- Email: [email protected]
Pressor and hemodilution responses compensate for acute hemorrhage in bluefish
Comp. Biochem. Physiol. Vol. 91A, No. 4, pp. 80%813, 1988
0300-9629/88 $3.00 + 0.00 © 1988 Pergamon Press plc
Printed in Great Britain
PRESSOR AND HEMODILUTION RESPONSES COMPENSATE FOR ACUTE HEMORRHAGE IN BLUEFISH CHRISTOPHER S. OGILVY,* P. GLENN TREMML* and ARTHUR B. DuBoIS John B. Pierce Foundation Laboratory and Yale University, 290 Congress Avenue, New Haven, CT 06519, USA. Telephone: (203) 562-9901
(Received 14 April 1988) Abstract---1. After hemorrhage of 21% blood volume (0.9% body weight) blood pressure (BP) and heart rate (H.R.) of unanesthetized bluefish (Pomatomus saltatrix) recovered within 5 min. 2. Phentolamine blocked this recovery. 3. Atropine increased control H.R. from 48 to 87 per min, and to 108 after hemorrhage, with delay of BP recovery to 10 min. 4. With small, repeated hemorrhages every 20 min, hemodilution and recovery of BP occurred between hemorrhages. Removal of 27% blood volume resulted in only temporary recovery. 5. Thirty rain after hemorrhage, plasma epinephrine was 5 × and norepinephrine 8 x control. 6. Thus, bluefish tolerate hemorrhage with initial vasoconstriction via alpha-adrenergic pathways, and hemodilution.
INTRODUCTION
MATERIALS AND METHODS
The immediate recovery of blood pressure after acute hemorrhage in mammals is due to vasoconstriction mediated through adrenergic pathways (Chien, 1958). A slower mechanism of recovery is the restoration o f volume which occurs over several hours (Starling, 1885-6). If hemorrhage is greater than 15-20% of blood volume, irreversible hypotensive shock occurs. In contrast to mammals, recovery after hemorrhage in dogfish has been shown to be due mostly to recovery of blood volume through hemodilution rather than to catecholamine mediated vasoconstriction (Carrol et al., 1984). In a related physiologic stress, smooth dogfish are unable to tolerate head-up tilting in air with subsequent hypotension whereas bluefish are able to maintain their blood pressure and survive tilting (Ogilvy and DuBois, 1982). The proposed mechanism for the difference in response to tilting was that bluefish had greater direct sympathetic innervation to the peripheral vasculature. In the present study we use phentolamine and atropine to block the sympathetic and parasympathetic pathways in an attempt to define the mechanisms of vascular recovery in response to the stress of acute hemorrhage in unanesthetized bluefish. In addition epinephrine and norepinephrine levels measured before and after hemorrhage are used to evaluate the presence of an adrenergic response. Small repeated hemorrhages were used to assess any possible role of hemodilution in response to hypovolemia.
*Present addresses: Dr Christopher S. Ogilvy, Neurosurgical Service, Massachusetts General Hospital, Boston, MA 02114; and Mr P. Glenn Tremml, University of Connecticut School of Medicine, Farmington, CT 06032, USA. 807
General handling Bluefish (Pomatomus saltatrix) were caught on a hook and line in waters around Woods Hole, MA. The fish were maintained in holding tanks and used within 7-10 days after capture. During experiments the unanesthetized fish were placed on a V-board in air with the gills perfused with aerated sea-water as described previously (Ogilvy and DuBois, 1982). An 18 or 20-gauge spinal needle was inserted through the floor of the mouth into the ventral aorta of the lightly restrained unanesthetized fish. The needle was connected through a 3-way stopcock to a Statham P-23-D pressure transducer and direct writing recorder (Grass Instrument Co., Quincy, MA). Blood samples were withdrawn and drugs were injected through the 3-way stopcock. Zero reference pressure was recorded from an open tube whose water level was set to the height of the tip of the aortic catheter with the fish on the V-board in air. The aortic catheter and needle were flushed with small amounts of Forster-Taggart teleost Ringer's solution. The solution is made by adding 7.84g NaC1, 0.186g KC1, 0.221g CaCI2.2H20, 0.203 g MgC12.6H20 and 0.069g NaH2PO 4 to a liter of distilled water. Bicarbonate buffer (1.26 g/l) is added prior to using the Ringer's solution. Several different experimental protocols were used and these are described below.
Control experiments Nine bluefish were used in a set of experiments designed to test if any significant change of blood pressure or heart rate occurred during immersion of fish in a tank of water. Zero reference pressure during immersion was the surface of the water in the tank. Acute hemorrhage was performed in one immersed bluefish. The remainder of the experiments were conducted with the fish on the V-board in air. Six fish were used to evaluate any significant change which might occur while on the V-board. The fish were placed on the V-board in air and their blood pressure and heart rate were followed for a period of at least 80 min. One ml blood samples were taken at the beginning and end of the time period. These samples were analysed for hematocrit, hemoglobin, osmolality, sodium, potassium, glucose, protein concentration, and in one fish, lactate.
808
CHRISTOPHER
S.
In experiments where pharmacologic blockade was used, 5 to 15 min were allowed to elapse for blood pressure and heart rate stabilization. In order to reveal any gradual change in heart rate or blood pressure, one fish was given 200/~ g of phentolamine and cardiovascular parameters were followed for 35 min. For determination of the normal blood volume in bluefish, blood volume was measured in 17 fish using Evans' blue dye hemodilution technique. A 2.5 ml syringe was weighed, 0.1ml of Evans' blue dye (Harvey Laboratories, Inc., Philadelphia, PA) was added and the syringe was weighed again. 2.5 ml of blood were withdrawn from the ventral aorta and mixed for 30 sec with the dye. The blood was reinjected into the ventral aorta followed by a small amount of Ringer's solution in order to flush in the dye. A 1.0 ml blood sample prior to dye injection (control) and a sample 10 min after dye injection were withdrawn and spun in a centrifuge. The optical density of the plasma was measured in a spectrophotometer in order to obtain the concentration of Evans' blue dye by comparison with known dilutions of dye in the control sample.
Acute 10 ml hemorrhage without pharmacologic blockade Experiments were conducted in five bluefish. After the needle had been placed in the ventral aorta the blood pressure and heart rate were followed for a 30 min control period. After the control period, 1.2ml of blood was withdrawn to clear the tube. Following this, 1.0 ml was removed and used for analysis of hematocrit, protein concentration and catecholamine levels, A total of 10ml of blood was withdrawn in 2 ml aliquots, with blood pressure measurements made between aliquots, over a 3 min period. The last ml was also used as a blood sample. The average weight of the five fish used in this protocol was 1.15 _-t-0.03 (SE) kg and the 10 ml removed represented 0.87% of body weight. Blood pressure and heart rate were then followed during a 30 min follow-up period with blood samples taken 15 and 30 min after the end of hemorrhage. The volume of samples removed during the follow-up period was replaced with blood withdrawn during hemorrhage. Epinephrine and norepinephrine levels were measured in duplicate on the blood sample before and 3 min after the start of hemorrhage, and 15 min and 30 min after the end of withdrawal of the blood, using a radioenzymatic assay (Cat-A-Kit, Upjohn Diagnostics) on frozen plasma samples.
OGILVY
al.
et
potassium, glucose and, in one fish, lactate concentrations were made during the control period prior to the initial hemorrhage and 20 min after the final hemorrhage. RESULTS
Control experiments Blood pressure a n d heart rate of nine bluefish m e a s u r e d before a n d d u r i n g immersion in water showed no significant difference. The average b l o o d pressure prior to immersion was 86/50 (SE, 8/8) m m H g with a heart rate of 48 (SE, 4) beats/min. While immersed, the blood pressure was 95/65 (SE, 8 / 9 ) m m H g a n d heart rate 63 (SE, 6) beats/min. A l t h o u g h heart rate increased, there was no significant difference in heart rate, systolic or diastolic blood pressures using the two-tailed t-test with each fish as its own control ( P > 0.20 for systolic BP a n d heart rate, 0.10 > P > 0.05 for diastolic BP). Acute h e m o r r h a g e in one immersed bluefish showed a blood pressure a n d heart rate response similar to fish hemo r r h a g e d while on the V - b o a r d (see below). Six fish used to evaluate any effect o f lying in air o n the V - b o a r d showed no significant change in hematocrit, h e m o g l o b i n or protein c o n c e n t r a t i o n over 80 min o f o b s e r v a t i o n (Fig. 1). Plasma sodium, potassium, a n d lactate c o n c e n t r a t i o n s and serum osmolality m e a s u r e d in these fish did not change (Table 1). The glucose level decreased over the 80 min observation period in these unfed fish. In one fish given 200/~g of p h e n t o l a m i n e without subsequent h e m o r r h a g e , b l o o d pressure a n d heart rate remained stable. Blood pressure prior to admin-
.
,
'c°n,;°,
'
'
'
+/
-1-
Acute 12.5 ml or 16 ml hemorrhage without pharmacologic blockade Three fish had blood volumes removed of 16, 12.5 and 12.5ml during acute hemorrhage. Blood volumes were measured in these fish using the Evans' blue dye technique described earlier. The volumes of blood removed represented an average of 27% their initial blood volume.
Acute 10 ml hemorrhage after pharmacologic blockade At least five fish were used in each condition studied. Two hundred /tg of phentolamine mesylate ("Regitine", Ciba Pharmaceutical Co.) 0.02mg of atropine sulfate injection (Eli Lilly and Co., Indianapolis, Ind.), or phentolamine followed by atropine, was administered after a 15 rain control period. An interval of 5 min was allowed between the phentolamine and atropine injections in experiments using both drugs. After the drug injections, 5 to 10 rain elapsed before hemorrhage. A total volume of 10 ml of blood was then withdrawn over 3 min in 2 ml aliquots with blood pressure measurements made between aliquots.
Repeated 4.5 ml hemorrhages Four bluefish were used in this protocol. 4.5 ml of blood was removed during 2 rain at intervals of 20 rain for 80 min (18 ml total blood removed). The initial ml of blood from each hemorrhage was used for measurement of hematocrit, hemoglobin and protein concentration. Osmolality, sodium,
i
I0
Control
-
E
_8
Control
C J Hornorrhogo
O-
II
20
I
I
20
I
[
I
40
I
'
60
I
I I ii
80
Time (rain) Fig. 1. Hematocrit, hemoglobin and protein concentration are shown for four bluefish with small repeated hemorrhages of 4.5 ml. The time of the hemorrhages is shown with a break in the line (arrows). Bars are the standard error of the group mean. Also shown are the results of control experiments of six bluefish placed on the V-board for 80 rain (solid circles).
Acute hemorrhage in bluefish
809
Table 1. Blood chemistries in control fish and in fish with 4.5 ml of blood removed every 20 min Control Small repeated hemorrhages (6 bluefish) (4 bluefish) Time (min) Time (min) 0 Na ÷ (mEq/I) 185 + 5 K + (mEq/I) 3.4 + 0.6 Osmolality 437 + 13 (mOsmol/kg) Glucose 199 + 34 (mg/dl) Lactate 5.13 (mmol/l) Values are means _+SE. * = P < 0.05.
80 185 + 6 4.1 _+1.0 428 + 14
0 197 + 3 3.4 + 0.1 414 _+6
80 210 +4* 3.9 _+0.4 434 _+10"
94 + 20*
171 + 24
89 + 10"
5.20 (l fish)
istration of phentolamine was 98/63 and heart rate was 39. Thirty-five min after the phentolamine injection blood pressure was 104/80 with a heart rate of 46. Using the Evans' blue dye technique, the blood volume of 17 bluefish with an average weight of 1.34 _+ 0.08 kg (SE) was 4.14 _ 0.23 (SE) % of body weight.
Acute lO ml blockade
hemorrhage
without pharmacologic
The control and recovery phases of blood pressure and heart rate in five bluefish to acute hemorrhage (10ml) are shown in Fig. 2. Immediately before hemorrhage the average blood pressure was 102/66 m m H g and heart rate 42 beats/min. The blood pressure decreased linearly during hemorrhage and heart rate increased. The linear fall in blood pressure for these 5 fish during hemorrhage is shown in detail in Fig. 3. Both the blood pressure and heart rate recovered close to control values within 5 min after hemorrhage. Table 2 shows epinephrine and nor-
140
I
. . . . . .
120
!1
E 100 E
4.99
4.78 (I fish)
epinephrine levels (ng/ml) at 3 min after onset of hemorrhage and 15 and 30 min after hemorrhage for the 5 bluefish shown in Fig. 1. The levels at each time after hemorrhage are divided by the control values, using each fish as its own control, in order to obtain an average ratio (_+ SE). The increase of the ratio is significant at 3, 15 and 30 min after hemorrhage using the two tailed t-test for matched data. The epinephrine rose to 5.4 times control (P < 0.005) and norepinephrine to 8.2 times control (P <0.005) 30 min after hemorrhage. There is a large variation in the resting epinephrine and norepinephrine levels, similar to the variation found in resting catecholamine levels of dogfish (Carrol et al., 1984; Opdyke et al., 1983). As is seen in Table 2 all of the individual fish had an upward trend of epinephrine and norepinephrine levels after hemorrhage. There is individual variation of the control epinephrine and norepinephrine concentrations. The catecholamine levels measured on two bluefish were excluded as aberrant from those of the five bluefish listed in Table 2. One of these two fish had little drop in BP during hemorrhage, and no rise in epinephrine or norepinephrine level. The other had a very low pulse pressure throughout the experiment and died 17 min after hemorrhage. The epinephrine and norepinephrine levels of the latter fish were high initially and very high at 15 min.
80
~ ~o
r20
n
~ 40
'
A m
Systolic
I
I
'
I00
20
~ 80 80
~ 6o ~
60 n .~
~lID 4 0 ~
m
20
g -
-20 "10
0
I0
20
30
Time (mini
Fig. 2. Blood pressure and heart rate are shown before (control) during (hemorrhage) and after removal of l0 ml of blood from five bluefish. The mean of five bluefish is shown. Bars are _ l SE of the group mean. After 30min of observation the blood removed was reinfused.
' -"~
~.~_
40
2O 4
6
8
IO
Volume ~1) of Blood Removed
Fig. 3. Blood pressure measured in five bluefish after removal of each 2ml aliquot of blood (10ml) during hemorrhage over a total period of 3 min. Bars are the SE of the group mean.
810
CHRISTOPHER S. OGILVY et al. Table 2. Plasma epinephrine and norepinephrine levels in five bluefish before (control) and after acute hemorrhage Fish no.
Control
3 min
15 min
30 min
Epinephrine concentration (ng/ml) 1 10.25 4 4.64 14 1.52 16 0.34 17 0.62
22.86 2.38 3.21 0.81 0.88
40.02 6.72 2.96 1.05 1.44
43.12 28.60 lost 2.52 2.45
Mean 3.47 Ratios 1.00 SE of ratios P value of ratio Norepinephrine concentration (ng/ml) 1 16.99 4 1.06 14 2.07 16 0.39 17 0.61
6.03 1.73 0,35 <0,01
10.44 2.54 0.43 <0.005
19.77 5.44 0.83 <0.005
36.38 0.90 5.98 0.78 0.52
93.51 2. I 1 2.03 1.39 0.69
93.08 12.90 lost 2.93 4.50
Mean Ratios SE of ratios P value of ratio
8.91 1.75 0.40 < 0.02
19.95 2.64 0.85 < 0.05
28.35 8.16 1.43 < 0.005
Acute 12.5 ml or 16ml pharmacologic blockade
4.22 1.00
hemorrhage
without
The three fish used for this protocol had an average control blood pressure of 87/65mmHg which dropped significantly during hemorrhage. Although the blood pressure did recover to 81/73 mmHg 15 min after hemorrhage it was 63/53 mmHg 30 min after hemorrhage. The fish remained hypotensive and subsequently died. The blood hematocrit, hemoglobin and protein concentration at 15 and 30 min after hemorrhage showed that hemodilution did not occur in these fish with the larger volume of blood removed.
Acute 10 ml blockade
140
,20
, ;I~ , Systolic
~
B
i
2C
I
'1
!
ii t
-? rn
' I ~
80
~ 60 ~
6e 4c
m 20
PhentolomlM
12oo1,~1
(0.02m~
RIIhfusion
i
120
A .E E 80 v)
A
E
Reinluslon
Atroph
of Whole BlOod
I Hem~'~hage I0 ml
0 I00
pharmacologic
I
E
,,n
after
The response of six bluefish to acute hemorrhage after pretreatment with 200/~ g phentolamine injected into the ventral aorta 10 min prior to hemorrhage is shown in Fig. 4. The phentolamine had no significant immediate effect on blood pressure, and the pressure during hemorrhage decreased as it had in fish without phentolamine. After hemorrhage there was tachycardia which gradually returned toward the normal rate over the course of 15--20 min. However, the blood pressure after hemorrhage failed to return to
"~'I- 120 "1-
hemorrhage
I00
of Whole Blood
Ilemorrho~lOmt
I
--
) 80 ..o ~ 60
60 40
g: 40
20 "1-
4o
I
-,0
,o
Time
(min)
i
3o
Fig. 4. The average blood pressure and heart rate response to hemorrhage (10 ml) of six bluefish pretreated with 200/z g of phentolamine is shown. The phentolamine was given 10 min prior to hemorrhage. Bars are the SE of the group mean.
:1:20 O. -:
'
-20
J
L
-I0
210
I0 Time
,
30
(rnin)
Fig. 5. Blood pressure and heart rate changes observed after hemorrhage (10 ml) in five fish pretreated with 0.02 mg of atropine. Bars are the SE of the group mean. The atropine was given 5 min prior to hemorrhage.
Acute hemorrhage in bluefish
10 ml of hemorrhage 5 min after the atropine injection (Fig. 6). The tachycardia and slight rise in blood pressure observed after the atropine in earlier experiments were again observed in this group of fish. Hemorrhage decreased blood pressure without changing the pulse rate. After hemorrhage the blood pressure did not return to control levels and the pulse rate remained elevated.
120 •1- I00
:1,
E 80
~- 4o "o o
N 2O
IIl~m~ I *H~ ~'r
120
Repeated 4.5 ml hemorrhages
Reinfusion
ofWholeBood
i
I00
I
80 ,~
60
~:
40
?-
20 -20 '
,
1
-I0
I0' Time
2'0
50
(rain)
Fig. 6. The average blood pressure and heart rate response after hemorrhage (10 ml) of six bluefish pretreated with 200/~g phentolamine and 0.02mg atropine. For blood pressure measurements each fish serves as its own control and bars are the SE of the mean difference. The blood removed during hemorrhage was reinfused 30 min after the end of hemorrhage. control levels in the six fish that had been given phentolamine. Figure 5 shows the change in blood pressure and heart rate observed in five bluefish after injection of 0.02 mg (20/~ g) of atropine followed by acute hemorrhage. Heart rate increased significantly (P < 0.02) from a control rate of 48 beats/min to 87 beats/min 5 min after atropine. The tachycardia increased further (P < 0.01) during hemorrhage to 108 beats/min and persisted. The blood pressure returned to control levels within 10 min after ,hemorrhage. Six bluefish were given 200 #g of phentolamine followed 5 min later by 0.02 mg of atropine and then 120 ,
~/ottolt¢ 60-: o_ 4o-t
Hemorrha~le
"~ _? n-,
t
t
t
20
0
2'o
811
'
4'o
'
e'o
'
80
T i m e (min)
Fig. 7. The average systolic and diastolic blood pressure response of four bluefish t o repeated small hemorrhages (arrows) of 4.5 ml every 20 min. Bars are the SE of the group mean.
Four bluefish had hemorrhages of 4.5 ml repeated at 20 min intervals for a total of 80 min. As shown in Fig. 7 with each hemorrhage the systolic pressure dropped an average of 13 mmHg and diastolic an average of 15mmHg acutely. During the 19min interval between hemorrhages there is recovery close to the blood pressure that existed before the hemorrhage. As with larger amounts of hemorrhage, the blood pressure recovered to near prehemorrhage level within 5 min after the end of hemorrhage. The hematocrit decreased progressively from a control value of 41.1 to a value of 24.7 vol% during the repeated small hemorrhages demonstrating a mean hemodilution of about 15% during each 20 min recovery (Fig. 1, top panel). Blood hemoglobin concentration and plasma protein concentration followed the same pattern (Fig. 1, lower panels). Values for plasma sodium, potassium, glucose and lactate concentrations as well as osmolality changes observed after small hemorrhages are shown in Table 1. As in the control fish, the glucose concentration dropped significantly (P < 0.02 by 2 tailed t-test). The sodium concentration (P < 0.05 by 2 tailed t-test) and osmolality (P = 0.03 by Wilcoxon signed-rank test for matched pairs) increased after gradual, repeated hemorrhage. DISCUSSION Factors that may influence blood pressure in the ventral aorta are resistance to blood flow in the gills or peripheral blood vessels, ventricular contractility, blood volume, cardiac output, and venous capacitance. It appears that bluefish, like mammals, have two mechanisms that compensate for blood loss. The initial recovery of arterial blood pressure in mammals after acute hemorrhage is due to vasoconstriction (Chien, 1958). Bluefish show a similar initial recovery of ventral aortic pressure following acute hemorrhage. The fact that this initial phase of recovery was blocked by phentolamine suggests that alpha adrenergic vasoconstriction occurs in bluefish as well as mammals. Our study has not attempted to measure venous capacitance, cardiac output, pressure in the dorsal aorta or ventricular contractility, and therefore we cannot rule out changes in these parameters during the recovery of blood pressure. A second mechanism of recovery after hemorrhage is hemodilution which occurs over a longer period of time and acts to restore blood volume. This hemodilution was observed during the small repeated hemorrhages as a fall in hematocrit, hemoglobin and protein concentration. The responses to acute hemorrhage observed in bluefish are in contrast to the results in dogfish reported by Carrol et al. (1984). In the dogfish there was a significant increase in epinephrine levels 15 min
812
CHRISTOPI-tER S. OGILVY el al.
after hemorrhage and an increase in norepinephrine 20 min after hemorrhage. The gradual blood pressure recovery after hemorrhage was unchanged after blockade with the aipha-adrenergic blocking agent phentolamine. The authors concluded that dogfish respond to hemorrhage primarily through restoration of blood volume and not vasoconstriction. Hemodilution in dogfish after hemorrhage has also been documented by Sudak and Wilber (1960). The ability of dogfish to hemodilute rapidly may be due to a high rate of leakage of fluids into the vascular bed. The capillaries of dogfish have been shown to have loosely overlapping endothelial cells without tight junctions or gap junctions, suggesting a high permeability throughout the vascular system (Rhodin, 1972). With this arrangement interstitial fluid may move readily into the vascular compartment when the blood pressure is lowered. The findings of this study are consistent with the vascular changes observed in bluefish and dogfish in response to tilting head up in air (Ogilvy and DuBois, 1982). When dogfish were tilted head up to 30 ° for 30min the pulse pressure and blood pressure fell. There was a concomitant increase in interstitial fluid pressure in the caudal region of the fish. Bluefish tolerated head-up tilting well and were able to maintain their blood pressure and pulse pressure presumably through constriction of the vascular bed. A similar mechanism was observed in salmon in response to exercise. Salmon have an increased blood pressure during swimming and it is abolished by the alpha-adrenergic blocking agent phenoxybenzamine (Randall and Stevens, 1967). The initial recovery of blood pressure in response to hemorrhage observed in our present study appears to be mediated via an alpha-adrenergic response. This conclusion is drawn from the observation that measured catecholamines increased and phentolamine blocked the recovery of blood pressure after hemorrhage. Nishimura et al. (1979) found that plasma renin activity increased significantly with hemorrhage in toad fish. In dogfish, Opdyke et al. (1981) found that catecholamines increased following injection of angiotensin II. In our experiments we have not measured renin activity and the possibility exists that increased renin leads to the increased catecholamines we measured. There is a transient tachycardia present in the bluefish immediately after hemorrhage. The heart rate returned to control values within 5 min after the end of hemorrhage. Pretreatment with atropine did prolong but did not block the vascular (blood pressure) recovery after hemorrhage in bluefish. A tachycardia occurred after atropine and increased again significantly following the hemorrhage. In addition, the blood pressure response observed after hemorrhage when bluefish were pretreated with phentolamine and atropine was similar to the blood pressure response after hemorrhage when fish were pretreated with phentolamine alone. This indicates that the tachycardia observed in the bled untreated fish (no pharmacologic blockade) was probably secondary to release of vagal inhibition in response to hypovolemia and plays only a minor role in the recovery from hemorrhage. Burnstock (1969) reviewed vagal inhibition of the heart rate of elas-
mobranchs and teleosts. In the trout, the heart rate seems to be free of vagal inhibition until the peripheral end of the vagus is stimulated, or acetylcholine given, or the fish made hypoxemic, all of which produce bradycardia (Wood and Shelton, 1980a, b). In bluefish, the resting heart rate is about 43 beats/min, but this rate became accelerated to 71 beats/min with tilting 30 degrees (Ogilvy and DuBois, 1982), or to 103 beats/min while swimming (DuBois et al., 1975, 1976) or to 70beats/min with hemorrhage in the present study. Atropine, 0.01-0.02mg in the ventral aorta, caused the heart rate of bluefish to accelerate and to be fixed and independent of swimming or tilting (DuBois et al., 1975). The blood volume removed during acute hemorrhage from the bluefish in this study was 10 ml which represents 0.87% of the total average body weight of 1.15 + 0.03 (SE) kg. Blood volume in bluefish is approximately 4% of total body weight (as measured using the Evans' blue dye technique). Therefore, 10ml represents 21% of blood volume. In hemorrhage experiments where blood volume was measured using Evans' blue dye, three fish were hemorrhaged an average of 27% of blood volume. These fish with the greater amount of blood withdrawn showed acute recovery of blood pressure but within 30 min after hemorrhage became hypotensive and died. The amount of hemorrhage which produces irreversible hypotensive shock in teleosts of this study appears to be between 20 and 30% of blood volume. In splenectomized dogs the blood pressure fell in a linear fashion with hemorrhage exceeding 15-20% of blood volume with gradual return of blood pressure towards control values 3(I-60 min after hemorrhage. In animals subjected to hemorrhage greater than 20% of blood volume, irreversible shock occurs (Chien, 1958). Thus, we conclude that in our fish, as in mammals, since blood pressure is dependent in part upon blood volume, essential organs such as the central nervous system may be so under-perfused that irreversible damage occurs with reduction in blood volumes greater than 20% of total blood volume. The contribution at rest of catecholamines to vasomotor tone is felt to be small in the bluefish based on the observation that no change in blood pressure or heart rate occurred following administration of phentolamine without subsequent hemorrhage. Randall and Stevens (1967) reached a similar conclusion regarding the resting vasomotor tone in sockeye salmon with little change in blood pressure observed after administration of phenoxybenzamine at rest. In contrast to this low level of resting tone in bluefish and salmon, the resting tone in rainbow trout was felt to be significant based on a drop in blood pressure following intravenous phenoxybenzamine at rest (Wood and Shelton, 1980). In small repeated hemorrhages hemodilution occurs (Fig. 1) with replacement of blood volume and near restoration of total pressure (Fig. 7). This prevents the hypoperfusion of essential organs. The reason for the observed rise in serum sodium concentration and osmolality after small repeated hemorrhages is not clear. A possible explanation might be that changes of gill permeability or increased oral intake of sea-water allow sodium to enter the vascu-
Acute hemorrhage in bluefish lar space in response to the induced hypovolemia. In both the control and hemorrhaged fish the glucose concentration fell significantly. Fed mammals have a significant increase in the glucose concentration in response to hemorrhage and this provides an osmotic force capable of mobilizing fluid into the vascular space. In hemorrhage after fasting, there is no increase in the glucose concentration (Mohsenin and DuBois, 1983). The fall in glucose concentration observed in the fish is somewhat unexpected. The exact cause of the decrease in concentration is difficult to fully analyse without simultaneous levels of catecholamines and insulin. Future experiments could be designed to further investigate this point. The large rise in plasma epinephrine and norepinephrine levels following hemorrhage indicates a response of the adrenergic system. Blockade with phentolamine indicates that the recovery of blood pressure which was observed in untreated fish after rapid hemorrhage was due to alpha-adrenergic stimulation. Acknowledgements--This work was supported in part by a grant HL 17407 from the National Institutes of Health. Parts of this work have been presented in preliminary form (A. B. DuBois and P. G. Tremml. Vascular responses to rapid and slow hemorrhage in bluefish and smooth dogfish. Fed. Proc. 43, 639, 1984). Kimberly O'Sullivan participated in the present studies as a research assistant. Dr Rosa Hendler (Yale New Haven Medical Center) measured the epinephrine and norepinephrine levels. REFERENCES
Burnstock G. (1969) Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol. Rev. 21, 247-324. Carrol R. G., Opdyke D. F. and Keller N. E. (1984) Vascular recovery following hemorrhage in the dogfish shark Squalus acanthias. Am. J. Physiol. 246, R825-R828.
CIB,P, 91/4A--L
813
Chien S. (1958) A quantitative evaluation of the circulatory adjustment of splenectomized dogs to hemorrhage. Am. J. Physiol. 193, 605-614. DuBois A. B., Camiol P. and Morris R. DB. (1975) Effect of gravity and exercise on the heart rate and blood pressure of bluefish (Pomatomus saltatrix). (Abstract) Biol. Bull. Woods Hole, Massachusetts 149, 425. DuBois A. B., Carniol P. J. and Morris R. DB. (1976) Blood pressure and heart rate in bluefish: the effects of exercise, gravity and atropine. (Abstract) Fed. Proc. 35, 796. Mohsenin V. and DuBois A. B. (1983) Intercompartmental fluid shifts due to glucose release during hemorrhage in rabbits. Am. J. Physiol. 245, H143-H149. Nishimura H., Lunde L. G. and Zucker A. (1979) Renin response to hemorrhage and hypotension in the aglomerular toad fish Opsanus tau. Am. J. Physiol. 237, H 105-HI 11. Ogilvy C. S. and DuBois A. B. (1982) Effect of tilting on blood pressure and interstitial fluid pressure of bluefish and smooth dogfish. Am. J. Physiol. 242, R70-R76. Opdyke D. F., Bullock J., Keller N. E. and Holmes K. (1983) Dual mechanisms for catecholamine secretion in the dogfish shark Squalus acanthias. Am. J. Physiol. 244, R641 -R645. Randall D. J. and Stevens E. D. (1967) The role of adrenergic receptors in cardiovascular changes associated with exercise in Salmon. Comp. Biochem. Physiol. 21, 415~;24. Rhodin J. A. (1972) Fine structures of elasmobranch arteries, capillaries and veins in the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 42A, 59ff34. Starling E. H. On the absorption of fluids from the connective tissue spaces. J. Physiol. 19, 312-326, 1885-1886. Sudak F. N. and Wilber C. G. (1960) Cardiovascular responses to hemorrhage in the dogfish. Biol. Bull. 119, 342. Wood C. M. and Shelton G. (1980a) Cardiovascular dynamics and adrenergic responses of the rainbow trout in vivo. J. exp. Biol. 87, 247-270. Wood C. M. and Shelton G. (1980b) Reflex control of heart rate and cardiac output in the rainbow trout. J. exp. Biol. 87, 271-284.
0300-9629/88 $3.00 + 0.00 © 1988 Pergamon Press plc
Printed in Great Britain
PRESSOR AND HEMODILUTION RESPONSES COMPENSATE FOR ACUTE HEMORRHAGE IN BLUEFISH CHRISTOPHER S. OGILVY,* P. GLENN TREMML* and ARTHUR B. DuBoIS John B. Pierce Foundation Laboratory and Yale University, 290 Congress Avenue, New Haven, CT 06519, USA. Telephone: (203) 562-9901
(Received 14 April 1988) Abstract---1. After hemorrhage of 21% blood volume (0.9% body weight) blood pressure (BP) and heart rate (H.R.) of unanesthetized bluefish (Pomatomus saltatrix) recovered within 5 min. 2. Phentolamine blocked this recovery. 3. Atropine increased control H.R. from 48 to 87 per min, and to 108 after hemorrhage, with delay of BP recovery to 10 min. 4. With small, repeated hemorrhages every 20 min, hemodilution and recovery of BP occurred between hemorrhages. Removal of 27% blood volume resulted in only temporary recovery. 5. Thirty rain after hemorrhage, plasma epinephrine was 5 × and norepinephrine 8 x control. 6. Thus, bluefish tolerate hemorrhage with initial vasoconstriction via alpha-adrenergic pathways, and hemodilution.
INTRODUCTION
MATERIALS AND METHODS
The immediate recovery of blood pressure after acute hemorrhage in mammals is due to vasoconstriction mediated through adrenergic pathways (Chien, 1958). A slower mechanism of recovery is the restoration o f volume which occurs over several hours (Starling, 1885-6). If hemorrhage is greater than 15-20% of blood volume, irreversible hypotensive shock occurs. In contrast to mammals, recovery after hemorrhage in dogfish has been shown to be due mostly to recovery of blood volume through hemodilution rather than to catecholamine mediated vasoconstriction (Carrol et al., 1984). In a related physiologic stress, smooth dogfish are unable to tolerate head-up tilting in air with subsequent hypotension whereas bluefish are able to maintain their blood pressure and survive tilting (Ogilvy and DuBois, 1982). The proposed mechanism for the difference in response to tilting was that bluefish had greater direct sympathetic innervation to the peripheral vasculature. In the present study we use phentolamine and atropine to block the sympathetic and parasympathetic pathways in an attempt to define the mechanisms of vascular recovery in response to the stress of acute hemorrhage in unanesthetized bluefish. In addition epinephrine and norepinephrine levels measured before and after hemorrhage are used to evaluate the presence of an adrenergic response. Small repeated hemorrhages were used to assess any possible role of hemodilution in response to hypovolemia.
*Present addresses: Dr Christopher S. Ogilvy, Neurosurgical Service, Massachusetts General Hospital, Boston, MA 02114; and Mr P. Glenn Tremml, University of Connecticut School of Medicine, Farmington, CT 06032, USA. 807
General handling Bluefish (Pomatomus saltatrix) were caught on a hook and line in waters around Woods Hole, MA. The fish were maintained in holding tanks and used within 7-10 days after capture. During experiments the unanesthetized fish were placed on a V-board in air with the gills perfused with aerated sea-water as described previously (Ogilvy and DuBois, 1982). An 18 or 20-gauge spinal needle was inserted through the floor of the mouth into the ventral aorta of the lightly restrained unanesthetized fish. The needle was connected through a 3-way stopcock to a Statham P-23-D pressure transducer and direct writing recorder (Grass Instrument Co., Quincy, MA). Blood samples were withdrawn and drugs were injected through the 3-way stopcock. Zero reference pressure was recorded from an open tube whose water level was set to the height of the tip of the aortic catheter with the fish on the V-board in air. The aortic catheter and needle were flushed with small amounts of Forster-Taggart teleost Ringer's solution. The solution is made by adding 7.84g NaC1, 0.186g KC1, 0.221g CaCI2.2H20, 0.203 g MgC12.6H20 and 0.069g NaH2PO 4 to a liter of distilled water. Bicarbonate buffer (1.26 g/l) is added prior to using the Ringer's solution. Several different experimental protocols were used and these are described below.
Control experiments Nine bluefish were used in a set of experiments designed to test if any significant change of blood pressure or heart rate occurred during immersion of fish in a tank of water. Zero reference pressure during immersion was the surface of the water in the tank. Acute hemorrhage was performed in one immersed bluefish. The remainder of the experiments were conducted with the fish on the V-board in air. Six fish were used to evaluate any significant change which might occur while on the V-board. The fish were placed on the V-board in air and their blood pressure and heart rate were followed for a period of at least 80 min. One ml blood samples were taken at the beginning and end of the time period. These samples were analysed for hematocrit, hemoglobin, osmolality, sodium, potassium, glucose, protein concentration, and in one fish, lactate.
808
CHRISTOPHER
S.
In experiments where pharmacologic blockade was used, 5 to 15 min were allowed to elapse for blood pressure and heart rate stabilization. In order to reveal any gradual change in heart rate or blood pressure, one fish was given 200/~ g of phentolamine and cardiovascular parameters were followed for 35 min. For determination of the normal blood volume in bluefish, blood volume was measured in 17 fish using Evans' blue dye hemodilution technique. A 2.5 ml syringe was weighed, 0.1ml of Evans' blue dye (Harvey Laboratories, Inc., Philadelphia, PA) was added and the syringe was weighed again. 2.5 ml of blood were withdrawn from the ventral aorta and mixed for 30 sec with the dye. The blood was reinjected into the ventral aorta followed by a small amount of Ringer's solution in order to flush in the dye. A 1.0 ml blood sample prior to dye injection (control) and a sample 10 min after dye injection were withdrawn and spun in a centrifuge. The optical density of the plasma was measured in a spectrophotometer in order to obtain the concentration of Evans' blue dye by comparison with known dilutions of dye in the control sample.
Acute 10 ml hemorrhage without pharmacologic blockade Experiments were conducted in five bluefish. After the needle had been placed in the ventral aorta the blood pressure and heart rate were followed for a 30 min control period. After the control period, 1.2ml of blood was withdrawn to clear the tube. Following this, 1.0 ml was removed and used for analysis of hematocrit, protein concentration and catecholamine levels, A total of 10ml of blood was withdrawn in 2 ml aliquots, with blood pressure measurements made between aliquots, over a 3 min period. The last ml was also used as a blood sample. The average weight of the five fish used in this protocol was 1.15 _-t-0.03 (SE) kg and the 10 ml removed represented 0.87% of body weight. Blood pressure and heart rate were then followed during a 30 min follow-up period with blood samples taken 15 and 30 min after the end of hemorrhage. The volume of samples removed during the follow-up period was replaced with blood withdrawn during hemorrhage. Epinephrine and norepinephrine levels were measured in duplicate on the blood sample before and 3 min after the start of hemorrhage, and 15 min and 30 min after the end of withdrawal of the blood, using a radioenzymatic assay (Cat-A-Kit, Upjohn Diagnostics) on frozen plasma samples.
OGILVY
al.
et
potassium, glucose and, in one fish, lactate concentrations were made during the control period prior to the initial hemorrhage and 20 min after the final hemorrhage. RESULTS
Control experiments Blood pressure a n d heart rate of nine bluefish m e a s u r e d before a n d d u r i n g immersion in water showed no significant difference. The average b l o o d pressure prior to immersion was 86/50 (SE, 8/8) m m H g with a heart rate of 48 (SE, 4) beats/min. While immersed, the blood pressure was 95/65 (SE, 8 / 9 ) m m H g a n d heart rate 63 (SE, 6) beats/min. A l t h o u g h heart rate increased, there was no significant difference in heart rate, systolic or diastolic blood pressures using the two-tailed t-test with each fish as its own control ( P > 0.20 for systolic BP a n d heart rate, 0.10 > P > 0.05 for diastolic BP). Acute h e m o r r h a g e in one immersed bluefish showed a blood pressure a n d heart rate response similar to fish hemo r r h a g e d while on the V - b o a r d (see below). Six fish used to evaluate any effect o f lying in air o n the V - b o a r d showed no significant change in hematocrit, h e m o g l o b i n or protein c o n c e n t r a t i o n over 80 min o f o b s e r v a t i o n (Fig. 1). Plasma sodium, potassium, a n d lactate c o n c e n t r a t i o n s and serum osmolality m e a s u r e d in these fish did not change (Table 1). The glucose level decreased over the 80 min observation period in these unfed fish. In one fish given 200/~g of p h e n t o l a m i n e without subsequent h e m o r r h a g e , b l o o d pressure a n d heart rate remained stable. Blood pressure prior to admin-
.
,
'c°n,;°,
'
'
'
+/
-1-
Acute 12.5 ml or 16 ml hemorrhage without pharmacologic blockade Three fish had blood volumes removed of 16, 12.5 and 12.5ml during acute hemorrhage. Blood volumes were measured in these fish using the Evans' blue dye technique described earlier. The volumes of blood removed represented an average of 27% their initial blood volume.
Acute 10 ml hemorrhage after pharmacologic blockade At least five fish were used in each condition studied. Two hundred /tg of phentolamine mesylate ("Regitine", Ciba Pharmaceutical Co.) 0.02mg of atropine sulfate injection (Eli Lilly and Co., Indianapolis, Ind.), or phentolamine followed by atropine, was administered after a 15 rain control period. An interval of 5 min was allowed between the phentolamine and atropine injections in experiments using both drugs. After the drug injections, 5 to 10 rain elapsed before hemorrhage. A total volume of 10 ml of blood was then withdrawn over 3 min in 2 ml aliquots with blood pressure measurements made between aliquots.
Repeated 4.5 ml hemorrhages Four bluefish were used in this protocol. 4.5 ml of blood was removed during 2 rain at intervals of 20 rain for 80 min (18 ml total blood removed). The initial ml of blood from each hemorrhage was used for measurement of hematocrit, hemoglobin and protein concentration. Osmolality, sodium,
i
I0
Control
-
E
_8
Control
C J Hornorrhogo
O-
II
20
I
I
20
I
[
I
40
I
'
60
I
I I ii
80
Time (rain) Fig. 1. Hematocrit, hemoglobin and protein concentration are shown for four bluefish with small repeated hemorrhages of 4.5 ml. The time of the hemorrhages is shown with a break in the line (arrows). Bars are the standard error of the group mean. Also shown are the results of control experiments of six bluefish placed on the V-board for 80 rain (solid circles).
Acute hemorrhage in bluefish
809
Table 1. Blood chemistries in control fish and in fish with 4.5 ml of blood removed every 20 min Control Small repeated hemorrhages (6 bluefish) (4 bluefish) Time (min) Time (min) 0 Na ÷ (mEq/I) 185 + 5 K + (mEq/I) 3.4 + 0.6 Osmolality 437 + 13 (mOsmol/kg) Glucose 199 + 34 (mg/dl) Lactate 5.13 (mmol/l) Values are means _+SE. * = P < 0.05.
80 185 + 6 4.1 _+1.0 428 + 14
0 197 + 3 3.4 + 0.1 414 _+6
80 210 +4* 3.9 _+0.4 434 _+10"
94 + 20*
171 + 24
89 + 10"
5.20 (l fish)
istration of phentolamine was 98/63 and heart rate was 39. Thirty-five min after the phentolamine injection blood pressure was 104/80 with a heart rate of 46. Using the Evans' blue dye technique, the blood volume of 17 bluefish with an average weight of 1.34 _+ 0.08 kg (SE) was 4.14 _ 0.23 (SE) % of body weight.
Acute lO ml blockade
hemorrhage
without pharmacologic
The control and recovery phases of blood pressure and heart rate in five bluefish to acute hemorrhage (10ml) are shown in Fig. 2. Immediately before hemorrhage the average blood pressure was 102/66 m m H g and heart rate 42 beats/min. The blood pressure decreased linearly during hemorrhage and heart rate increased. The linear fall in blood pressure for these 5 fish during hemorrhage is shown in detail in Fig. 3. Both the blood pressure and heart rate recovered close to control values within 5 min after hemorrhage. Table 2 shows epinephrine and nor-
140
I
. . . . . .
120
!1
E 100 E
4.99
4.78 (I fish)
epinephrine levels (ng/ml) at 3 min after onset of hemorrhage and 15 and 30 min after hemorrhage for the 5 bluefish shown in Fig. 1. The levels at each time after hemorrhage are divided by the control values, using each fish as its own control, in order to obtain an average ratio (_+ SE). The increase of the ratio is significant at 3, 15 and 30 min after hemorrhage using the two tailed t-test for matched data. The epinephrine rose to 5.4 times control (P < 0.005) and norepinephrine to 8.2 times control (P <0.005) 30 min after hemorrhage. There is a large variation in the resting epinephrine and norepinephrine levels, similar to the variation found in resting catecholamine levels of dogfish (Carrol et al., 1984; Opdyke et al., 1983). As is seen in Table 2 all of the individual fish had an upward trend of epinephrine and norepinephrine levels after hemorrhage. There is individual variation of the control epinephrine and norepinephrine concentrations. The catecholamine levels measured on two bluefish were excluded as aberrant from those of the five bluefish listed in Table 2. One of these two fish had little drop in BP during hemorrhage, and no rise in epinephrine or norepinephrine level. The other had a very low pulse pressure throughout the experiment and died 17 min after hemorrhage. The epinephrine and norepinephrine levels of the latter fish were high initially and very high at 15 min.
80
~ ~o
r20
n
~ 40
'
A m
Systolic
I
I
'
I00
20
~ 80 80
~ 6o ~
60 n .~
~lID 4 0 ~
m
20
g -
-20 "10
0
I0
20
30
Time (mini
Fig. 2. Blood pressure and heart rate are shown before (control) during (hemorrhage) and after removal of l0 ml of blood from five bluefish. The mean of five bluefish is shown. Bars are _ l SE of the group mean. After 30min of observation the blood removed was reinfused.
' -"~
~.~_
40
2O 4
6
8
IO
Volume ~1) of Blood Removed
Fig. 3. Blood pressure measured in five bluefish after removal of each 2ml aliquot of blood (10ml) during hemorrhage over a total period of 3 min. Bars are the SE of the group mean.
810
CHRISTOPHER S. OGILVY et al. Table 2. Plasma epinephrine and norepinephrine levels in five bluefish before (control) and after acute hemorrhage Fish no.
Control
3 min
15 min
30 min
Epinephrine concentration (ng/ml) 1 10.25 4 4.64 14 1.52 16 0.34 17 0.62
22.86 2.38 3.21 0.81 0.88
40.02 6.72 2.96 1.05 1.44
43.12 28.60 lost 2.52 2.45
Mean 3.47 Ratios 1.00 SE of ratios P value of ratio Norepinephrine concentration (ng/ml) 1 16.99 4 1.06 14 2.07 16 0.39 17 0.61
6.03 1.73 0,35 <0,01
10.44 2.54 0.43 <0.005
19.77 5.44 0.83 <0.005
36.38 0.90 5.98 0.78 0.52
93.51 2. I 1 2.03 1.39 0.69
93.08 12.90 lost 2.93 4.50
Mean Ratios SE of ratios P value of ratio
8.91 1.75 0.40 < 0.02
19.95 2.64 0.85 < 0.05
28.35 8.16 1.43 < 0.005
Acute 12.5 ml or 16ml pharmacologic blockade
4.22 1.00
hemorrhage
without
The three fish used for this protocol had an average control blood pressure of 87/65mmHg which dropped significantly during hemorrhage. Although the blood pressure did recover to 81/73 mmHg 15 min after hemorrhage it was 63/53 mmHg 30 min after hemorrhage. The fish remained hypotensive and subsequently died. The blood hematocrit, hemoglobin and protein concentration at 15 and 30 min after hemorrhage showed that hemodilution did not occur in these fish with the larger volume of blood removed.
Acute 10 ml blockade
140
,20
, ;I~ , Systolic
~
B
i
2C
I
'1
!
ii t
-? rn
' I ~
80
~ 60 ~
6e 4c
m 20
PhentolomlM
12oo1,~1
(0.02m~
RIIhfusion
i
120
A .E E 80 v)
A
E
Reinluslon
Atroph
of Whole BlOod
I Hem~'~hage I0 ml
0 I00
pharmacologic
I
E
,,n
after
The response of six bluefish to acute hemorrhage after pretreatment with 200/~ g phentolamine injected into the ventral aorta 10 min prior to hemorrhage is shown in Fig. 4. The phentolamine had no significant immediate effect on blood pressure, and the pressure during hemorrhage decreased as it had in fish without phentolamine. After hemorrhage there was tachycardia which gradually returned toward the normal rate over the course of 15--20 min. However, the blood pressure after hemorrhage failed to return to
"~'I- 120 "1-
hemorrhage
I00
of Whole Blood
Ilemorrho~lOmt
I
--
) 80 ..o ~ 60
60 40
g: 40
20 "1-
4o
I
-,0
,o
Time
(min)
i
3o
Fig. 4. The average blood pressure and heart rate response to hemorrhage (10 ml) of six bluefish pretreated with 200/z g of phentolamine is shown. The phentolamine was given 10 min prior to hemorrhage. Bars are the SE of the group mean.
:1:20 O. -:
'
-20
J
L
-I0
210
I0 Time
,
30
(rnin)
Fig. 5. Blood pressure and heart rate changes observed after hemorrhage (10 ml) in five fish pretreated with 0.02 mg of atropine. Bars are the SE of the group mean. The atropine was given 5 min prior to hemorrhage.
Acute hemorrhage in bluefish
10 ml of hemorrhage 5 min after the atropine injection (Fig. 6). The tachycardia and slight rise in blood pressure observed after the atropine in earlier experiments were again observed in this group of fish. Hemorrhage decreased blood pressure without changing the pulse rate. After hemorrhage the blood pressure did not return to control levels and the pulse rate remained elevated.
120 •1- I00
:1,
E 80
~- 4o "o o
N 2O
IIl~m~ I *H~ ~'r
120
Repeated 4.5 ml hemorrhages
Reinfusion
ofWholeBood
i
I00
I
80 ,~
60
~:
40
?-
20 -20 '
,
1
-I0
I0' Time
2'0
50
(rain)
Fig. 6. The average blood pressure and heart rate response after hemorrhage (10 ml) of six bluefish pretreated with 200/~g phentolamine and 0.02mg atropine. For blood pressure measurements each fish serves as its own control and bars are the SE of the mean difference. The blood removed during hemorrhage was reinfused 30 min after the end of hemorrhage. control levels in the six fish that had been given phentolamine. Figure 5 shows the change in blood pressure and heart rate observed in five bluefish after injection of 0.02 mg (20/~ g) of atropine followed by acute hemorrhage. Heart rate increased significantly (P < 0.02) from a control rate of 48 beats/min to 87 beats/min 5 min after atropine. The tachycardia increased further (P < 0.01) during hemorrhage to 108 beats/min and persisted. The blood pressure returned to control levels within 10 min after ,hemorrhage. Six bluefish were given 200 #g of phentolamine followed 5 min later by 0.02 mg of atropine and then 120 ,
~/ottolt¢ 60-: o_ 4o-t
Hemorrha~le
"~ _? n-,
t
t
t
20
0
2'o
811
'
4'o
'
e'o
'
80
T i m e (min)
Fig. 7. The average systolic and diastolic blood pressure response of four bluefish t o repeated small hemorrhages (arrows) of 4.5 ml every 20 min. Bars are the SE of the group mean.
Four bluefish had hemorrhages of 4.5 ml repeated at 20 min intervals for a total of 80 min. As shown in Fig. 7 with each hemorrhage the systolic pressure dropped an average of 13 mmHg and diastolic an average of 15mmHg acutely. During the 19min interval between hemorrhages there is recovery close to the blood pressure that existed before the hemorrhage. As with larger amounts of hemorrhage, the blood pressure recovered to near prehemorrhage level within 5 min after the end of hemorrhage. The hematocrit decreased progressively from a control value of 41.1 to a value of 24.7 vol% during the repeated small hemorrhages demonstrating a mean hemodilution of about 15% during each 20 min recovery (Fig. 1, top panel). Blood hemoglobin concentration and plasma protein concentration followed the same pattern (Fig. 1, lower panels). Values for plasma sodium, potassium, glucose and lactate concentrations as well as osmolality changes observed after small hemorrhages are shown in Table 1. As in the control fish, the glucose concentration dropped significantly (P < 0.02 by 2 tailed t-test). The sodium concentration (P < 0.05 by 2 tailed t-test) and osmolality (P = 0.03 by Wilcoxon signed-rank test for matched pairs) increased after gradual, repeated hemorrhage. DISCUSSION Factors that may influence blood pressure in the ventral aorta are resistance to blood flow in the gills or peripheral blood vessels, ventricular contractility, blood volume, cardiac output, and venous capacitance. It appears that bluefish, like mammals, have two mechanisms that compensate for blood loss. The initial recovery of arterial blood pressure in mammals after acute hemorrhage is due to vasoconstriction (Chien, 1958). Bluefish show a similar initial recovery of ventral aortic pressure following acute hemorrhage. The fact that this initial phase of recovery was blocked by phentolamine suggests that alpha adrenergic vasoconstriction occurs in bluefish as well as mammals. Our study has not attempted to measure venous capacitance, cardiac output, pressure in the dorsal aorta or ventricular contractility, and therefore we cannot rule out changes in these parameters during the recovery of blood pressure. A second mechanism of recovery after hemorrhage is hemodilution which occurs over a longer period of time and acts to restore blood volume. This hemodilution was observed during the small repeated hemorrhages as a fall in hematocrit, hemoglobin and protein concentration. The responses to acute hemorrhage observed in bluefish are in contrast to the results in dogfish reported by Carrol et al. (1984). In the dogfish there was a significant increase in epinephrine levels 15 min
812
CHRISTOPI-tER S. OGILVY el al.
after hemorrhage and an increase in norepinephrine 20 min after hemorrhage. The gradual blood pressure recovery after hemorrhage was unchanged after blockade with the aipha-adrenergic blocking agent phentolamine. The authors concluded that dogfish respond to hemorrhage primarily through restoration of blood volume and not vasoconstriction. Hemodilution in dogfish after hemorrhage has also been documented by Sudak and Wilber (1960). The ability of dogfish to hemodilute rapidly may be due to a high rate of leakage of fluids into the vascular bed. The capillaries of dogfish have been shown to have loosely overlapping endothelial cells without tight junctions or gap junctions, suggesting a high permeability throughout the vascular system (Rhodin, 1972). With this arrangement interstitial fluid may move readily into the vascular compartment when the blood pressure is lowered. The findings of this study are consistent with the vascular changes observed in bluefish and dogfish in response to tilting head up in air (Ogilvy and DuBois, 1982). When dogfish were tilted head up to 30 ° for 30min the pulse pressure and blood pressure fell. There was a concomitant increase in interstitial fluid pressure in the caudal region of the fish. Bluefish tolerated head-up tilting well and were able to maintain their blood pressure and pulse pressure presumably through constriction of the vascular bed. A similar mechanism was observed in salmon in response to exercise. Salmon have an increased blood pressure during swimming and it is abolished by the alpha-adrenergic blocking agent phenoxybenzamine (Randall and Stevens, 1967). The initial recovery of blood pressure in response to hemorrhage observed in our present study appears to be mediated via an alpha-adrenergic response. This conclusion is drawn from the observation that measured catecholamines increased and phentolamine blocked the recovery of blood pressure after hemorrhage. Nishimura et al. (1979) found that plasma renin activity increased significantly with hemorrhage in toad fish. In dogfish, Opdyke et al. (1981) found that catecholamines increased following injection of angiotensin II. In our experiments we have not measured renin activity and the possibility exists that increased renin leads to the increased catecholamines we measured. There is a transient tachycardia present in the bluefish immediately after hemorrhage. The heart rate returned to control values within 5 min after the end of hemorrhage. Pretreatment with atropine did prolong but did not block the vascular (blood pressure) recovery after hemorrhage in bluefish. A tachycardia occurred after atropine and increased again significantly following the hemorrhage. In addition, the blood pressure response observed after hemorrhage when bluefish were pretreated with phentolamine and atropine was similar to the blood pressure response after hemorrhage when fish were pretreated with phentolamine alone. This indicates that the tachycardia observed in the bled untreated fish (no pharmacologic blockade) was probably secondary to release of vagal inhibition in response to hypovolemia and plays only a minor role in the recovery from hemorrhage. Burnstock (1969) reviewed vagal inhibition of the heart rate of elas-
mobranchs and teleosts. In the trout, the heart rate seems to be free of vagal inhibition until the peripheral end of the vagus is stimulated, or acetylcholine given, or the fish made hypoxemic, all of which produce bradycardia (Wood and Shelton, 1980a, b). In bluefish, the resting heart rate is about 43 beats/min, but this rate became accelerated to 71 beats/min with tilting 30 degrees (Ogilvy and DuBois, 1982), or to 103 beats/min while swimming (DuBois et al., 1975, 1976) or to 70beats/min with hemorrhage in the present study. Atropine, 0.01-0.02mg in the ventral aorta, caused the heart rate of bluefish to accelerate and to be fixed and independent of swimming or tilting (DuBois et al., 1975). The blood volume removed during acute hemorrhage from the bluefish in this study was 10 ml which represents 0.87% of the total average body weight of 1.15 + 0.03 (SE) kg. Blood volume in bluefish is approximately 4% of total body weight (as measured using the Evans' blue dye technique). Therefore, 10ml represents 21% of blood volume. In hemorrhage experiments where blood volume was measured using Evans' blue dye, three fish were hemorrhaged an average of 27% of blood volume. These fish with the greater amount of blood withdrawn showed acute recovery of blood pressure but within 30 min after hemorrhage became hypotensive and died. The amount of hemorrhage which produces irreversible hypotensive shock in teleosts of this study appears to be between 20 and 30% of blood volume. In splenectomized dogs the blood pressure fell in a linear fashion with hemorrhage exceeding 15-20% of blood volume with gradual return of blood pressure towards control values 3(I-60 min after hemorrhage. In animals subjected to hemorrhage greater than 20% of blood volume, irreversible shock occurs (Chien, 1958). Thus, we conclude that in our fish, as in mammals, since blood pressure is dependent in part upon blood volume, essential organs such as the central nervous system may be so under-perfused that irreversible damage occurs with reduction in blood volumes greater than 20% of total blood volume. The contribution at rest of catecholamines to vasomotor tone is felt to be small in the bluefish based on the observation that no change in blood pressure or heart rate occurred following administration of phentolamine without subsequent hemorrhage. Randall and Stevens (1967) reached a similar conclusion regarding the resting vasomotor tone in sockeye salmon with little change in blood pressure observed after administration of phenoxybenzamine at rest. In contrast to this low level of resting tone in bluefish and salmon, the resting tone in rainbow trout was felt to be significant based on a drop in blood pressure following intravenous phenoxybenzamine at rest (Wood and Shelton, 1980). In small repeated hemorrhages hemodilution occurs (Fig. 1) with replacement of blood volume and near restoration of total pressure (Fig. 7). This prevents the hypoperfusion of essential organs. The reason for the observed rise in serum sodium concentration and osmolality after small repeated hemorrhages is not clear. A possible explanation might be that changes of gill permeability or increased oral intake of sea-water allow sodium to enter the vascu-
Acute hemorrhage in bluefish lar space in response to the induced hypovolemia. In both the control and hemorrhaged fish the glucose concentration fell significantly. Fed mammals have a significant increase in the glucose concentration in response to hemorrhage and this provides an osmotic force capable of mobilizing fluid into the vascular space. In hemorrhage after fasting, there is no increase in the glucose concentration (Mohsenin and DuBois, 1983). The fall in glucose concentration observed in the fish is somewhat unexpected. The exact cause of the decrease in concentration is difficult to fully analyse without simultaneous levels of catecholamines and insulin. Future experiments could be designed to further investigate this point. The large rise in plasma epinephrine and norepinephrine levels following hemorrhage indicates a response of the adrenergic system. Blockade with phentolamine indicates that the recovery of blood pressure which was observed in untreated fish after rapid hemorrhage was due to alpha-adrenergic stimulation. Acknowledgements--This work was supported in part by a grant HL 17407 from the National Institutes of Health. Parts of this work have been presented in preliminary form (A. B. DuBois and P. G. Tremml. Vascular responses to rapid and slow hemorrhage in bluefish and smooth dogfish. Fed. Proc. 43, 639, 1984). Kimberly O'Sullivan participated in the present studies as a research assistant. Dr Rosa Hendler (Yale New Haven Medical Center) measured the epinephrine and norepinephrine levels. REFERENCES
Burnstock G. (1969) Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol. Rev. 21, 247-324. Carrol R. G., Opdyke D. F. and Keller N. E. (1984) Vascular recovery following hemorrhage in the dogfish shark Squalus acanthias. Am. J. Physiol. 246, R825-R828.
CIB,P, 91/4A--L
813
Chien S. (1958) A quantitative evaluation of the circulatory adjustment of splenectomized dogs to hemorrhage. Am. J. Physiol. 193, 605-614. DuBois A. B., Camiol P. and Morris R. DB. (1975) Effect of gravity and exercise on the heart rate and blood pressure of bluefish (Pomatomus saltatrix). (Abstract) Biol. Bull. Woods Hole, Massachusetts 149, 425. DuBois A. B., Carniol P. J. and Morris R. DB. (1976) Blood pressure and heart rate in bluefish: the effects of exercise, gravity and atropine. (Abstract) Fed. Proc. 35, 796. Mohsenin V. and DuBois A. B. (1983) Intercompartmental fluid shifts due to glucose release during hemorrhage in rabbits. Am. J. Physiol. 245, H143-H149. Nishimura H., Lunde L. G. and Zucker A. (1979) Renin response to hemorrhage and hypotension in the aglomerular toad fish Opsanus tau. Am. J. Physiol. 237, H 105-HI 11. Ogilvy C. S. and DuBois A. B. (1982) Effect of tilting on blood pressure and interstitial fluid pressure of bluefish and smooth dogfish. Am. J. Physiol. 242, R70-R76. Opdyke D. F., Bullock J., Keller N. E. and Holmes K. (1983) Dual mechanisms for catecholamine secretion in the dogfish shark Squalus acanthias. Am. J. Physiol. 244, R641 -R645. Randall D. J. and Stevens E. D. (1967) The role of adrenergic receptors in cardiovascular changes associated with exercise in Salmon. Comp. Biochem. Physiol. 21, 415~;24. Rhodin J. A. (1972) Fine structures of elasmobranch arteries, capillaries and veins in the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 42A, 59ff34. Starling E. H. On the absorption of fluids from the connective tissue spaces. J. Physiol. 19, 312-326, 1885-1886. Sudak F. N. and Wilber C. G. (1960) Cardiovascular responses to hemorrhage in the dogfish. Biol. Bull. 119, 342. Wood C. M. and Shelton G. (1980a) Cardiovascular dynamics and adrenergic responses of the rainbow trout in vivo. J. exp. Biol. 87, 247-270. Wood C. M. and Shelton G. (1980b) Reflex control of heart rate and cardiac output in the rainbow trout. J. exp. Biol. 87, 271-284.