As a scuba diver ascends to the surface, inert gases, which were absorbed into the blood and tissue under high ambient pressure during the dive, are released into the blood as venous gas emboli (VGE) and have traditionally served as a quantifiable indicator for decompression stress. Recently, other methods have been investigated to measure additional parameters associated with decompression sickness (DCS) and stress, including platelet count (25), aggregation (22), and microparticles (MP) (24). MP are cell-derived vesicles containing a lipid bilayer, protein aggregates, and other debris with a diameter of 0.1–1.0 μm. MP are produced as a result of apoptosis, oxidative stress, or cellular activation/calcium influx (11,12,15). MP are present in the peripheral blood of healthy individuals; they increase with traumatic and inflammatory disorders and may serve as intercellular messengers because they can contain cytokines or other signaling proteins, messenger RNA, and microRNA (19). MP are characterized by the surface expression of antigenic markers from parent cells, and many also have surface-bound annexin V because, as they are formed, negatively charged phosphatidylserine residues become exposed. Enlarged MP (1–3 μm), resulting from expansion of an inert gas core during decompression, have been shown to cause vascular injuries in mice (36), and it has been demonstrated that these MP provide a nucleation site for inert gas uptake (31) during decompression. Additional human studies showed an increase in size and number of MP after an open-water dive (29) and increased endothelial MP in a simulated dive (33). In addition to scuba diving, exercise has been shown to increase circulating MP for several hours after a single bout (5,18,27), and one study demonstrated a near return to baseline after 24 h (20).
The effect of exercise before diving on VGE measured postdiving was investigated in several studies. Some of these studies used simulated dry chamber dives (14), which may not accurately replicate the stress associated with open-water diving (21). Currently, exercise is considered by some to be a form of protective preconditioning for scuba diving (9,13,23), although these claims have been based on animal survival studies (35) or decreased VGE in humans after aerobic exercise. Proposed mechanisms include altered hemodynamics (2), increased nitric oxide concentration, and improved vasoreactivity (9). Exercise protocols tested before diving varied in intensity from 60% age-predicted HRmax (3) to intervals at 90% HRmax (8), with running as the mode of exercise. Studies examined interactions of exercise with diving within 2 (2–4) or 24 h (8) before diving.
Because some forms of exercise have been shown to increase circulating MP and MP may be associated with increased decompression stress (36), exercise may affect the bubble load by producing an increased number of circulating cell fragments and micronuclei that may absorb inert gas upon decompression. To further test this hypothesis, in the current study, we had divers exercise (EX) before an open-water dive in conditions that would elicit MP responses lasting for at least 2 h and compared these results with those of a control dive (CON) under the same conditions but without exercise. Finally, in our previous studies with volunteers undergoing repetitive dives (28) or dives with two different breathing gas mixtures and two levels of exertion during dives (29), blood was collected only up to 2 h postdive. Therefore, this study was planned to assess associations between MP number and neutrophil activation up to 24 h postdive, in parallel to collected murine data.
MATERIALS AND METHODS
Nineteen divers including 14 males (age (mean ± SD), 38 ± 6 yr) and five females (31 ± 8 yr) with 14.1 ± 9 yr of diving experience participated in this study. Maximal oxygen consumption (V˙O2max) for males was 36.5 ± 3.4 mL·kg−1·min−1 and 36.1 ± 4.2 mL·kg−1·min−1 for females. The mean body mass index for males was 27.4 ± 2.5 kg·m−2 and 22.5 ± 2.4 kg·m−2 for females. Divers were apparently healthy at the beginning of the study and were recently given clearance to dive by a physician. No subjects reported previous cases of DCS. After receiving a written explanation and an oral briefing of the methods and potential risks, all subjects gave their written informed consent. This study was approved by the University of Split School of Medicine ethics committee, and all procedures were conducted in accordance with the Declaration of Helsinki.
Exercise selection protocol.
Testing was conducted on three different exercise protocols to monitor MP response. The goal was to compare a treadmill protocol, which has been proven to be beneficial to divers related to VGE production in a previous study (8), with a similar protocol performed on a cycle ergometer and an anaerobic protocol. Wingate cycling was chosen for the anaerobic trial because of the relative safety compared with that in equivalent anaerobic efforts performed on a treadmill. These trials were conducted 2 months before the beginning of the study to determine the exercise protocol that should be used before diving. Briefly, the following protocols were performed each with four subjects: 1) intervals on a bicycle ergometer consisting of 3 min at V˙O2max power (W) and 1.5 min of active recovery, which were repeated until the subject could no longer maintain 90% of V˙O2max power (cumulative time, 15–18 min), 2) repeated Wingate tests on a bicycle ergometer consisting of 4 × 30 s of maximal efforts with 4 min of active recovery (cumulative time, 23 min including warm-up), and 3) treadmill intervals consisting of 3 min at a velocity that elicited 90% of HRmax determined by previous testing (explained in detail in the following section) and 2 min at 50% repeated eight times (cumulative time, 60 min). Treadmill interval exercise (protocol 3) was selected for this study for two reasons: it is the same protocol used in an earlier exercise and a simulated dive study (8) has shown the protocol to decrease VGE, and the MP response was more pronounced and prolonged compared with that in the cycling protocols (data not shown).
V˙O2max and pulmonary function testing were performed on all divers at least 3 d before the diving experiment. Before testing, height, weight, and percent body fat for each subject were determined. Body density was estimated by measurement of subcutaneous skinfold thickness with a caliper (Harpenden skinfold caliper; Baty International, West Sussex, England) for the calculation of body composition. Pulmonary function assessment included forced vital capacity and maximal voluntary ventilation tests. The V˙O2max test was an incremental test conducted on a treadmill (COSMED T165 sport; COSMED, Rome, Italy) beginning at 3 km·h−1 and 2% grade and increasing 1 km·h−1 every minute until voluntary termination or when at least two of the three following requirements were met: 1) a plateau of V˙O2 (<150 mL absolute increase) or HR with an increase in workload, 2) RER greater than 1.1, and 3) HR in excess of 90% of age-predicted (220 − age) maximal values. Once these criteria were met, the highest recorded V˙O2 was selected as the subject’s maximal value.
Subjects completed a 20-min self-selected warm-up immediately followed by 40 min of intervals as described by protocol 3 of the pilot studies, for a total of 60 min of treadmill running. The treadmills were controlled by volunteers to allow the divers to focus on running and to ensure that the correct exercise prescription was delivered.
Dive and VGE analysis
This study was performed at a military installation of the Croatian Navy Force. The dive site was located near the base, within a short (approximately 30 m) distance from the location where the experiments would take place. The site was chosen because of the minimal transit time between finishing the dive and beginning initial transthoracic echocardiography analysis. All divers performed the dive at a depth of 18 m seawater with a bottom time of 41 min. This dive profile was selected with a dive planning software built into Galileo dive computers (Uwatec Galileo Sol; Johnson Outdoors, Inc., Racine, WI), which were also used to verify subject adherence to the dive protocol. Decompression was performed at a rate of 9 m seawater·min−1, with direct ascent to the surface. Sea temperature at the bottom was approximately 11°C, and the outside temperature was approximately 19°C. Throughout the dive, divers performed swimming of subjectively moderate intensity and HR was monitored via dive computers. Within 8–15 min after surfacing, the divers were placed in the supine position where a dual-frequency (1.5–3.3 MHz) ultrasonic probe connected to a Vivid q echographic scanner (GE, Milwaukee, WI) was used to obtain a clear, apical, four-chamber view of the heart. VGE were monitored at 15, 40, 80, and 120 min after surfacing, and bubble scores were recorded and graded on a scale of 0–5, with 4 being subdivided into 4A, 4B, and 4C, according to the method described by Eftedal and Brubakk (10) and later modified by Ljubkovic et al. (17). In addition to monitoring scores at rest, VGE were graded after two different movements, arm and leg contractions, to mobilize bubbles that may be lodged in the venous circulation.
EX and CON dive protocols.
Subjects were randomly selected to begin the CON or EX dive first, with at least 3 d between the dives. The divers performed 1 h of treadmill running beginning 3 h before descent. Upon surfacing, VGE were graded for 120 min after surfacing, as described previously. Blood for complete blood count (CBC) and MP analysis was drawn, as will be described in the next section. The CON dive was performed exactly as the EX dive, only without the exercise. Water was allowed ad libitum throughout the study, and a light meal was provided in between exercise and diving.
Whole blood samples were obtained from the subjects before exercise (EX dive protocol only) approximately 30 min before diving and 15 min after surfacing. Blood was stored in a cooler and transported to the Department of Biochemistry, University Hospital Split, for analysis within 2 h of obtaining the sample. CBC values were measured with the Abbott Cell-Dyne 4000 cell counter (Abbott Laboratories, Abbott Park, IL).
MP chemicals and procedures.
Venous blood was collected by a trained phlebotomist 30 min before and at 15 min, 2 h, 4 h, and 24 h after the dives. Blood was drawn into Cyto-Chex BCT test tubes that contain a proprietary preservative (Streck, Inc., Medimark Europe, Grenoble, France). The volume drawn per sample (two tubes) was approximately 5 mL.
Unless otherwise noted, chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Annexin binding buffer and the following antibodies were purchased from BD Pharmingen (San Jose, CA): fluorescein isothiocyanate-conjugated anti-annexin V and fluorescein isothiocyanate-conjugated anti-human myeloperoxidase (MPO). R-phycoerythrin–conjugated anti-human CD41 was purchased from eBioscience (San Diego, CA); PerCP Cy5.5-conjugated anti-human CD66b from BioLegend (San Diego, CA) and Alexa 647-conjugated anti-human CD18 were purchased from AbD Serotec (Raleigh, NC).
Standard procedures for MP and neutrophil acquisition and processing.
Blood samples in tubes containing a preservative were sent by express mail to the University of Pennsylvania where all analyses were performed within 24 h after arrival, approximately 2–6 d from the time of collection. As described previously, MP and neutrophil characteristics remain unchanged when samples stored at either 4°C or at room temperature are processed in a time span of 3 wk from the time of collection (16). All reagents and solutions used for MP analysis were sterile and filtered (0.2-μm filter). The blood was centrifuged for 5 min at 1500g; the supernatant EDTA was made at a concentration of 0.2 M and then centrifuged at 15,000g for 30 min. Aliquots of the 15,000g supernatant were stained with antibodies for analysis by flow cytometry and confocal microscopy.
Flow cytometry was performed with a 10-color FACSCanto (Becton Dickinson, San Jose, CA) using a standard acquisition software. Gates were set to include 0.3- to 5.0-μm particles, with exclusion of background corresponding to debris usually present in buffers. MP were stained with annexin V antibody and were analyzed as previously described, including microbeads with diameters of 0.3 μm (Sigma, Inc.), 1.0 μm, 3.0 μm, and 5 μm (Spherotech, Inc., Lake Forest, IL) to assess the size of particles. We define MP as annexin V-positive particles with diameters up to 1 μm and enlarged MP as those between 1 and 3 μm. Analysis of neutrophils was performed on fixed blood samples, as previously described (30). Platelet activation was assessed by staining samples with CD41 and annexin V antibodies in samples that included microbeads with diameters of 3 and 5 μm. Platelets were identified as particles between these microbead size limits that were CD41-positive and annexin V-negative, and activation was assessed as surface expression of CD63 and CD62b, analogous to procedures described by others (32).
Parametric data are expressed as mean ± SD, and nonparametric data are expressed as median and 25th and 75th percentile. MP counts and platelet and neutrophil activation were analyzed by repeated-measures ANOVA followed by the Bonferroni correction. Bubble scores were analyzed with the Wilcoxon signed-rank test. Comparisons between dive samples were made with the Student’s t-test, and correlations were assessed with the Spearman rank order test, with the Bonferroni correction to account for multiple comparisons. Statistics were calculated using the Statistica 7.0 software (StatSoft, Inc., Tulsa, OK).
Diving and intravascular bubbles.
There were no adverse effects reported from any of the subjects after exercise and scuba diving activities. Data for the 14 males and five females are grouped together. The median bubble score, taken at rest, was unchanged between the EX and the CON dives. The median score for both dives was 3 at the 15-, 40-, and 80-min measurements and 2 at the 120-min measurement (Table 1). However, overall, VGE grades were significantly higher in the EX group at the 40- and 80-min measurements at rest and at 80- and 120-min measurements during the voluntary leg contractions (P = 0.047, 0.004, 0.003, and 0.043, respectively).
Platelets, leukocytes, and hematocrit.
Platelet counts (Table 2) were significantly reduced after the EX dive compared with those after the CON dive at all measured time points after surfacing (15 min, P = 0.013; 2 h, P = 0.037; 4 h, P = 0.002; and 24 h, P = 0.003). Leukocytes were significantly increased by exercise when measured before diving (P = 0.0002) and were also significantly increased by diving during both the EX and CON dives (P = 0.000 and 0.002) (Table 2). Hematocrit (Hct) was significantly increased compared with predive values after surfacing after the CON and EX dives at 15-min, 2-h, and 4-h time points and returned to baseline after 24 h in the EX dive but not in the CON dive (Table 2). Both the CON and EX dives follow the same pattern; there was no significant difference in the mean values of Hct other than that before diving, where it was significantly elevated after exercise (P = 0.013) (Table 2).
Platelet activation was assessed as surface expression of CD63 and CD62P, as described in Methods. The percent of all platelets expressing these activation-associated markers is shown in Figure 1A and B, and geometric mean fluorescence is shown in Figure 1C and D. Exercise increased platelet activation; however, activation was significantly lower postdiving when compared with that after the CON dive.
Neutrophil activation was assessed as surface expression of CD18 and MPO on CD66b-positive cells (Fig. 2A and B). Activation occurred after the CON and EX dives, with a significant difference at all postdiving measurements of CD18 and at 15 min, 2 h, and 24 h for MPO. Platelet–neutrophil interactions were evaluated as the presence of CD41 on CD66b-positive cells (Fig. 2C). Notably, there was an increase in CD41 mean fluorescence after predive exercise and it remained elevated after the dive. After the CON dive, fluorescence was increased compared with both predive values and compared with values at all time points of the EX dive.
Circulating MP and enlarged MP counts related to the EX and CON dives are displayed in Figure 3A and B, respectively. There was no significant difference in annexin V-positive MP (0.3–1 μm) before exercise and before diving on the day of the CON dive, indicating no difference in baseline among divers before the two different dive conditions. MP increased significantly at all time points up to 4 h in both dives. There was a significant difference in MP number between EX and CON dives at all four postdive measurements, with lower response in the EX group. Elevated MP values persisted for 24 h after both dive conditions but were only significant after the CON dive. Elevations of large MP occurred after the CON dive in a pattern similar to the 0.3- to 1-μm MP but not after the postexercise dive.
MP expressing the platelet-specific CD41a surface protein are displayed in Figure 4A, and those expressing the neutrophil-specific CD66b are displayed in Figure 4B. These subtypes of MP increased after both CON and postexercise diving and peaked by the 2-h sample. Significantly, fewer MP expressing CD41 and CD66b were present at all sampling time points after surfacing (15 min and 2, 4, and 24 h) in the EX dive compared with those after the CON dive. MP expressing CD41 and CD66b remained elevated 24 h postdive in both groups.
Correlations between variables.
Correlations between variables were tested on the basis of previous research and hypotheses. After the CON dive, a positive correlation was found between enlarged MP and BG (at rest and during leg compressions) 15 min after surfacing (r = 0.636 and 0.561, P = 0.003 and 0.013, respectively). After predive exercise, a positive correlation was found between platelet count at 120 min after surfacing and large MP (r = 0.526, P = 0.012).
We set out to further investigate the interactions between MP and VGE and explore the effect of predive exercise because of recent animal and human data pointing to decompression stress as an acute vascular injury with an inflammatory response. Results from the study provide the following observations with exercise 2 h before diving compared with a nonexercising CON group: 1) no change in median VGE but increased 75th percentiles and maximal bubble scores at 40 and 80 min after surfacing after the EX dive compared with those after the CON dive, 2) smaller increases in platelet counts at all time measurements, 3) increased leukocyte counts prediving, 4) fewer circulating MP, 5) a smaller increase in CD41- and CD66b-positive MP, 6) evidence of platelet activation prediving but less activation postdiving, and 7) less evidence of platelet–neutrophil interactions. These observations provide additional information in the relation between exercise, MP, and VGE.
Whereas previous studies have demonstrated a beneficial effect of exercise on the basis of a reduction of VGE (3,9,13), we did not observe similar outcomes. These data are contrary to previous studies where exercise before diving was found to result in a clear reduction in postdive bubbles (3,4,8,9,13). There are many factors that influence VGE production outside exercise, including fatigue, environmental conditions, and, more recently, acclimatization to repeated bouts of scuba diving (1). It is possible that any one of these could have contributed to differences between studies and overshadowed any potential changes in VGE activity resulting from the exercise intervention. Moreover, the effect of using alternate bubble grading techniques, scales, and equipment on the magnitude of these differences cannot be ruled out as well.
It has also been proposed that alterations in hemodynamics are related to dehydration as a potential mechanism of changes in VGE after exercise (2). In the present study, subjects were allowed water ad libitum at all times, including the exercise sessions. Although Hct was progressively higher at each time point after surfacing after both dives, the elevations were not significantly different between the groups (Table 2). Significant only within each group, these changes appear within normal values, and there are other aspects of exercise that can lead to increased hemo concentration. This maintenance of hydration status could partially explain the lack of significant changes in VGE grades between the two trials.
Platelet counts were significantly reduced after the EX dive compared with those after the CON dive at all measurements and are displayed in Table 2. It is important to note that the predive values (baseline) are significantly higher in the CON dive than the preexercise values in the EX dive (Table 2). That is, the subjects began the day of experiments (before any intervention including diving and exercise) with a higher platelet count on the control day compared with that in the exercise day and the difference seems to be related to the different baseline values. It is not clear what is responsible for these different baseline values. Markers of platelet activation including surface coexpression of CD62P/CD41 and CD63/CD41 glycoproteins show significant reductions in postdiving increase after EX compared with those after the CON dive (Fig. 1). Increased platelet activation is associated with DCS in the rat model (26), and platelet activation in humans is associated with damaging thrombotic events.
It is clear that exercise before diving has an effect on annexin V-positive MP count and subtype expression after diving. First, this experiment provides new data on MP, platelet, and leukocyte responses up to 24 h after surfacing. Whereas exercise caused an increase in MP, consistent with reports by others, paradoxically, elevations in the absolute number of circulating MP are significantly less when diving is preceded by exercise (Fig. 3A), warranting further studies on the protective effect of exercise. Total MP counts remained elevated 24 h after diving, with larger increases above baseline seen in the CON dive. Enlarged MP (1–3 μm) (Fig. 3B) were evaluated as well. Although absolute numbers were different between the two dives, the proportion of enlarged to regular MP was similar. We found a relation between enlarged MP and ultrasonic scannable VGE under some diving conditions in a previous study (29), although the correlation was weak and not always in the same direction. Although results from a murine study suggest that MP containing an intranitrogen dioxide phase serve as nucleation points for inert gas released from the tissues during decompression, we have not made similar observations in humans under these conditions. It was hypothesized that uptake of inert gas facilitates expansion of MP to enlarged MP (1–3 μm in diameter) that may be visualized ultrasonically. In the present study, a significant positive correlation was seen between large MP and BG 15 min after surfacing (r = 0.636, P = 0.003) only in the CON dive. Regardless of the relation between MP and VGE, there remains strong evidence that these large MP are related to vascular damage. Naive mice injected with these particles exhibit signs and symptoms of DCS, and recompression, resulting in a reduction in size of these particles, ameliorates these symptoms (30).
Platelet and neutrophil activation and interactions are associated with DCS in mice. Platelet-derived MP (Fig. 4A) play a key role in neutrophil activation (30), and the reduction after the EX dive could be related to a reduction in neutrophil activation observable as decreased CD66b (Fig. 4B) and MPO expression (Fig. 2B). A decrease in CD66b expressing MP was also associated with a decrease in vascular injuries in mice (30), although further investigation is needed to determine whether these risks are present and subsequently ameliorated by exercise before open-water diving in humans.
It is unclear whether reductions in MP counts after dives preceded by exercise compared with those after CON dives are related to an increased rate of uptake or a decrease in production. Currently, little is known about MP clearance and elimination. Phagocytosis is believed to be the primary means of MP clearance, as has been demonstrated by Distler et al. in vitro (7). It is believed that this is a result of the interaction between MP surface expression of phosphatidylserine and splenic macrophages. An alternative clearance pathway consisting of MP binding via surface expression of Del-1 was associated with the ability of endothelial cells to internalize MP, describing a potentially more widespread clearance from vascular beds (6). This implies a complex relation between exercise, diving, and endothelial function, which is worth further investigation. Intense exercise can stimulate an acute increase of circulating leukocytes followed by a decrease as they move from the blood to the periphery in preparation to support a “fight or flight” response (34). It is possible that sympathetic activation associated with this immune response could be responsible for increased uptake and destruction of MP; our data show a significantly increased leukocyte count before and 15 min after diving in the EX group compared with that in the CON group. Regardless of the mechanism, more research is needed to determine whether the reduction in MP and concomitant diminution in neutrophil activation associated with exercise are related to the protective effect of exercise, which has been shown to reduce the incidence of DCS in mice.
In conclusion, our results provide additional insight into the interaction of exercise and scuba diving as well as the relation between VGE and MP. This study shows a clear reduction in MP counts and platelet and neutrophil activation with exercise before diving relative to diving alone, although these changes are not associated with VGE in a statistically significant manner. In the present study, these changes in MP have been clinically asymptomatic. Surprisingly, even a dive that would be considered mild by even recreational standards (18-m seawater depth, 41-min bottom time) produced approximately twice as many MP as what many people would consider a challenging exercise session (60 min total, 8 × 3 min intervals of high-intensity running). Additional work on the dose–response relations between exercise variables such as mode, intensity, and timing relative to diving is also needed.
Although exercise is not considered dangerous, some manageable level of damage is necessary to disrupt homeostasis enough to drive supercompensation. MP seem to be an additional part of this disruption for both exercise and diving. There may be a certain threshold that, once exceeded, would lead to negative effects in humans, as it has been demonstrated in mice. Additional studies should include MP analysis of divers that report to hyperbaric centers for treatment of DCS to determine when these levels become clinically significant. As with changes in cardiovascular parameters, endothelial function, and VGE after diving, MP represent an additional physiological consequence that remains mostly asymptomatic. However, these alterations can also contribute to DCS and diving injuries, and observing thresholds at which this may occur and how this relates to a diver’s current health status, as well as the long-term effects, are still to be determined.
We would like to thank Mislav Lozo, Ivana Banic, and Ivana Dragicevic for their hard work, professionalism, and technical expertise.
The funding for this work was provided by a grant from the Office of Naval Research. This work was supported by the FP7-PEOPLE-2010-ITN (264816 PHYPODE) and the Croatian Ministry of Science, Education, and Sports (grant no. 216-2160133-0130 to Z. D.).
The authors declare no conflict of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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