G. Zapol, Graham. C. Liggins, and Warren. M. Zapol. Splenic contraction, catecholamine release, and blood volume redistribution during diving in the Weddell ...
Splenic volume
contraction, redistribution
catecholamine release, and blood during diving in the Weddell seal
WILLIAM E. HURFORD, PETER W. HOCHACHKA, ROBERT C. SCHNEIDER, GREGORY P. GUYTON, KEVIN S. STANEK, DAVID G. ZAPOL, GRAHAM C. LIGGINS, AND WARREN M. ZAPOL Department of Anesthesia, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 2A9, Canada; and Postgraduate School of Obstetrics and Gynaecology, National Women’s Hospital, Auckland 3, New Zealand Hurford, William E., Peter W. Hochachka, Robert C. Schneider, Gregory P. Guyton, Kevin S. Stanek, David G. Zapol, Graham C. Liggins, and Warren M. Zapol. Splenic contraction, catecholamine release, and blood volume redistribution during diving in the Weddell seal. J. Appl. PhysioZ. 80(l): 298306, 1996.-The spleen of the Weddell seal (Leptonychotes weddeE) may contract and inject red blood cells (RBCs) into the peripheral circulation during diving, but evidence for this hypothesis is indirect. Accordingly, we measured splenic dimensions by ultrasonography, plasma catecholamine concentrations, hemoglobin concentration, and hematocrit in five Weddell seals before and after intravenous epinephrine during halothane anesthesia and while awake at the surface after voluntary dives. Spleen size was reduced immediately after epinephrine injection or after the seal surfaced. Within the first 2 min after the seal surfaced, cephalocaudal splenic length was 71 t 2% (mean t SD; P < 0.05) and splenic thickness was 71 2 4% (P < 0.05) of the maximal resting values. Splenic size increased (halftime = 6-9 min) after the seal surfaced and was inversely correlated with plasma epinephrine and norepinephrine concentrations. Hemoglobin concentration increased from 17.5 t 5.3 g/d1 (measured during general anesthesia) to 21.9 t 3.7 g/d1 (measured in the first 2 min after surfacing). At these same times, the hematocrit increased from 44 t 12 to 55 t 8% These values decreased (half-time = 12-16 min) after the seal surfaced. We estimate 20.1 liters of RBCs were sequestered at rest, presumably in the spleen, and released either on epinephrine injection or during diving. Catecholamine release and splenic contraction appear to be an integral part of the voluntary diving response of Weddell seals. spleen; blood hematocrit; ultrasonography; Leptonychotes weddelli; breath-hold diving
diving
mammals;
trial mammals (1, 2, 16, 34), and injects red blood cells (RBCs) into the peripheral circulation during diving (31). Evidence for this hypothesis in seals is limited to indirect calculations based on hematocrit changes and observations of the size of the spleen at autopsy or under anesthesia. Actual measurements of splenic contraction have not been performed, and the rate at which the spleen contracts and fills is unknown. We therefore used ultrasonography to directly measure splenic dimensions. Because the Weddell seal has a very large spleen, approximately two-thirds of the seal’s RBCs could be sequestered in the spleen while the seal rested at the surface (31). Direct RBC volume measurements in the Weddell seal, however, are based on data obtained from two seals (25). To more accurately estimate the quantity of sequestered RBCs, we performed direct measurements of RBC and plasma volumes by using indicatordilution techniques. Our overall hypothesis was that increased sympathetic nervous system activity during diving, which would be reflected by increased plasma concentrations of epinephrine and norepinephrine, would cause the spleen to contract (19). This contraction would increase the hemoglobin concentration and hematocrit of circulating blood. After the seal surfaced from diving, plasma catecholamine concentrations should decrease. This would be followed by splenic relaxation and reductions in circulating hemoglobin concentration and hematocrit. To test this hypothesis, we studied changes of plasma epinephrine and norepinephrine concentrations, spleen size, hemoglobin concentration, and hematocrit while the seals were anesthetized and unstimulated or after intravenous epinephrine administration and while awake after the seal surfaced from voluntary isolated hole dives.
SEAL (Leptonychotes weddelli) is capable of dives to depths of X00 m and ~1 h in duration. Dives of up to 20 min in duration usually take place under aerobic conditions, suggesting that highly refined strategies control the storage and utilization of oxygen (21, 22). Because the seal’s lungs collapse during diving, the oxygen available to the diving seal is largely limited to stores in blood and myoglobin (13). In this study, we sought to explore the variation of blood oxygen stores during voluntary isolated hole diving. The circulating hemoglobin concentration of Weddell seals increases by 60% during the first lo-12 min of a dive (31). It has been hypothesized that the spleen of the Weddell seal contracts, similarly to the contraction observed in exercising horses (27,28) and other terres-
SeaZ characteristics and fieZd techniques. All studies were performed under US National Marine Fisheries Service Marine Mammal Permit no. 600 and approved by the Massachusetts General Hospital Subcommittee on Research Animal Studies. Five subadult male Weddell seals [estimated body wt 365 t 50 (SD) kg; Ref. 91 inhabiting the annual sea ice near Ross Island, Antarctica (168” E, 78” S) were captured and sledged to a field camp constructed for isolated hole diving (23, 31). The location was chosen to be far from natural ice cracks so that a seal released at this site would be likely to return to the drilled holes to breathe. At the site, two
298
the American
THE WEDDELL
0161-7567/96
$5.00
Copyright
o 1996
METHODS
Physiological
Society
SPLENIC
CONTRACTION
DURING
1.3-m-diam holes were drilled through the 2-m-thick annual sea ice of McMurdo Sound. A fish hut, with a hole in its floor, served as a field laboratory and was placed over one hole. The second hole provided a route for the seal to enter the water and haul out. This hole was blocked during the study so that the seal had to surface at the hole inside the field laboratory to breathe between dives. The animal was isolated in a sled for at least 8 h to permit gastric emptying before anesthesia. General anesthesia was induced with ketamine (0.15-0.25 mg/kg) administered via an extradural intravertebral vein and maintained by mask inhalation of l-4% halothane in oxygen administered via a to-and-fro circuit. In all seals, an extradural intravertebral vein was cannulated with a 20-cm double-lumen intravenous catheter placed in the midline -10 cm above the iliac crest. The catheter was tunneled subcutaneously and sutured to the skin. In two seals, a small skin incision was made over a foreflipper artery and an arterial catheter (model 93A-741H7.5F, Baxter Healthcare, Irvine, CA) was advanced into the aorta via an arteriotomy. Extension tubing (2 m) was attached to the vascular catheters and filled with absolute ethanol to prevent freezing until the seal returned to the sea. The free end of each extension tubing was fitted with a three-way stopcock and attached to a small plastic float. Whenever the seal was at the breathing hole, the float would bring the catheters and stopcocks to the seawater surface. The catheter tubing could then be quickly retrieved and blood samples withdrawn without disturbing the seal in the breathing hole. The first 10 ml of each blood sample withdrawn from the catheter were discarded. Patency of the catheters was maintained by flushing with a solution of 10% ethanol and heparin (100 U/ml) in 0.9% saline periodically and after each blood withdrawal. A custom-built microprocessor (Wildlife Computers, Woodinville, WA) that logged time, depth, swimming velocity, and near-infrared muscle absorbance (15) was glued (Loctite 422 adhesive) to the fur of each seal. After full recovery from anesthesia, the seal was released into the water via the outside hole. At the conclusion of each study, the outside hole was reopened. After the seal hauled out, it was reanesthetized and the catheters and monitors were removed. After recovery, the seal was returned to its capture site and released. Ukrasonography. Splenic size was measured with a realtime 2.5- or 3.5-MHz mechanical sector transducer and a portable ultrasound scanner (Ausonics MI 1000, Universal Medical Systems, Bedford Hills, NY). Real-time images were recorded on VHS videotape. Standardized views were obtained along the left posterolateral cephalocaudal axis beginning caudal to the foreflipper. A ruled l-cm-thick 70-cm-long neoprene strip was glued to the seal’s fur. This strip guided the transducer’s movement and permitted reproducible imaging of the seal while anesthetized in the field laboratory and subsequently while awake in the ice hole. The seawater provided excellent acoustic coupling, and bubble reflections were absent. The cephalocaudal length was measured directly. Because of the ventral orientation of the spleen tip, this was often not the maximal dimension of the spleen. The maximal splenic transverse thickness on the video images was measured by using the ultrasound scanner’s software calipers. Plasma volume and RBC volume measurements. Plasma volume was measured by the dilution of indocyanine green (Sigma Chemical, St. Louis, MO). While the seal was anesthetized, 40-100 mg of indocyanine green were rapidly injected via the extradural intravertebral vein catheter. Timed blood samples were withdrawn via a second extradural intravertebra1 vein catheter over the next 24 min (0, 3,6, 9, 12, 18, and
VOLUNTARY
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299
24 min). The absorbance of indocyanine green in plasma was measured spectrophotometrically against a plasma blank at 805 nm. Lipemic specimens were not analyzed. Plasma volume was calculated by extrapolating the indocyanine green clearance to time 0. Autologous RBCs were labeled with “‘Cr by standard techniques (red cell tagging kit 374, Mallinckrodt Medical, St. Louis, MO). While the seal was anesthetized, 35 ml of blood were withdrawn, anticoagulated with 10 ml anticoagulant citrate-dextrose solution, and labeled in vitro with 333 uCi (12.2 MBq) of sodium .51Cr After the labeling was terminated by the addition of 50 mg of ascorbic acid, 25 ml of labeled RBC suspension were reinjected intravenously. Timed blood samples were withdrawn over the next 60 min (3,6,9,12,18, 21, 25, 30, 40, and 60 min) and again -24 h after labeling. Whole blood and plasma samples of withdrawn blood and the injected RBC suspension were counted for a minimum of 10,000 counts corrected for background (1275 Minigamma, LKB Wallac, Turku, Finland). RBC volume was calculated from standard formulas by using the values for whole blood and plasma activity of terminal-phase blood samples. Hemoglobin was determined by the cyanmethemoglobin method (kit 525A, Sigma). Hematocrit was determined by microcentrifugation (model MHCT II, Adams). An extradural vein was used as our blood sampling site in this study. Conceivably, blood within these veins might be subject to hemoconcentration or sludging compared with arterial samples. We therefore compared aortic and extradural vein hemoglobin concentrations and hematocrit in two seals (seaZs 4 and 5; 25 paired samples). No systematic difference between the two sampling sites was apparent (P > 0.15 by paired t-test; r > 0.9 for comparisons of both hemoglobin concentration and hematocrit). The coefficient of variation between the two sampling sites was 0.026. Plasma epinephrine and norepinephrine concentrations. Blood samples for plasma epinephrine and norepinephrine concentrations were withdrawn from indwelling catheters and immediately centrifuged, and the plasma was decanted and stored at -80°C until assayed. Catecholamine concentrations were determined by using standard high-performance liquid chromatography techniques (Endolab, Christchurch, New Zealand). Experimental protocol. Measurements of hemoglobin concentration, hematocrit, plasma epinephrine and norepinephrine concentrations, plasma volume, and splenic dimensions initially were performed while the seal was anesthetized. To simulate sympathetic stimulation, 1 or 2 mg epinephrine (Elkins-Sinn, Cherry Hill, NJ) were injected via the extradural vein catheter. Hemoglobin concentration, hematocrit, and splenic dimensions were measured 3, 6, 9, and 12 min before and 3, 6, 10, 15, 25, and 45 min after the epinephrine injection. Blood samples for the determination of RBC volume were withdrawn after the epinephrine injection to maximize mixing of labeled cells with the splenic blood pool. Measurements obtained after the seal surfaced from diving included hemoglobin concentration, hematocrit, plasma epinephrine and norepinephrine concentrations, and splenic dimensions. The five Weddell seals included in this report were also the subjects of parallel protocols that studied hormonal regulatory adjustments and myoglobin saturation during voluntary diving. The results of these protocols are published elsewhere (15, 19). Hochachka et al. (19) describe and analyze in detail the plasma catecholamine measurements included in the present study. Statistics. Multiple comparisons among values obtained under anesthesia and at different times after the seal surfaced from diving were performed by using an analysis of
300
SPLENIC
Table 1. Dive, sampling,
Seal No.
1 2 3 4 5 Average Values
are means
CONTRACTION
VOLUNTARY
DIVING
and blood volume data
No. of Dives
No. of Samples
Dive Depth,
12 4 28 10 16 14*9
29 8 65 19 35 31221
NA 102 * 85 2 NA 108 t 95 k
? SD. RBC,
DURING
red blood
m
59 78 74 74
Total RBC Volume, liters
Dive Duration, min
12.2 16.3 18.2 22.5 14 16.7
* + + + + +
5.8 10.6 12.1 10.3 10.7 10.8
36.8 41.0 35.4 40.5 35.3 37.8 5 2.8
Total Plasma Volume, liters
Total Blood Volume, liters
29.8 29.0 28.6 32.0 31.1 30.12 1.4
66.6 70.0 64.0 72.5 66.4 67.9 + 3.3
cell; NA, not available.
variance with repeated measures. Because the number of measurements per seal obtained during each phase varied, reporting simple averages and contrasts derived from pooled data might be misleading. Accordingly, mean values for each phase were calculated for each seal. These mean values were then averaged to calculate group values of means t SD for each time period. Each value was also expressed as a percentage of its maximal value. This was done to reduce the error in determining rates of change. When single comparisons were made, a paired t-test was used. Changes over time of hemoglobin concentration, hematocrit, and spleen size were best fitted to monoexponential models by using linear regression techniques. Half-times (tl/,> for the rates of change were calculated from the linear regression model and are expressed as 2 95% confidence intervals. P < 0.05 was considered significant. RESULTS
General characteristics. Data from 70 dives of the 5 seals were available for analysis (Table 1). In three of these seals the depth of dives was successfully recorded. The depth of the dives averaged 95 2 74 m (range 8-264 m), and the dives lasted 16.7 ? 10.8 min (range 1.8-42.7 min). Thirty-eight of the 70 dives (54%) lasted >17 min. Splenic size. Immediately after the seal surfaced, the dimensions of the spleen were -65% of resting surface values (Table 2; Figs. 1 and 2). During the first 2 min after the seal surfaced, the average values recorded were 23 t 2 cm (71 t 2% of maximal resting values; P < 0.05) for cephalocaudal length and 8.1 t 0.5 cm (71 t 4% of maximal resting values; P < 0.05) for splenic thickness (Table 2). Subsequently, the size of the spleen increased. The tl/, values for the increase of splenic length and thickness after the seal surfaced were 8 t 7 (mean _ + 95% confidence limits) and 7 t 4 min, respectively (Fig. 2, A and B), and were statistically identical. Hemoglobin concentration, hematocrit, and catecholamine concentrations. On surfacing, the hemoglobin concentration and hematocrit were increased -67% above the baseline obtained during anesthesia (Fig. 3, A and B). The hemoglobin concentration increased from 17.5 5 5.3 g/d1 (measured under anesthesia) to 21.9 t 3.7 g/d1 (measured during the first 2 min after the seal surfaced; Table 2; P < 0.05). At the same times, the hematocrit increased from 44 t 12 to 55 2 8% (Table 2; P < 0.05). These values decreased after the seal . surfaced. The tl/, was 13 2 7 min (mean t 95% con-
fidence limits) and 12 t 6 min (mean it 95% confidence limits) for reductions of hemoglobin and hematocrit, respectively (Fig. 3, A and B). Plasma epinephrine and norepinephrine concentrations were increased during the first 2 min after the seal surfaced compared with samples taken later after the seal surfaced or during general anesthesia (Table 2; P < 0.05 for all comparisons). Changes of splenic size after the seal surfaced were inversely correlated with circulating plasma catecholamine concentrations. Figure 4 shows the relationship between percent maximal splenic thickness and the plasma epinephrine concentration ( [epinephrine] > (%maximal splenic thickness = 160 - 17ln([epinephrine]); r = 0.66, P < 0.05). Similar correlations were demonstrable between the percent maximal splenic length and plasma epinephrine concentrations (%maximal splenic length = 177 - 18.71n[epinephrine]; r = 0.64, P < 0.05) and between splenic size Table 2. Hemoglobin, hematocrit, and splenic dimensions during anesthesia and after surfacing from diving in five Weddell seals Time After Anesthetized
Hematocrit,
%j
44212
60) Hematocrit, %maximal Hemoglobin concentration, g/d1 Hemoglobin, %maximal Plasma epinephrine, rig/I Plasma norepinephrine, rig/I Cephalocaudal splenic length, cm Splenic length, %maximal Splenic thickness, cm Splenic thickness, %maxima1
73 + 14 (60) 17.5 + 5.3 (63)
o-2
55 + 8::: (34) 93k 4::: (34) 21.9 + 3.7:‘: (34)
72 + 14
91-+ 5:i:
(63) 140 5 56 (13) 3612 97 (13) 36-+11 (3)
(34) 204 t 45:‘: (27) 988 t 318:‘: (27) 23 + 2:;: (15)
9352 (3) 11.6 -+ 0.7 (4) 9927 (4)
71 2 2:i: (15) 8.12 0.5 (31) 7124 (31)
Surfacing, 2-10
50 + 11*i (34) 84 + 9:‘:~I20.7?
(34) 5.1”:j(34)
85 + CJK-/(34) 151 ?I 37”r
min >lO
42 +- lp$ (44) 70 + 12** (44) 17.2 + 5.13 (44)
662 + 332i(32) 25 + 4:::
70 k 12$ (44) 111 + 21t (41) 485 + 296t (41) 32 k 3:1:$
(14)
(14)
78 + 4:i:
97 + 4”: (14) 11.1 + 0.6*$
(32)
(14) 9.3 ? 0.6 (34) 7855 (34)
(18)
95 + 5:‘:f (18)
Values are means ? SD; nos. in parentheses are no. of samples. Significantly different (P < 0.05 by analysis of variance) from: ‘i’ anesthetized; f 0- to 2-min value; $ earlier surface values.
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CONTRACTION
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VOLUNTARY
301
DIVING
Response to intravenous epinephrine. In four seals, l-2 mg epinephrine were administered via the extradural vein catheter while the animal was anesthetized. This injection produced a peak plasma epinephrine concentration of 58,000 Ifr 28,000 rig/l (>400-fold increase over resting values). Plasma norepinephrine concentrations were unchanged (maximum concentration 509 t 232 rig/l). The size of the spleen, measured successfully in three seals, decreased within 3 min after epinephrine administration. Within 6 min after the epinephrine injection, splenic length decreased from 34 t 12 to 26 I 11 cm; thickness decreased from 10.6 t 1.0 to 9.2 +- 1.8 cm (Fig. 5). Hematocrit slowly increased from 48 ? 10% preinjection to 54 t 8% 15 min after the administration of epinephrine. Hemoglobin increased similarly from 18.8 t 3.6 to 21.3 t 2.4 gldl.
a
0
Seal 1
+
Seal2 Seal3
0
Seal4
4
Seal
5
0 1
Fig. 1. Representative left posterolateral intercostal sonograms of seal 5 1.5 (A) and 17 min (B) after it surfaced from 134-m dive lasting 26.8 min. Splenic thickness increased from 7.4 to 12.5 cm during this time. S, spleen. Dark vertical stripe overlying splenic image in B is caused by sound reflected by overlying rib.
changes and plasma norepinephrine concentrations ([norepinephrinel) (%maximal splenic thickness = 126 - &6ln[norepinephrinel, r = 0.42; %maximal splenic length = 175 - 14.2ln[norepinephrinel, r = 0.65; P < 0.05 for both correlations). Plasma epinephrine, but not norepinephrine, concentrations were also correlated with changes of hemoglobin concentration ([hemoglobin]) (%maximal [hemoglobin] = -6.4 + 17.5lnlepinephrinel; r = 0.58, P < 0.05) and hematocrit (%maximal hematocrit = -0.6 + 165ln[epinephrine]; r = 0.57, P < 0.05) after the seal surfaced. Compared with dives lasting ~17 min, longer dives demonstrated a greater degree of splenic contraction and a larger increase of hemoglobin concentration and hematocrit in the first 2 min after the seal surfaced (Table 3; P < 0.05 for all comparisons). Plasma epinephrine and norepinephrine concentrations during the first 2 min after the seal surfaced were greater in dives lasting >17 min. For all sonographic, hematologic, and plasma catecholamine measurements obtained later than 2 min after the seal surfaced, no differences between dives ~17 or >17 min were found.
10
100
200
10
100
200
B
1
Minutes Fig. 2. A: splenic length after seals surfaced. Line indicates regression for pooled data: 32.0 - 10.8e( m1n/13 3); r = 0.79, half-time (tJ = 9 2 7 min (mean i 95% confidence limits). When splenic length is expressed as percentage of maximal value for each seal, regression line for the pooled data is 102.7 - 33.7e(-m1ti11 7); r = 0.78, tM= 8 2 7 min. B: splenic thickness after seal surfaced. Line indicates regression for pooled data: 11.6 - 4.2e(-mm1103); r = 0.83, tx= 7 t 4 min. When splenic thickness is expressed as percentage of maximal value for each seal, regression line for pooled data is 94.3 - 36.0e(-m’D/9 5); r = 0.83, tx= 7 2 4 min.
302
SPLENIC
30
CONTRACTION
DURING
VOLUNTARY
z
r
DIVING
loo-
iE s w cn 8 5 0 .-
0
no
b +
90 -
+*
+
l
0
Seal
1
0
Seal
2
+
Seal
3
Cl
Seal
4
+
Seal
5
+
80-
\
+
70-
t
50 I
0
lb0
260
Epinephrine
360
400
(rig/L)
Fig. 4. Relationship between plasma epinephrine concentrations ([epinephrine] > and percent maximal splenic thickness measured after seals surfaced from voluntary isolated hole diving. Line indicates regression for pooled data: 160 - 17ln[epinephrine]; r = 0.66, P < 0.05.
a 3o
0
20- 0 lo-
1
Seal
1
+
Seal2 Seal 3
cl
Seal
4
l
Seal
5
a
a.
0
17.7 liters of RBCs were circulating at rest {circulating RBC volume = plasma volume X [hematocrit/(l hematocrit)]). This suggests that -20.1 liters of RBCs (30% of total blood volume) were sequestered while the seal rested at the surface (Fig. 6).
a a
f .a
#a
0, 0
.a
DISCUSSION 10
100
200
This study provides direct sonographic evidence that the spleen of the Weddell seal is an active and major
Minutes Fig. 3. A: venous hemoglobin concentration changes after seals surfaced. Line indicates regression for pooled data: 13.8 + g . 29 - tnitd22.3); r = 0.56, tl/,= 16 ? 14 min. When venous hemoglobin concentration is expressed as percentage of maximal value for each seal, regression line for pooled data is 61.0 + 33.1e(-t11i”/1s.3); r = 0.73, t?/, = 13 + 7 min. B: venous hematocrit changes after seals surfaced. Line indicates regression for pooled data: 33.9 + 22.6e’ min/22.4); r = 0.60, tl/, = 16 t 12 min. When venous hematocrit is expressed as percentage of maximal value for each seal, regression line for pooled data is 61.6 + 34.3e’ mmin/17.1); r = 0.78, tl/, = 12 t 6 min.
Blood volume measurements. Erythrocyte volume, measured directly by 51Cr labeling of all five seals, was 37.8 t 2.8 liters (Table 1, Fig. 6). Plasma volume, measured directly by indocyanine green dilution (9 measurements of 5 seals), was 30.1 2 1.4 liters. Summing these two values yields a value for total body blood volume of 67.9 t 3.3 liters or -186 ml blood/kg body mass. These values are equivalent to a calculated total hematocrit (assuming no splenic sequestration) of 56 t 2%. This value is virtually identical to the average maximal hematocrit measured within the first 2 min after the seal surfaced (55 t 8%). Minimal and maximal average hemoglobin concentration values were 15.3 t 4.8 and 24.3 t 3.2 g/d& and the values were 37 2 11 and 59 5 8% for hematocrit. Based on a measured average plasma volume of 30.1 liters and a minimum hematocrit of 37%, we estimate that
Table 3. Hemoglobin, hematocrit, plasma catecholamine concentrations, and splenic dimensions after surfacing from short (4 7 min) and long (217 min) dives in five Weddell seals
Hematocrit,
%
Hematocrit,
%maximal
Hemoglobin g/d1 Hemoglobin,
concentration,
Plasma
%maximal
epinephrine,
rig/l
Plasma norepinephrine, rig/l Cephalocaudal splenic length, cm Splenic length, %maximal Splenic
thickness,
cm
Splenic ma1
thickness,
%maxi-
Values samples surfaced
Dives Cl7 min
Dives 217 min
4929 (17) 8826 (17) 19.7 -+ 4.2 (17) 8625 (17) 190 + 71 (15) 8002373 (15) 26-+3 (7) 80 t 10 (7) 9.12 1.3 (14) 7429 (14)
57+7 (17) 95+4 (17) 22.5 ~3.4 (17) 9226 (17) 263583
are means + SD; nos. in parentheses and measurements were taken within from a dive.
P Value
0.007 0.0003 0.043 0.005 0.021
(12) 13262571
0.008
(12) 2153 (8) 6327 (8) 8.2 k 1.1 (17) 6529 (17)
0.007 0.0018 0.043 0.009
are no. of samples. All 2 minutes after seals
SPLENIC
E 2
CONTRACTION
DURING
20 1
Minutes Fig. 5. Mean splenic length and thickness, venous hemoglobin concentration, and hematocrit in anesthetized Weddell seals before and after intravenous injection of l-2 mg epinephrine [time 0 (solid vertical line), time of injection]. Splenic length and thickness (n = 3 seals) decreased immediately after intravenous injection of epinephrine. This was followed by increase of hemoglobin concentration and hematocrit (n = 4 seals). Error bars omitted for clarity.
reservoir of RBCs and contracts on stimulation by exogenous epinephrine infusion. In the awake voluntarily diving seal, the size of the spleen increased within minutes of surfacing. This increase was followed by reductions of the circulating hemoglobin concentration and hematocrit. Plasma catecholamine levels were increased above resting values in the first 2 min after a dive and decreased to resting levels within 10 min of surfacing. Decreases of plasma catecholamine concentrations correlated with increases of spleen size after the seal surfaced. In the anesthetized seal, reductions of spleen size were induced by the intravenous injection of epinephrine and were followed by increases of the circulating hemoglobin concentration and hematocrit. These findings suggest that catecholamine release, splenic contraction, and graded increases of the hemoA
Bl1 Plasma
60
1
l-4 3OL
Plasma 3OL RBC 18L
RBC 2OL
RBC 38L
0
Fig. 6. Blood volumes of Weddell seal during resting conditions (A) and after splenic contraction (B). Plasma volume was determined from dilution of indocyanine green, and red blood cell (RBC) volume was calculated from dilution of 51Cr-labeled RBCs in 5 Weddell seals (mean body mass 365 kg). During resting conditions, 20 liters of RBCs were sequestered, presumably in the spleen. This is equivalent to 53% of all RBCs and-30% of total blood-volume. During diving, these cells entered circulation and peripheral hematocrit increased from 37 to 56%. Circulating blood volume increased from 14 to 20% of body mass.
VOLUNTARY
DIVING
303
globin concentration and hematocrit are integral components of the diving response of Weddell seals (15,19). To our knowledge, plasma catecholamine concentrations in voluntarily diving Weddell seals have not been reported previously. Because an intravenous injection of epinephrine produced decreases of spleen size and subsequent increases of hemoglobin concentration and hematocrit, it is likely that sympathetic stimulation of smooth muscle cells within the spleen, its vasculature, and/or the splenic capsule produced the splenic size reductions and the hematologic changes that we observed during free diving. The correlation of plasma catecholamine concentrations with changes of splenic size after the seal surfaced was nonlinear. Progressively higher catecholamine concentrations correlated with only minor further reductions of spleen size. Such a correlation would be likely because once the spleen was almost completely contracted, further increases of catecholamine levels would be expected to produce minimal changes (19). Extremely large concentrations of epinephrine resulting from intravenous administration, for example, produced similar changes of spleen size and increases of hemoglobin concentration and hematocrit to those measured after voluntary dives. Plasma norepinephrine concentrations did not appear to be correlated with changes of the hemoglobin concentration or hematocrit after the seal surfaced. This suggests that circulating epinephrine may be an important hormone in the control of splenic contraction and RBC redistribution in the free-diving seal. Additional mechanisms undoubtedly regulate splenic size and RBC distribution in the Weddell seal. The variations of plasma catecholamine concentrations that we measured do not completely account for the changes of hemoglobin concentration, hematocrit, or spleen size (r values ranged between 0.42 and 0.66 in our study). It is likely that direct neural stimulation of splenic contraction is important, as evidenced by the rich innervation of the Weddell seal spleen (33). Direct neural activity was not examined in our study and would not be accurately reflected by circulating catecholamine concentrations. Kooyman et al. (23) reported that hematocrit was increased above resting values in the Weddell seal immediately after diving. They reported a mean resting arterial hemoglobin concentration of 17.4 ? 1.2 g/dl. Immediately after the seal surfaced, the hemoglobin concentration was at least 20 g/d1 and after a 59-min dive was noted to be 25.5 g/dl. These increases appeared to be related to the duration of the preceding dive. Qvist et al. (31), using a microcomputer-controlled blood sampler, demonstrated that the hemoglobin concentration and hematocrit of voluntarily diving Weddell seals increased nearly 60% during the first lo-12 min of the dive and was maximal on surfacing. Qvist et al. suggested that these changes might be due to splenic sequestration of RBCs during periods of rest and hypothesized that the spleen might contract and release RBCs during the dive. In the present study, we learned that hemoglobin concentration and hematocrit decreased after the seal surfaced, with tl/, values of
304
SPLENIC
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DURING
12-13 min. Values were lowest during periods of rest (at least 10 min after the seal surfaced) or sleep at the surface. These hematologic changes are probably independent of exercise, hypoxia, or changes of hydrostatic pressure or position during diving because similar hematocrit variations have been observed in elephant seal pups during periods of sleep apnea (7). Because the changes of spleen size, hemoglobin concentration, and hematocrit were time dependent, minimal changes of these parameters would be expected if time at the surface was very limited, as is commonly observed during bouts of foraging dives (8). In terrestrial mammals such as dogs, cats, sheep, goats, pigs, and horses, large increases of hematocrit occur during exercise or physical restraint (1,2, 16,27, 34). The induced polycythemia is almost completely abolished by splenectomy, providing evidence that the spleen is the major reservoir for concentrated RBC storage in these animals. Intravenous administration of epinephrine or norepinephrine to goats (1) and intravenous epinephrine administration to pigs (16) and horses (27) similarly increase the hematocrit. This effect was abolished by splenectomy in these studies. In humans, splenic contraction has been reported to occur in response to exercise (14,24), after the subcutaneous injection of epinephrine (32), and after bouts of breathhold diving (20). Concomitant increases of venous hemoglobin concentration and hematocrit have been reported after both human exercise and repetitive breath-hold diving (14,20,24). During anesthesia, the size of the Weddell seal’s spleen, the hemoglobin concentration, and hematocrit were slightly greater than awake resting values (See Table 2). While plasma catecholamine concentrations did not differ statistically between the awake and anesthetized state, our results suggest that sympathetic tone during anesthesia was slightly increased, perhaps because of the light stage of anesthesia employed. Our findings are consistent with the suggestions of Ponganis et al. (30) that values for spleen size may be less than maximal under general anesthesia and might vary with anesthetic state. Spleen as a RBC reservoir. The large relative size of the Weddell’s spleen in relation to other species has been discussed in several reports (3, 5, 6, 30, 31). Castellini and Castellini (6) suggested that the increased splenic mass of seals is a consequence of their large blood volume and is correlated among various mammalian species with the blood volume-to-body mass ratio. Splenic mass has been measured directly or indirectly in several Phocid species and ranges from 0.18 to 0.16% of body mass for the Crabeater seal (Lobodon carcinophagus) and Ross seal (Ommatophoca rossi) (4) to -7-10% of body mass for the Northern elephant seal (Mirounga angustirostris) (6) and Weddell seal (6, 31). These values are calculated from the weights of autopsy specimens or from estimates of RBC sequestration derived from measurements of plasma volume and peripheral blood hematocrit changes. Direct in vivo splenic size measurements have been reported by Ponganis et al (30). Using computed tomog-
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raphy of restrained or anesthetized animals, they noted spleen size to be -0.8-30/c, of body mass in three harbor seals (Phoca vitulina) and two California seal lions (Zalophus cal i f ornianus). Measurements of splenic contractility were not reported. That the spleen of the seal may act as a reservoir for RBCs has been suggested previously (5,9,31). Schumacher and Welsch (33) conducted detailed structural studies of the Weddell seal spleen. They classified the Weddell seal spleen as a storage spleen by demonstrating a well-developed trabecular system within the spleen and a capsule rich in innervated smooth muscle cells. The red pulp was innervated by numerous cholinergic and adrenergic nerve fibers (33). The presence of a storage spleen may permit an animal to intermittently maintain a larger circulating blood volume than otherwise possible and to alter the circulating hematocrit in response to neural and/or circulating catecholamine stimulation. The pig, for example, sequesters 20-25%) of its total body RBC volume within the spleen, turning over sequestered cells at a rate of -7%/min (16). Horses have been estimated to sequester up to 54%) of their total RBC volume within the spleen (28). After recovery from a splenectomy, however, the horse’s total blood volume is reduced and the hematocrit, in response to exercise or epinephrine administration, remains relatively fixed at levels markedly below peak presplenectomy exercise values (27). The degree of resting RBC sequestration can be estimated from the difference between the circulating and total RBC volumes. Estimates of total blood volume solely derived from measurements of plasma volume and the observed peripheral hematocrit values could be underestimations because complete splenic contraction cannot be ensured. We therefore chose to directly and independently measure RBC and plasma volumes in the present study. The five seals we studied had an average total blood volume of 68 liters (186 ml/kg body mass) and an average plasma volume of 30 liters. We estimate that -20 liters of RBCs (30% of the total blood volume) were stored within the spleen at rest. Our estimates may be slightly low because one of the seals (seal 1) was anemic at baseline compared with the other four seals we studied (see Fig. 2, A and B). Blood volume has been determined by exsanguination in several Phocid species. The blood volume of the Crabeater seal and the Ross seal is -10% of body mass (4). The blood vo 1ume of the Northern elephant seal and the Southern elephant seal (Mirounga Leonina) is 12 and 15% of body mass, respectively (5,12). Direct blood volume measurements of the Weddell seal have been limited to the values obtained by Lenfant and co-workers (25) in two adult Weddell seals anesthetized with halothane. They reported a mean blood volume of 55.7 liters or 148 ml/kg body mass. These values are slightly smaller than the 67.9 liters or 186 ml/kg body mass that we measured and probably represent underestimates. The spleen may not have been completely contracted at the time of study and the mixing of labeled cells may have been incomplete when
SPLENIC
CONTRACTION
DURING
the blood samples were withdrawn. Complete mixing of labeled cells may be delayed in animals with storage spleens. For example, Anderson and Rogers (1) observed slow mixing of Cr-labeled RBCs in goats. Such delayed mixing was not observed in a splenectomized goat or animals receiving an epinephrine injection immediately before receiving the labeled cells (1). In the present study, blood sampling was performed after the intravenous injection of epinephrine. In addition, because Cr-labeled RBCs are stable for over 24 h, we were able to ensure complete mixing by following the time-activity curve of the blood for many hours after injection. Stable values from the terminal portion of the time-activity curve were used to calculate the blood volume (see METHODS). Ponganis and co-workers (29) reported the plasma volume of Weddell seals to be -7% of body mass and the circulating blood volume to be 14.5% of body mass when anesthetized and 21% of body mass immediately after the seal surfaced. In their report, plasma volume was calculated from the dilution of Evans blue and blood volume was estimated from plasma volume measurements and the venous hematocrit. The values we measured in our present study (plasma volume, 8% of body mass; circulating blood volume when anesthetized, 14% of body mass; circulating blood volume after the seal surfaced, 20% of body mass) are in close agreement with those reported by Ponganis et al. Blood released from the spleen may be accommodated by the large capacity of the inferior vena cava and hepatic sinuses (17). Elsner and co-workers (12) reported that the inferior vena cavae of Northern elephant seals had reservoir volumes of 20-25 liters (-20%) of total bl oo d vo 1ume). Inferior vena cava blood had an increased oxygen content compared with arterial blood in the latter half of forced dives (10). This finding suggests that oxygenated blood had entered the vena cava, presumably from the spleen, during the dive. A striated muscle sphincter is located in the inferior vena cava at the level of the diaphragm in several Pinnipedae (17). Constriction of this sphincter might control the rate of blood returning to the heart and the central circulation (10, 17). The inferior vena cava and the hepatic sinuses may thus serve as reservoirs for RBCs. The delay of several minutes that we observed between the changes of spleen size and changes of peripheral venous hemoglobin concentration or hematocrit probably represents the slow rate of equilibration among the splenic, vena caval, and peripheral circulatory blood pools. Blood oxygen stores and the role of the splenic reseruoir. Polycythemia may assist the uptake of oxygen and unloading of carbon dioxide during the brief surface time between dives (31). This would minimize the surface time necessary for respiratory gas exchange during diving bouts. The reduction of hematocrit occurring when surface time is prolonged, such as during rest or sleep, would prevent problems such as thrombosis or sludging associated with sustained polycythemia and the increased viscosity of Weddell seal blood (11, 18, 26). Splenic contraction at depth would inject
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Table 4. Calculation of blood oxygen stores in the Weddell seal Blood
Spleen Arterial Venous Total
Oxygen
Stores
(present
data)
20.1 liters x 400 g Hb/l x 1.34 ml 02/g Hb 0.33 x 478 dl X 15.3 g Hb/dl X 1.34 ml 02/g Hb x (0.95 - 0.2) 0.67 x 478 dl x (15.3 g Hb/dl X 1.34 ml 02/g Hb x 0.95) - 5 ml OJdl
02,
liters
8.1 2.4
4.6 15.1
Values are averages for 5 Weddell seals studied (approximate weight 365 + 50 kg). Values derived from the present study: splenic blood volume 20.1 liters; circulating blood volume 47.8 liters; average minimum resting hemoglobin (Hb) concentration is 15.3 g/dl. The following assumptions are from Ponganis et al. (30): 1 liter packed cells (PC) contains 400 g Hb; Oz-carrying capacity is 1.34 ml On/g Hb; 33% circulating blood volume is arterial, and 67%) is venous; initial arterial saturation is 95% and end-arterial saturation is 200/o; and venous extraction is initial arterial 02 content - 5 ml O&O0 ml. Adapted from Ponganis et al. (30).
oxygenated RBCs, previously sequestered at ambient pressure, into the central circulation (12). This can increase peripheral oxygen delivery and dilute high blood nitrogen tensions occurring during descent (31). When the seal surfaced, the increased hemoglobin concentration would increase the ability to take up oxygen and release carbon dioxide at the surface between dives. Ponganis and co-workers (30) have estimated the oxygen-carrying capacity of arterial and venous blood in the Weddell seal. Employing their assumptions for the oxygen-carrying capacity and arterial oxygen saturation of hemoglobin, and the partitioning of circulating blood volume into arterial and venous components in the seals we studied (approximate weight 365 kg), we estimate that splenic blood contained 8.1 liters of oxygen bound to hemoglobin when the seal rested at the surface (Table 4). We estimate the seal’s total blood oxygen-carrying capacity at rest to be 15.1 liters or 42 ml OJkg body mass. These values are in excellent agreement with the estimate of 44 ml OJkg body mass previously reported by Ponganis et al. Limitations of this study. Our data provide direct evidence that the marked increases of peripheral hemoglobin concentration and hematocrit occurring during diving are primarily due to splenic sequestration. Splenic sequestration could be examined further by imaging the changes of radiolabeled RBC activity over the splenic bed or by repeating the experiments after a splenectomy is performed. In our present study, sufficient radioactivity to permit external counting or imaging was not present after the RBCs were labeled with “lCr This was due to the limited amount of activity that we administered and to signal attenuation by the seal’s overlying 10 cm of blubber. Performing a splenectomy in the Weddell seal was considered inappropriate because satisfactory healing of a laparotomy incision would be unlikely under Antarctic field conditions. Ultrasonic imaging suggested that the spleen decreased -35% in linear dimensions during diving. Ultrasonic splenic imaging of an awake unrestrained
306
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CONTRACTION
DURING
Weddell seal swimming within an ice hole is obviously problematic. Because of the orientation of the spleen and the position of the seal within the dive hole, the linear dimensions that we measured by ultrasound probably are not maximal. This is especially . likely for length measurements becaus #eth e caudal portion of the spleen was obscured by the left kidney. We also were only able to image the sple en i n two dimensions. It is poss ible that contractio n of the spleen . was not uniform in all axes. The rel .ative change of these di .men sions, however, should be directly proportional to the maximal change of each respective linear dimension. The splenic size changes that we measured after the seal surfaced provide direct evidence that splenic contraction is an integral part of the voluntary diving response of the Weddell seal.
15.
16.
17.
18.
19.
The authors thank Antarctic Support Associates and US Navy Squadron VXE-6 for field assistance in Antarctica. These studies were funded by the National Science Foundation Division of Polar Programs Grant DPP 91-18192. Address for reprint requests: W. E. Hurford, Dept. of Anesthesia, Massachusetts General Hospital, Boston, MA 02114.
20.
Received
21.
22 May
1995; accepted
in final
form
21 August
1995.
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