined with a strain gauge. Exhaled nitric oxide was measured using chemiluminescence. Results: Breathing of Weddell seals was characterized by an apneustic pattern with end-inspiratory pauses with functional residual capacity at the end of inspiration. Respiratory ow rate and tidal volume peaked within the rst 3 min after surfacing. Lung volume reductions before and increases after diving were approximately 20% of the lung volume at rest. Thoracic circumference changed by less than 2% during diving. The excess CO2 eliminated after dives correlated closely with the duration of the preceding dive. Nitric oxide was not present in the expired gas. Conclusion: Our data suggest that most of the changes in lung volume during diving result from compression and decompression of the gas remaining in the respiratory tract. Cranial shifts of the diaphragm and translocation of blood into the thorax rather than a reduction of thoracic circumference appear to compensate for lung collapse. The time to normalise gas exchange after surfacing was mainly determined by the accumulation of CO2 during the dive. These ndings underline the remarkable adaptations of the Weddell seal for restoring lung volume and gas exchange after diving. 2008 Elsevier B.V. All rights reserved.

Article history: Accepted 15 April 2008 Keywords: Marine mammals Respiratory function Lung volume Lung collapse Apnea NO

1. Introduction The extreme diving capabilities of Weddell seals (Leptonychotes weddelli) are due to their high storage capacity for oxygen (Qvist et al., 1986; Guyton et al., 1995; Kooyman and Ponganis, 1998) and its remarkably economical utilization mediated by the diving response (Scholander, 1940; Butler and Jones, 1997). In addition the management of pulmonary gas is paramount for efcient foraging in these animals. They exhale before and immediately after the dive, characteristics of their breathing pattern are obviously important for the handling of the considerable amount of pulmonary nitrogen as

Corresponding author at: Am Neuen Garten 41, D 14469 Potsdam, Germany. Tel.: +49 331 249003; fax: +49 331 249005. E-mail address: KonradJ.Falke@charite.de (K.J. Falke). 1569-9048/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.04.007

well as the regulation of buoyancy. So far only very few quantitative studies on the pattern of respiration before and the restoration of lung volume after free dives have been performed (Kooyman et al., 1971, 1973; Parkos and Wahrenbrock, 1987). As originally suggested by Scholander (1940) and in line with Kooyman et al. (1972) study in restrained seals, we have previously demonstrated indirect evidence (by determining blood nitrogen tensions) that the lungs of freely diving Weddell seals undergo alveolar collapse during descent between 25 and 50 m, a phenomenon stopping pulmonary gas exchange and, thus protecting these animals from the unwanted effects of excess nitrogen absorption (Falke et al., 1985). However, directly measured data on the mechanical events associated with lung compression, decompression and full recruitment at surfacing after each dive have been extremely scanty. In a freely diving trained dolphin, visible deformation of the chest wall began to occur between 10 and 60 m depth, and a photograph taken at 300 m

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depth showed marked thoracic compression from just behind the ippers and posteriorly (Ridgway et al., 1969). Following up on a suggestion of the late Professor Hermann Rahn (personal communication), we attempted to correlate the changes in lung volume during diving with measurements of thoracic circumference. The aerobic diving limit (ADL) of Weddell seals is sufcient (1520 min) for them to perform most underwater activities under aerobic metabolic conditions (Kooyman et al., 1983; Qvist et al., 1986; Castellini et al., 1992; Ponganis et al., 1993). In prolonged dives, however, the arterial blood oxygen tension (PaO2 ) may fall below 20 mmHg (Qvist et al., 1986) suggesting that low PaO2 is not necessarily the primary respiratory drive or the most important factor in the regulation of diving behavior (Stephenson, 2005). Instead, a profound impact of CO2 on respiration and diving (Pasche, 1976; Parkos and Wahrenbrock, 1987) has been reported but CO2 elimination was not quantied. In humans, breath-holding causes a pronounced increase of gaseous nitric oxide concentrations in the upper respiratory tract (Gustafsson et al., 1991; Persson et al., 1990). By analogy we hypothesized that after the prolonged apnea of diving Weddell seals may also accumulate NO in the airways, and because of its potent selective pulmonary vasodilatory effects (Frostell et al., 1991) it might contribute to the re-establishment of pulmonary perfusion after recruitment of the lungs at surfacing. In the present study, we aimed to extend previous observations (Kooyman et al., 1971, 1972, 1973; Kooyman, 1981b; Parkos and Wahrenbrock, 1987; Falke et al., 1985; Qvist et al., 1986) on the respiratory functions of freely diving Weddell seals rstly focusing on the respiratory pattern using measurements of respiratory ows as well as lung volumes and its shifts between dives. We quantied the changes in lung volume before, after and between dives expecting that the seal would decrease its lung volume signicantly before and restore it to a large extent during the rst few breaths after surfacing. We also expected that lung collapse during descent, and recruitment during ascent, would be associated with distinct corresponding changes in thoracic circumference, a hypothesis based on the above mentioned observation made in a dolphin by Ridgeway in 1969. Secondly we measured exhaled CO2 and hypothesized that CO ) should correlate with the durapost dive CO2 elimination (V 2 tion of the dive, a phenomenon which to our knowledge has not been previously reported in unrestrained Weddell seals. Thirdly, we hypothesized that NO should be present in the expired gas of seals in particular after the long breath-hold associated with long dives. Thus, we aimed to determine the concentration of endogenous NO in exhaled respiratory gas upon resurfacing. Due to the constraints of our permit and due to the specic Antarctic environmental conditions our studies were restricted to three animals, consequently, our results are presented individually. These data were extremely difcult to obtain, hence, they should be considered a pilot study and hopefully will serve to stimulate future studies. 2. Materials and methods 2.1. Animal handling and instrumentation All seal studies were conducted under US NMFS marine mammal permit #600, and approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. The general experimental procedure has been well described (Kooyman et al., 1971, 1973; Falke et al., 1985; Qvist et al., 1986; Parkos and Wahrenbrock, 1987). Five sub-adult male Weddell seals with weights estimated from 280 to 340 kg were captured near the Dellbridge Islands, the Erebus Glacier Tongue, and Arrival Heights on Ross Island (approximately 78 South, 168 East) and were moved to a eld site equipped with

a portable research laboratory located above a one m diameter man made ice hole where pulmonary function studies could be carried out between free dives. After the induction of anesthesia a previously calibrated (using a ruler), 3 mm thick silicone covered mercury strain gauge (custom made by K. Stanek) was tted around the seals chest caudal to the fore ippers near the maximum circumference. The strain gauge produced an electrical resistance proportional to the thoracic circumference. The resistance was converted to a voltage using a Wheatstone bridge circuit. The signal was amplied, converted to a digital format with 8 bit resolution and transferred to the random-access memory of a time-depth recording computer (Wildlife Computers, Woodinville, WA, USA) which was xed to a rubber patch glued to the seals dorsal fur. Measured values of thoracic circumference were stored every second, and values of seawater depth every 5 s. After complete recovery from anesthesia the seal was released into a temporarily opened second ice hole next to the mobile laboratory. At the termination of the studies, the seals were allowed to exit the water, and all monitoring equipment was removed from the animals. The seal was then released near its site of capture. The latex patches were left to fall off during molting. 2.2. Studies of respiratory physiology For the measurement of ventilatory parameters, the ice hole inside the laboratory was closed with a plywood sheet leaving a central hole (20 cm diameter) covered with an acrylic dome to which a two way giant non-rebreathing valve (Series 7200, Hans Rudolph, Inc., Kansas City, MO, USA) was attached closely resembling the setup described by Parkos, Wahrenbrock and Pasche (Parkos and Wahrenbrock, 1987; Pasche, 1976). We inserted an appropriately sized pneumotachograph (PT 18, Jaeger GmbH, Wuerzburg, Germany) between the acrylic dome and the nonrebreathing valve (see Fig. 1). The dome, the pneumotachograph and the non-rebreathing valve increased the seals anatomical dead space by approximately 2.5 L. For re-breathing maneuvers the nonrebreathing valve was replaced by a large three-way stopcock and a meteorological latex balloon of 180 cm length and a capacity of

Fig. 1. Schematic representation of experimental setup using a pneumotachograph to measure inspiratory and expiratory respiratory ow between free dives of Weddell seals while resting in a man made ice hole (modied from Parkos et al.). For re-breathing maneuvers the Rudolph valve was replaced with a large latex balloon tted with an appropriately sized three way stopcock.

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50 L, hanging from the ceiling of the hut. Hence, the gas volumes in the bag and their concentrations were determined at room temperature. Due to the specic heating conditions of the experimental hut there was a large temperature gradient between the bottom and the ceiling of the hut, thus the room temperature could not be precisely determined. Consequently gas volumes were not corrected to STPD or BTPS.1 A small suction line was attached between the acrylic dome and the pneumotachograph to measure CO2 concentration during breathing, using a standard infrared gas analyzer (Normocap, Datex Instruments, Helsinki, Finland). Respiratory ow rate and CO2 concentration were recorded online and stored in a personal computer, utilizing software specically designed for our studies. Tidal volume and ventilation were obtained by integration of the ow signal. The pneumotachograph was calibrated before and after each set of measurements using a 2 L acrylic syringe simulating expected ow ranges. The CO2 analyzer was calibrated using a gas mixture containing 5% CO2 in dry air. Mean expired CO2 concentration was either obtained by electronic integration of the CO2 signal or in mixed expired gas collected in the latex balloon. Lung volume was determined by washout of the nitrogen which had remained in the lung during diving or during breathing at rest into a closed system with a xed volume of 100% O2 (the latex balloon was lled to 20, 30 or 40 L). Nitrogen concentrations at the end of re-breathing were calculated based on measurements of O2 concentrations using an electrochemical monitor (Ohio 500, Ohio Medical Corporation, USA) and CO2 concentrations (Normocap, Datex Instruments, Helsinki, Finland) in appropriate gas samples obtained from the latex balloon. To calculate the lung volume at the beginning of oxygen re-breathing (VL start ), either at surfacing after diving or at rest, and at the end of re-breathing procedures (VL end ) equations according to Kooyman et al. (1971) were used.2 Nitric oxide (NO) concentration in exhaled gas was measured using chemiluminescence analysis, a process in which NO is converted to NO2 in the presence of an excess of ozone. This chemical reaction is associated with the production of infrared radiation, the intensity of which is a measure of the NO concentration. The instrument used (CLD 700 AL, ECO-Physics, Duernten, Switzerland) possessed a lower detection limit of 0.5 parts per billion (ppb) and a rise time of 2 s. The adjustment of the zero-level is accomplished via interruption of a beam of light between the reaction chamber and a photomultiplier; hence, NO free gas was not required for calibration. In order to scale the display, a test gas with a NO content of 90 ppm (AGA, Bottrop, Germany) was used. Considering the high linearity of the apparatus, this calibration is sufcient for all concentrations below 100 parts per million (ppm). Analog output of the instrument was converted to digital signals which were stored continuously by our data acquisition system for later analysis. 2.3. Data presentation and statistical methods Because of the small number of animals we studied we will present mostly individual data. However, in our investigation of the differences in thoracic circumference at various depths we

Table 1 Biometric data Seal C D E Total length [cm] 229 231 224 Maximum circumference [cm] 158 180 160 Nose to maximum circumference [cm] 91 79 89 Estimated weight [kg] 260 345 261

employed Wilcoxons signed rank test for matched pairs (two tailed). A correlation analysis of CO2 excess production at surfacing after diving with the duration or depth of the dive was performed using the Spearman rank correlation coefcient. All calculations were carried out using SPSS statistical software (version 6.0). Results with p-values of less than 0.05 were considered as statistically signicant. Data in the text and tables are expressed as mean S.D. 3. Results 3.1. Biometric data We captured 5 Weddell seals (AE), but physiological data could only be obtained from 3 seals CE. Presented in Table 1 are their body weights (estimated according to Castellini and Kooyman, 1990).3 Because application of the strain gauge was a new technique we obtained reliable thoracic circumference measurements only in 2 seals (D and E). 3.2. Pattern of ventilation We collected data during a total of 32 phases of breathing between dives. Respiratory frequency, mean and peak expiratory and inspiratory ow rates, mean and maximal volumes as well as the ventilation of seals CE are shown in Table 2. Data in the left column of numbers (Resting) were obtained while the seal rested in the ice hole (mean values S.D.). The columns to the right (headed After dive, 1 to 15 post) show individual values from the rst to the 15th minute after the termination of a single dive, one selected for presentation from each of the three seals because of their long duration. The column on the far right (Before dive) shows values from the last minute preceding the following dive. In these three seals, we observed wide ranges of respiratory parameters with minute ventilation near and above 100 L per minute during the recovery phase from the long dives. Some of the post dive values that were collected from seals C and E were obtained during re-breathing maneuvers immediately after surfacing lasting 2 or 1 min, respectively. This was associated with a signicant degree of CO2 retention. In seal D the values were obtained during normal breathing without re-breathing. Representative individual respiratory ow patterns, tidal volumes and exhaled CO2 concentrations are displayed in Fig. 2A and B. Fig. 2A shows respiratory ow pattern, tidal volumes and expired CO2 concentrations while resting in the ice hole with the seal almost fully immersed in sea water and immediately prior to a dive. Characteristically, the respiratory cycle begins with an expiration which abruptly terminates at a very high ow rate and which is immediately followed by an inspiration. A post-inspiratory pause precedes the next respiratory cycle. Under these conditions inhaled and exhaled tidal volumes are identical, but the shape of the ow curves differed and generally peak expiratory exceeded peak inspi-

1 In these measurements the effects of temperature variation need to be considered as a source of error. Pneumotachographic measurements were conducted at or near BTPS, while gases collected in the rebreathing bag for determination of lung volume were obtained at ambient temperature (near 0 C at the oor and frequently near 30 C at the top of the laboratory). However, since the gas volumes at the beginning and at the end were determined at the same environmental conditions this should not have affected the calculation of the dilutional lung volume to a relevant degree. 2 See online Supplement I.

3 For seal A the performance of the computer was unsatisfactory. Seal B escaped from the site of investigation before useful data were obtained.

88 Table 2 Individual respiratory parameters Seal C Resting 81 Breaths [per min] Exp. ow, mean [L/s] Exp. ow, peak [L/s] Insp. ow, mean [L/s] Insp. ow, peak [L/s] Tidal volume, mean [L] Tidal volume max [L] Ventilation [L/min] End-tidal CO2 [%] Exp. CO2 mean [%] CO2 [cm3 /min] V Seal D 10 4.9 7.0 4.8 6.2 3.9 4.5 38.9 4.8 2.5 981 2.0 0.5 0.7a 0.7 0.8a 0.4 0.6 7.7 0.4a 0.1 223

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After dive #7 (duration 43 min; unknown depth) 1 post 2 post 3 post 17 5.4 6.0 6.4 7.2 5.1 5.6 87.4 7.2 3.9 3443 4 post 17 5.5 6.0 6.8 7.2 5.6 6.0 95.6 7.8 4.0 3822 5 post 16 5.6 6.2 6.8 7.2 5.6 6.0 90.3 7.5 3.8 3437 9 post 16 6.0 8.1 5.7 6.7 5.4 6.3 86.3 5.5 3.1 2661 15 post 9 5.3 7.5 4.9 5.5 4.1 4.7 37.1 4.2 2.6 967

Before dive #4 1 pre 11 4.6 6.8 4.7 5.4 3.9 5.4 43.0 4.0 2.1 910 Before dive #1

13 12 4.6 4.2 8.1 6.0 4.1 4.1 6.1 6.5 4.0 4.8 4.5 5.2 52.5 57.3 Re-breathing 4.1c 4.0c 2156 2270

Resting 77

After dive #2 (duration 33 min; depth 98 m) 1 post 2 post 13 6.0 6.2 6.7 7.4 8.9 9.1 116.3 8.4 4.7 5397 3 post 13 6.2 6.2 6.8 7.4 9.1 9.3 117.8 7.9 4.4 5102 4 post 13 6.1 6.2 6.6 7.1 8.9 9.1 115.3 6.8 4.0 4588 5 post 12 6.1 6.4 6.4 7.0 8.4 8.7 101.7 5.9 3.5 3553 9 post 7 5.2 6.0 5.1 5.5 5.0 7.1 45.1 5.0 2.8 1256 15 post 5 4.3 5.8 3.9 5.4 5.0 5.4 25.2 4.8 2.5 640

1 pre 9 5.3 6.3 5.1 7.9 7.0 8.4 66.2 4.2 2.8 1864 Before dive #3

Breaths [per min] Exp. ow, mean [L/s] Exp. ow, peak [L/s] Insp. ow, mean [L/s] Insp. ow, peak [L/s] Tidal volume mean [L] Tidal volume max [L] Ventilation [L/min] End-tidal CO2 [%] Exp. CO2 mean [%] CO2 [cm3 /min] V Seal E

4 4.5 6.3 4.1 4.9 5.1 5.5 23.2 5.2 3.0 695

1 0.4 0.3a 0.4 0.5a 0.6 0.6 7.3 0.1a 0.4 256

14 6.3 8.4 6.1 6.5 8.4 9.2 118.3 8.2 4.8 5625

Resting 67

After dive #5 (duration 29 min; depth 194 m) 1 post 2 post 13 5.5 7.2 7.2 7.6 8.5 8.9 110.6 9.5 5.1 5617 3 post 12 5.4 7.0 6.8 7.5 8.3 8.5 99.2 8.9 4.6 4555 4 post 12 5.3 6.7 6.5 6.9 8.1 8.4 97.0 7.5 4.2 4026 5 post 12 5.1 6.2 6.1 6.5 7.4 8.0 89.0 6.6 3.7 3298 9 post 6 4.3 6.0 4.8 5.4 5.9 6.9 35.2 5.2 3.0 1046 15 post

1 pre 11 4.8 6.1 4.1 4.9 6.3 7.9 69.8 4.2 2.5 1714

Breaths [per min] Exp. ow, mean [L/s] Exp. ow, peak [L/s] Insp. ow, mean [L/s] Insp. ow, peak [L/s] Tidal volume mean [L] Tidal volume max [L] Ventilation [L/min] End-tidal CO2 [%] Exp. CO2 mean [%] CO2 [cm3 /min] V

6 4.0 5.8 4.2 4.6 5.6 6.3 33.2 5.1 3.1 1000

2 0.6 0.6b 0.7 0.4b 0.7 0.8 15.0 0.6b 0.2 432

13 5.1 7.2 5.9 7.2 7.6 8.7 98.9 Re-breathing 5.3c 5199

Resting values are given as mean S.D. pooled from all dives of each seal in minutes (Seal C, 81 min; Seal D, 77 min; Seal E, 67 min) except if labeled as superscript a = maximum of n = 6 readings, b = maximum of n = 7 readings c = extrapolated because of re-breathing.

ratory ow (see also Table 2). For the last breath prior to diving, in this example the seal takes only a small inspiration presumably to reduce the gas volume of the lung (and therefore buoyancy) before diving. After each dive (see Fig. 2B) the rst respiratory maneuver of the seal is a small expiration of the gas which has remained in the airways during the dive. This initial expiration usually has a large peak ow velocity, demonstrating that it may be released at a high pressure. In addition Fig. 2B shows that within the rst few breaths after the dive the inspiratory tidal volumes exceed the expiratory tidal volumes, indicating rapid relling of the pulmonary gas volume after diving. This phenomenon of relling the lungs after a dive and reducing lung volume before a dive is again demonstrated in Fig. 3 showing tidal volume continuously measured between two sequential dives to depths of 50 and 150 m. This gure also provides a running difference between inspiratory and expiratory tidal volume, thereby quantifying lung volume changes after and before the dives. Of note, major changes in lung volume only occur during the rst few inhalations after surfacing and with the last exhalation before diving (Figs. 2 and 3). The mean reductions in lung gas volume (determined by pneumotachograph) caused by exhalation before diving were in seal C 1.6 L (n = 1), in seal D 0.6 to 2.1 L (n = 3), and in seal E 3.2 to 5.5 L (n = 5).

3.3. Lung volume estimation by N2 dilution Values for diving and resting lung volume in seal C were 11.1 and 13.3 L, in seal D 11.8 and 13.1 L, and in seal E 16.3 and 18.6 L, respectively. The mean difference between resting volume and diving volume was found to be 1.9 0.6 L, a magnitude similar to the values obtained using the pneumotachograph. When determined at rest, lung volume was comparable at the start and end of the closed circuit re-breathing procedure (15.0 L vs. 17.4 L). However, when determined after surfacing from diving, lung volume increased markedly during the re-breathing procedure (VL start : 13.1 2.8 L; VL end : 24.4 12.4 L). All measurements that were carried out simultaneously in seal E are exemplied graphically in Fig. 4. It shows the CO2 elimination rate, measurements of lung volume and changes in lung volume (equal to tidal volume), thoracic circumference, and depth over several sequential free dives. After dive #2 a re-breathing maneuver was undertaken in order to determine the lungs gas volume by N2 dilution. In the example shown this led to the previously mentioned marked increase of lung volume which was conrmed by the dilutional volume determination. Lung volume determined by N2 dilution at the beginning of re-breathing was 16 L and at the end 37 L, probably near the seals total lung capacity.

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Fig. 3. As an example, the shifts in lung volume are shown between 2 dives. The lung volume and the tidal volume are shown in the upper panel. The absolute lung volume in the upper panel was taken from the N2 dilution whereas the changes in volume and the tidal volume were obtained from the pneumotachograph. In the lower panel, Vinsp Vexp is shown demonstrating the increase in lung volume immediately after the dive (left), and the reduction of the lung volume occurring with the last expiration before the next dive (right). It demonstrates that only 2025% of the alterations in lung volume occur due to ventilation. A much larger proportion of the diving lung volume is compressed.

Fig. 2. (A) Respiratory gas ow, lung volume, tidal ventilation and exhaled CO2 concentration between and during the last respiratory cycles (seal E) before diving for 30 min to a maximum depth of 194 m. Lung volume reduction prior to diving of 4 L (a, below interrupted line) was restricted to the last breath. Positive ow values correspond to the expiratory phase and negative to the inspiratory phase of the respiratory cycle. Due to the measuring technique, the CO2 signal is delayed by approximately 1.5 s as compared with the gas ow recordings. (B) Respiratory gas ow, lung volume (VL ), tidal volume and exhaled CO2 concentration of seal E during the rst breaths taken after surfacing from a dive of 12 min duration. The seal rst exhales a small gas volume (rst small dip of VL ) a part of which had remained in the airways and then re-inates the lungs with the rst two inhaled breaths. However the net-increase in lung volume by inhalation remains surprisingly small (a) and corresponds to the decrease of VL measured in the last breath before diving (see Fig. 3). Hence, most of the restoration of lung volume must be due to decompression of the gas (b) which had remained in the airways during the dive. It was a general observation that the exhaled CO2 concentration of the rst few breaths after returning to the surface was quite low and similar to resting levels.

3.4. Thoracic circumference changes The thoracic circumference changes of seal E during diving as measured by the strain gauge are shown in Table 3. We observed steep changes of circumference with decreases at the beginning of the dive and increases at the return to the surface (Table 3 and Fig. 4). Comparing the measurements immediately after the beginning of the dive at 0 m (see Table 3) and at maximum depth, the mean difference in circumference was 2.58 0.55 cm. The main characteristics of circumferential data recordings are revealed by inspection of an example in seal E (Fig. 4) which contains additional results of accompanying respiratory measurements covering a period of several dives and rest phases. At approximately 50 m depth (dive #2) when the lung is collapsed, there was a minimal reduction of 1 cm in the thoracic circumference. Even at 150 m depth (dive #2) the decrease was only in the range of 23 cm (Table 3). The unexpected substantial increase in lung volume from 16 to 27 L (reproduced by dilutional lung volume determination)

Fig. 4. Computer tracings demonstrating the results of all measurements that were carried out simultaneously (seal E), from the top down: breath by breath CO2 elimi CO2 ), lung volume (VL ), its changes and tidal breaths, thoracic circumference nation (V (Circ), and depth during and at rest of several free dives. After dive #2 a re-breathing maneuver was undertaken in order to determine the N2 dilutional lung volume. In contrast to the volume recordings after dives #1 and 3 when no re-breathing took place this was associated with a doubling of the lung volume presumably due to the inability of the seal to remove the CO2 . This is a true lung volume change (and no artifact) because it was conrmed by the independent determination of VL by the N2 dilution technique, calculating the volume at the beginning VL start = 16 L and at the end VL end = 37 L of the re-breathing maneuver, a level probably near the total lung capacity. It is interesting because it supports our notion that changes in lung volume (here in the opposite direction as compared to descent in the water) are associated with very small almost not recognizable thoracic circumference changes (see panel 3 from the top).

90 Table 3 Thoracic circumference of seal E at various depths Dive number Circumference Before dive [cm] 2 3 5 9 Mean S.D. 175.2 173.6 174.3 172.9 174.0 1.0

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Maximum depth of dive [m] At 0 m [cm] 171.8 172.0 171.7 171.1 171.7 0.4 At 10 m [cm] 171.3 171.1 170.9 170.4 170.9 0.4 At 20 m [cm] 170.4 170.4 170.4 169.6 170.2 0.4 At maximum depth [cm] 169.6 169.6 168.3 168.8 169.1 0.6 147 157 196 152 163.0 22.4

which occurred during a re-breathing maneuver following dive #2 also resulted in only a 1 cm increase of thoracic circumference (Fig. 4, immediately following dive #2). Our ndings were further substantiated by comparing the circumference values measured when the curve stabilized shortly after the beginning of the dives (with the seal at 20 m of depth) with those measured at the deepest point of the dive. Including data from 11 dives (3 from seal D and 8 from seal E) with a mean depth of 176 55 m (all greater than 82 m) we could demonstrate only a small albeit signicant decrease in thoracic circumference (surface, 166.5 5.3 cm vs. depth, 165.3 5.3 cm, p = 0.003). 3.5. CO2 exhalation studies The end-tidal as well as the mean expired CO2 concentrations CO ) are shown and the mean rate of respiratory CO2 elimination (V 2 in Table 2. Breathing at rest, the end-tidal (=maximal) expiratory CO2 concentrations ranged between 4.8 0.35 and 5.1 0.6% while the basal CO2 elimination rate varied between 695 256 and 1000 432 mL min1 . After surfacing commonly three exhalations were required to wash out the articial dead space and to reach CO2 concentrations reecting alveolar gas. The end-tidal CO2 concentrations as well as the breath-by-breath CO2 elimination increased to maxima within the rst 23 min after surfacing, up to 89% and 5.6 L min1 , respectively (Table 2) and approached normal resting values within 15 min (Figs. 4 and 5). After a total of 12 dives we observed a signicant and marked linear correlation (R = 0.73, p = 0.007) between the CO2 eliminated in excess of the basal elimination rate during rest and the duration of the preceding dives with values obtained up to >40 min of dive duration (Fig. 6). 3.6. Nitric oxide in exhaled gas In the expiratory gas of all 5 seals, we could not detect any exhaled NO, indicating that NO was either completely absent from
Fig. 6. CO2 elimination in excess of the resting basal rate during diving (retained CO2 ) as determined after surfacing and plotted against the duration of the preceding dive. A signicant and close correlation is demonstrated (R = 0. 986, p < 0.001).

the exhaled gas or remained at a level below our detection limit of 0.5 ppb (parts per billion of gas volume). In ambient air we measured an average NO concentration of 12 ppb. 4. Discussion 4.1. Respiratory pattern and volumes As a modication of the ingenious setup for pulmonary functions studies in freely diving Weddell seals developed by Kooyman et al. (1971, 1973), in which ventilation parameters were determined using classical spirometers and collecting bags, we employed a pneumotachograph to allow breath-by-breath measurement of respiratory ow rates and derived tidal volume by electrical signal integration. Resting values for breaths per minute, tidal and minute volumes as well as lung volumes were within the ranges previously reported in unrestrained Weddell seals (Kooyman et al., 1971, 1973; Castellini et al., 1992; Parkos and Wahrenbrock, 1987). After prolonged dives, ventilation increased up to 120 L min1 , considerably less than in Kooymans original study (1971). This probably reects our use of sub-adult animals (estimated body weight, 260345 kg) as compared to adult seals in the previous report (mean body weight, 425 kg). The pneumotachographic respiratory ow pattern (Fig. 2) demonstrates that in Weddell seals in contrast to humans the respiratory cycle begins with an expiration which is immediately followed by an inspiration prior to a phase of rest (apneustic breathing) as previously observed by Scholander (1940, p. 21). Under these circumstances the resting lung volume corresponding to the functional residual capacity does not lie at the end-expiratory but at the end-inspiratory level of the lung volume. This larger resting lung volume increases buoyancy and allows the seal to oat near the surface with few or no active ipper movements. The large lung volume can only be maintained against the high surrounding seawater pressure because the seals nostrils function as highly effective positive

Fig. 5. Breath by breath CO2 elimination in a single seal after a dive to 48 m depth for 12 min duration. The VCO2 of the rst three breath is underestimated due to the articial dead space of the breathing dome. The dotted line indicates the CO2 elimination during rest. It was a common characteristic of our measurements that the maximum amount of CO2 was exhaled within the rst 23 min after surfacing.

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pressure valves closing after inspiration (Scholander, 1940, p. 23). An interesting question is the extent to which expiration is performed as an active manoeuvre. The weight of the chest wall and blubber as well as the pressure exerted by the surrounding sea water would imply that expiration is mainly passive. The observed expiratory ow pattern, however, differs from humans whose passive expiration is characterized by a decelerating ow. In contrast we observed forceful increases and decreases in airway gas ow with a plateau phase between them (Fig. 2), suggesting active muscular forces are operating during expiration. This is in line with the observation of Kooyman and Cornell (1981) of an extremely high expiratory peak ow rate of 162 L s1 in a dolphin trained to make a maximum expiratory effort. It is well known (see Figs. 2A and 3) that Weddell seals usually exhale before diving (Kooyman et al., 1971), presumably in order to decrease buoyancy and, thus facilitate their descent in the water. It was an important goal of our studies to quantify the changes in lung volume immediately before and after dives. In contrast to our initial hypothesis, the degree of these lung volume changes was relatively small. In the example shown in Fig. 1A this exhalation before diving represents a lung volume reduction of approximately only 20%. After the dive when the seal surfaces the rst expiration is of a similar magnitude (see Figs. 2B and 3). This represents a component of the gas, which has remained compressed in the upper airway during the dive after lung collapse and is usually released with considerable positive pressure. Presumably this gas, which is considered a portion of the diving lung volume, re-expands with the decreasing water pressure as the seal approaches the surface and, hence, due to its evolving positive pressure contributes to the recruitment of the previously collapsed lung. We also note that complete restoration of lung volume to the pre-dive level occurs quickly within the rst 24 breaths after surfacing, most of it taking place in the rst breath (see Figs. 1B and 2). In the example shown in Fig. 1B, 16 L of gas are compressed to 2.7 L (b) at 50 m depth which is forced into the upper airways indicating that lung collapse occurs approximately at this depth or slightly beyond, an interpretation corresponding well with earlier observations (Kooyman et al., 1971; Falke et al., 1985).

taken into account.4 There is evidence from human (Craig, 1968; Schaefer et al., 1968) and animal diving observations (Cozzi et al., 2005; Ninomiya et al., 2005) that translocation of blood from the peripheral to intrathoracic/-pulmonary vessels may also play a space lling function when the seals lung collapses. It should, however, be stressed that thoracic circumference is probably not only affected by passive compression of the chest wall, but also by muscular activity. Since the strain gauge was placed near the ippers, we believe that the sharp changes in measured circumference immediately at the beginning and at the end of a dive (see Fig. 4) could well be caused by activation of the ippers. 4.3. CO2 concentration and elimination Due to the conguration of our measurement device (dome and pneumotachograph) the anatomical dead space was articially increased by approximately 2.5 L. Assuming PCO2 being the primary drive for ventilation (Parkos and Wahrenbrock, 1987) this additional dead space should slightly increase minute ventilation and decrease mean expired CO2 concentration accordingly. Due to this CO effect the end-tidal CO2 underestimated alveolar PCO and the V
2 2

4.2. Circumference measurements Lung collapse in diving Weddell seals has been explained by a compression of the chest wall (Kooyman, 1981a,b; Kooyman and Ponganis 1998), similar to observations in freely diving dolphins (Ridgway et al., 1969; Ridgway and Howard, 1979). According to our previous studies, complete lung collapse should occur at 2550 m below the surface (Falke et al., 1985). Surprisingly at this range of depths we consistently observed only a very small decrease in thoracic circumference of about 23 cm (i.e., less than 2%). In principle, irregular deformation of the thorax could occur without marked changes at the level of our circumference measurements, but it is more reasonable to assume that the lack of major changes of thoracic circumference is due to additional mechanisms going along with the marked decrease in intrathoracic volume such as an upward movement of the diaphragm as well as the displacement of blood from extrathoracic to the intrathoracic vasculature (Kooyman, 1981a,b). A calculation using a cone as a model of the seals thorax (with the tip being the nose and the base the equivalent of the diaphragm) revealed that the diaphragm would be shifted upwards within a plausible range of 512 cm if the determined diving lung volumes and the observed small changes in thoracic circumference were

of the rst three breath after surfacing (Table 2 and Figs. 2B and 5). Taking into account the large lung and tidal volumes of the animals in particular after surfacing the additional dead space was, however, readily washed out within three breaths as demonstrated by the original CO2 recordings (partially shown in Fig. 2B). In our opinion it becomes insignicant during subsequent exhalations and during steady state conditions (resting) in respect to the determination of CO which was the main focus of our investigation. Our endthe V 2 tidal CO2 concentrations were in a range close to these previous observations of end-tidal CO2 and blood gas tension levels (Parkos and Wahrenbrock, 1987; Qvist et al., 1986). Because we had to rely on the estimated body weights of just CO per kg body weight. Nevthree animals we hesitated to present V 2 ertheless we found an excellent and statistically highly signicant correlation between dive duration and post-dive CO2 elimination above the basal rate. Breath by breath analysis of CO2 elimination showed that the elimination of retained CO2 reached a maximum CO above resting values. after 14 min with a 38-fold increase in V 2 It is notable that the time course of the CO2 elimination prole resembles that of the end-tidal O2 concentration observed by Ponganis et al. (1993). 4.4. Nitric oxide Gaseous nitric oxide (NO) a potent selective pulmonary vasodilator produced in the epithelium of the lower and upper respiratory tract (Busch et al., 2000), and primarily in the paranasal sinuses (Lundberg et al., 1995; Deja et al., 2003) is auto-inhaled in humans (Gerlach et al., 1994; Lundberg et al., 1996) and many terrestrial animals (Gustafsson et al., 1991; Jenkins and Langlois, 1995; Lewandowski et al., 1996; Schedin et al., 1997). After a human breath-hold the exhaled concentration may be as high as 30 ppb (Kimberly et al., 1996). Accordingly, we hypothesized that gaseous NO should accumulate in the upper respiratory tract of diving Weddell seals and by its auto-inhalation might contribute to the restoration of pulmonary blood ow at the end of the dive. However, we could not detect any NO in the expiratory gas of Weddell seals. Due to our lower detection limit of 0.5 ppb we cannot rule out even lower concentrations in the parts per trillion range. After termination of our studies, based on measurements in baboons it was

The detailed description of the model calculation, see online Supplement II.

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K.J. Falke et al. / Respiratory Physiology & Neurobiology 162 (2008) 8592 King, J.E., 1983. Seals of the world. British Museum and Oxford University Press, Oxford, p. 153. Kimberly, B., Nejadnik, B., Giraud, G.D., Holden, W.E., 1996. Nasal contribution to exhaled nitric oxide at rest and during breathholding in humans. Am. J. Respir. Crit. Care Med. 153, 829836. Kooyman, G.L., 1981a. Crabeater seal. In: Ridgway, S.H., Harrison, R.J. (Eds.), Handbook of Marine Mammals. Academic Press, London, New York, Toronto, Sydney, San Francisco, p. 227. Kooyman, G.L., 1981b. Weddell Seal, Consummate Diver. Cambridge University Press, Cambridge, London, New York, New Rochelle, Melbourne, Sidney, pp. 6274. Kooyman, G.L., Cornell, L.H., 1981. Flow properties of expiration and inspiration in a trained bottle-nosed porpoise. Physiol. Zool. 54, 5561. Kooyman, G.L., Ponganis, P.J., 1998. The physiological basis of diving to depth: birds and mammals. Annu. Rev. Physiol. 60, 1932. Kooyman, G.L., Kerem, D.H., Campbell, W.B., Wright, J.J., 1971. Pulmonary function in freely diving Weddell seals, Leptonychotes weddelli. Respir. Physiol. 12, 271 282. Kooyman, G.L., Schroeder, J.P., Denison, D.M., Hammond, D.D., Wright, J.J., Bergman, W.P., 1972. Blood nitrogen tensions of seals during simulated deep dives. Am. J. Physiol. 223, 10161020. Kooyman, G.L., Kerem, D.H., Campbell, W.B., Wright, J.J., 1973. Pulmonary gas exchange in freely diving Weddell seals, Leptonychotes weddelli. Respir. Physiol. 17, 283290. Kooyman, G.L., Castellini, R.W., Davis, R.W., Maue, R.A., 1983. Aerobic diving limits of immature Weddell seals. J. Comp. Physiol. 151, 171174. Lewandowski, K., Busch, T., Lewandowski, M., Keske, U., Gerlach, H., Falke, K.J., 1996. Evidence of nitric oxide in the exhaled gas of Asian elephants (Elephas maximus). Respir. Physiol. 106, 9198. Lewandowski, K., Busch, T., Lohbrunner, H., Rensing, S., Keske, U., Gerlach, H., Falke, K.J., 1998. Low nitric oxide concentrations in exhaled gas and nasal airways of mammals without paranasal sinuses. J. Appl. Physiol. 85, 405410. Lundberg, J.O., Farkas-Szallasi, T., Weitzberg, E., Rinder, J., Lidholm, J., Anggaard, A., Hokfelt, T., Lundberg, J.M., Alving, K., 1995. High nitric oxide production in human paranasal sinuses. Nat. Med. 1, 370373. Lundberg, J.O., Settergren, G., Gelinder, S., Lundberg, J.M., Alving, K., Weitzberg, E., 1996. Inhalation of nasally derived nitric oxide modulates pulmonary function in humans. Acta Physiol. Scand. 158, 343347. Negus, V., 1958. The Comparative Anatomy and Physiology of the Nose and Paranasal Sinuses. Livingstone, Edinburgh, pp. 285319. Ninomiya, H., Inomata, T., Shirouzu, H., Katsumata, E., 2005. Microanatomy of the terminal air spaces of Bairds beaked whale (Berardius bairdii) lungs. J. Vet. Med. Sci. 67, 473479. Parkos, C.A., Wahrenbrock, E.A., 1987. Acute effects of hypercapnia and hypoxia on minute ventilation in unrestrained Weddell seals. Respir. Physiol. 67, 197207. Pasche, A., 1976. The effect of hypercapnia on respiratory characteristics and diving behaviour of freely diving seals. Respir. Physiol. 26, 183194. Persson, M.G., Gustafsson, L.E., Wiklund, N.P., Moncada, S., Hedqvist, P., 1990. Endogenous nitric oxide as a probable modulator of pulmonary circulation and hypoxic pressor response in vivo. Acta Physiol. Scand. 140, 449457. Ponganis, P.J., Kooyman, G.L., Castellini, M.A., 1993. Determinants of aerobic dive limit of Weddell seals: analysis of diving metaboloc rates, postdive end tidal Po2 s, and blood and muscle oxygen stores. Physiol. Zool. 66, 732749. Qvist, J., Hill, R.D., Schneider, R.C., Falke, K.J., Liggins, G.C., Guppy, M., Elliot, R.L., Hochachka, P.W., Zapol, W.M., 1986. Hemoglobin concentrations and blood gas tensions of free-diving Weddell seals. J. Appl. Physiol. 61, 15601569. Ridgway, S.H., Howard, R., 1979. Dolphin lung collapse and intramuscular circulation during free diving: evidence from nitrogen washout. Science 206, 1182 1183. Ridgway, S.H., Scronce, B.L., Kanwisher, J., 1969. Respiration and deep diving in the bottlenose porpoise. Science 166, 16511654. Schaefer, K.E., Allison, R.D., Dougherty Jr., J.H., Carey, C.R., Walker, R., Yost, F., Parker, D., 1968. Pulmonary and circulatory adjustments determining the limits of depths in breathhold diving. Science 162, 10201023. Schedin, U., Roken, B.O., Nyman, G., Frostell, C., Gustafsson, L.E., 1997. Endogeneous nitric oxide in the airways of different animal species. Acta Anaesthesiol. Scand. 41, 11331141. Scholander, P.F., 1940. Experimental Investigations on the Respiratory Function in Diving Mammals and Birds, vol. 22. Hvalradets Skrifter, Oslo, pp. 1131. Stephenson, R., 2005. Physiological control of diving behaviour in the Weddell seal Leptonychotes weddelli: a model based on cardiorespiratory control theory. J. Exp. Biol. 208, 19711991.

hypothesized (Lewandowski et al., 1998) that terrestrial animals without paranasal sinuses do not have NO concentrations above a few ppb in their respiratory gas. Hence, we believe that the main explanation for the absence of NO in the exhaled gas of Weddell seals is their lack of open and aerated paranasal sinuses (Negus, 1958; King, 1983). Other factors may include scavenging of NO by blood hemoglobin which increases to levels around 25 g% during diving in Weddell seals (Qvist et al., 1986). Acknowledgements These studies were funded by National Science Foundation grant OPP 91-18192 and also supported by the Deutsche Forschungsgemeinschaft, grant DFG Fa 139/4. We thank Bernhard Huettel, the German representative of ECO Physics (Duernten, Switzerland) who provided the equipment for the NO measurements and Axel Mohnhaupt who provided the computer software. Special thanks goes to Professor Peter Scheid for his very valuable support in nalizing the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.resp.2008.04.007. References
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