AMIVXICAN
JOURNAL
OF PHYSIOLOGI-
Vol. 228, No. 2, February
Regulation
1975.
Printed
in U.S.A.
of arterial
in the common LYLE A. HOHNKE Defiartment of Physiology,
green
blood
pressure
iguana
Uiziuersity of California .
School of Medicine,
HOHN KE, LYLE A. Regula tiolz of arterial blood pressure in the commongreen iguana. Am. J. Physiol. 228(2) : 386-391. 1975.-Arterial blood pressure (ABP) responses to graded hemorrhage and passive head-up tilt were studied in restrained, anesthetized, and unanesthetized iguanas. The ABP fell slowly in response to hemorrhage up to a critical deficit of 35 + 19 yO of the estimated blood volume; the rate of ABP fall then increased nearly 40-fold to continued hemorrhage. Increased heart rate and decreased femoral arterial blood flow accompanied progressive hemorrhage. Propranolol (2-3 pg/kg) did not appreciably alter arterial pressure-hemorrhage curves but hemorrhage-induced increases in heart rate were diminished nearly 50%. Atropine had little effect on either the blood pressure or heart rate changes induced by hemorrhage. During passive tilts of U-90’ carotid arterial pressure
90024
the results are interpreted on the basis of reflex compensatory changes that may operate to assist systemic arterial pressure control. METHODS
AND
MATERIALS
Iguanas were obtained commercially. The animals, ranging in weight from 3 12 to 4,200 g, were kept in a partitioned room on a 12-h light-dark cycle. Heat lamps, 4 feet above the floor, and a stainless steel container, approximately 18 inches x 18 inches x 6 inches filled with water, were provided at opposite ends of the straw-covered area. Canned dog food, lettuce, and bananas were made available regularly to the animals. Animals were anesthetized for surgery with pentobarbital (Somnopental), 15 mg/kg ip, with additional anesthetic throughout the day as necessary. Other drugs administered via the indwelling venous catheter were propranolol (2-4 pg/kg), atropine (0.01-O. I mg/kg), and isoproterenol U-10 Pglkg). Direct recordings of pressures were made with indwelling saline-filled catheters connected to Statham pressure transducers. The femoral artery, femoral vein, and jugular vein were catheterized for arterial pressure, drug injection, and central venous pressure, respectiveIy. Temperatures were monitored rectally with a digital thermometer and experiments not run at room temperature were controlled by adjustable infrared heat lamps. Blood flow measurements were made with noncatheterizing electromagnetic flow probes (Micron) on the femoral artery. Flow probes were calibrated with vessel segments by gravity-induced saline flow controlled by an adjustable clamp. In vivo zero flows were obtained by occluding the vessel with forceps or a miniature hydraulic occluder (5). Flow probes were connected to a Biotronix electromagnetic flowmeter and all recordings were made with a Grass model 7 polygraph recorder. Blood volume (BV) determinations were made by dye dilution (8) with estimated values for the plasma volume (PV): BV = PV,/l - (Hct X 0.92). During both hemorrhage and tilt experiments, animals were restrained on a platform elevated about 12 inches above the surgical table. Experiments were performed at least 18-24 h after surgery on both anesthetized and unanesthetized animals. Surgery was well tolerated and good chronic preparations were used until physiological deterioration became evident.
fell 33 yO before returning to control levels (2 min). Heart rate increased and femoral arterial blood Aow and central venous pressure fell in response to head-up tilts. It is concluded that hemorrhage and passive head-up tilting can induce reflex cardiovascular changes that assist ABP regulation in iguanas. hemorrhage;
Los Angeles, Cd~urnia
passive tilt; reptiles; blood volume
THE TRANSITION OF ANIMAL LIFE from an aquatic to a terrestrial environment was associated with significant changes in circulatory requirements. In particular, lung development and dependence on aerial respiration required progressive separation of pulmonary and systemic circulations. A structurally double circulation first appears in the reptilian class Crocodilia; Squamata (lizards) possess a single ventricle that is tripartite, but confluent. The ventricular continuity of the squamates has invited numerous studies on the patterns of circulation of systemic and pulmonary venous blood (2, 10, 12, 15-l 8). As a result of such studies it has become clear that distributional patterns for systemic and pulmonary venous return may vary with the functional state of the animal, e.g., during diving and thermal stress. TFhe unique cardiac architecture of squamates fostered much of this work and has led to a considerable revision of views on reptilian circulation held earlier by comparative anatomists. Very little insight has been provided, however, on the ability of these animals to regulate cardiovascular functions such as arterial blood pressure and on reflexes, if any, that operate to maintain these functions. The present study was undertaken to partially characterize arterial blood pressure regulation in the common green iguana Iguana iguana. Pressure changes were induced by graded hemorrhage or passive tilts and
386
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BLOOD
PRESSURE
REGULATION
IN
387
REPTILES
Graded hemorrhage consisted of removing a fixed percentage (l-5 “r) of the estimated blood volume at fixed intervals (2-4 min); blood was later restored to the animal along the same volume and time course. The volume of blood removed was stored at room temperature in heparinized syringes and mixed well before reinjection. Passive head-up tilting was performed by elevating one end of a platform with a rope and pulley arrangement. Selected tilts were secured in IO-15 s and resDonses monitored several minutes before making further lchanges. All transducers were mounted on the tilt platform and b&e-line shifts were checked before making the measurements. Statistical analyses were done with a calculator (Monroe model 1766) with programs for computing standard deviations, t statistics, and correlation coefficients* Arterial blood pressure responses to hemorrhage and reinfusion were analyzed by the statistical jackknife technique (1, 13) on a PDP-10 computer.
A. Mean
70
Arterial Pressure (mm
80
6 O
Hg)
30
BHeart
l
-m
Hemorrhage
l
80-
Rote
(beotdmin)
7 ‘-
RESULTS Blood
Volume
Estimated blood volumes and their relationship to total body weight are shown in Table 1. Single determinations were run for each animal after all surgery was complete but before hemorrhage and reinfusion experiments were begun.
c. Central Pressure
(mm
Femoral
Normal responses. Responses to graded hemorrhage in seven iguanas were studied in 17 separate experiments; femoral arterial blood flow was measured in three of these experiments on two different animals. Figure 1 shows the response pattern of a hemorrhage experiment that included a determination of femoral arterial blood flow. Arterial pressure changes induced by hemorrhage and reinjection were divided arbitrarily into two regions for mathematical analysis. During early stages of hemorrhage (region I) arterial pressure fell slowly but then decreased dramatically (region II) upon additional blood loss. The point of greatest slope change was estimated mathematically by applying the statistical jackknife estimate to both hemorrhage and reinfusion data. This point, designated the critical blood volume deficit (CBVD), represents a level of blood loss beyond which continued hemorrhage will significantly enhance decreases in arterial pressure.
TABLE
1. Estimated Animal
VII VIII Mean
=t SD
No.
Hg)
D* IO
Graded Hemorrhage
~__~-
Venous
blood volumes of common green iguana iTeight,
g
Estim;$dm:Iood
,
1,188 960 315 2,815 1,400
63 18 116
1,870 4,200 3,625
105 244 191
Blood VoJ, ml x 1OO Body Wt, g
62
5.5%
1
Fiow (ml/mm) IO Blood FIG.
fusion
20
30 Volume
1. Cardiovascular responses to graded in a conscious, restrained iguana.
40
50 Deficit
hemorrhage
60
70
( ml)
and
rein-
The CBVD values are summarized in Table 2 in milliliters and as a percent of total estimated blood volume for all animals studied. The CBVD did not differ significantly during hemorrhage (H) and reinfusion (R) in any one experiment and the values were treated together to calculate the mean & SD for untreated animals. Unusually high CBVD values may reflect low estimates of circulating blood volume rather than real differences in compensatory response. The enhanced decrease in arterial pressure during the latter stages of hemorrhage is reflected in the higher absolute value of the regression coefficient for region II compared with region I. The mean regression value for region II was approximately 40 times greater than in region I (a
< .OOl).
5.2 6.6 5.7 4.1 5.4 5.6 5.8 5.3
76
Blood
*
0.7
Associated with hemorrhagic-induced lowering of blood pressure were increases in heart rate and decreases in central venous pressure and femoral arterial blood flow (Fig. 1) The leveling off of heart rate during late hemorrhage was not a consistent observation in all experiments. Femoral resistance, estimated from femoral blood flow and arterial-venous pressure differences, increased nearly twofold initially but decreased during the final stages of hemor-
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388
L. A. HQHNKE
TABLE 2. Summary of hemorrhage and reinfusion exj2eriments in Iguana iguana
A;““1
Hemor rhipienor . fusion
.
H H H H R H R
Critic al ? Iood VoI Deficit, ml
Pre~o~~lof Estimated Blood Vol
24.0* 1.9t 40*0+21.0 3.8rt
0.3
25.0* 34.ozt 28.0&
2.3 0.2 1.4
35.&k
4.4
A.
Regression
Coefficient
Region
Region
38 62
-0.003
22
+2.4
I
-0.58
11
Treatmerit*
M0Cln Arterial Pressure (mm
-2.75 -1.92
ControI
Hgl
-26.0
22 29 24 30
-0.30 -0.60 -0.40 -0.37
-1.7 -655 -1.6 -2.3
H R
20.0*
5.0
ll.Ozt
2.8
26 15
-0.80 -0.07
-2.5 -2.2
H R H R
47.OM6.0 50.01t 0.6 47.0* 5.5 57.ozt31 .o
71 76 71 86
-0.34 -0.09 -0.39 -0.42
-1.6
H R H
58.0& 56.011~ 64.0&
3.0 3.0 3.0
24 23 29
-1.6 3-0.7 +1.6
- 13.5 - 16.0 -17.5
H R H R H R H R
71.0~ 96.Ozt 41 .o* 50.0& 36.OzU3.0 52.0& 53.0+ 50.0zt19.0
1.6 9.6 7-9 9.6
41 50 21 26 19 27 28 26
-to.31 -0.22 -0.25 -0.00 -0.00 +0.15 -0.1.5 -0.24
-2.4 -2.4 -1.3 -1.3 -0.75 -1.8 -1.4 -0.75
-2.6 -2,3 -1.3
/ Propranolol (3 Y/K)
B.
P P
80
Mean Arterial A
Pressure (mm
Mean “A
7.4 9.8
j= SD$ =
atropine; P = propranolol. $ Untreated animals only.
35 *19x
- .13
-5.2
=t*77$
&6.8$
t 60 70 confidence
Hg’b
A
30
A A+P A + P
20
I o0
bounds.
rhage in two of three experiments. In a third experiment femoral resistance increased nearly fourfold without any subsequent decreases. After reinfusion femoral resistance was nearly equal to the prehemorrhage state except in one experiment in which femoral resistance returned to approximately one-half of the prehemorrhage level. The smaller femoral resistance in the latter experiment resulted in an increase in femoral arterial blood flow that was nearly 2.5 times greater (3.8 ml/ min) than the prehemorrhage rate (1.6 ml/min). Effect of atropine and propranolol on arterial PreSSwe and heart rate. Propranolol did not significantly change the jackknife estimate of the CBVD or the general profile of the arterial pressure response to hemorrhage (Fig. ZA). Although the jackknife estimate of the CBVD was not significantly changed by propranolol administration, the presence of two distinct slopes is less evident and a single regression line through all points correlated well with the data (r = 0.92). Atropine or atropine plus propranolol affected arterial responses to hemorrhage similarly (Fig. ZB). Heart rate decreased nearly 50 % in the propranololtreated animal (Fig. 3A) but the slope remained nearly unchanged. Slight increases in heart rate occurred after atropine treatment; this became more apparent as the degree of hemorrhage progressed since the slope increased
Atropine (IO0 Y/K)
+
Propranolol
I
I
I
I
I
(3 Y/K) I
I
c
IO
20
30
40
50
60
70
80
Mood
Volume
Deficit
FIG. 2. Arterial blood pressure responses to hemorrhage fusion in a conscious, restrained iguana after treatment pranolol (3 &kg) and/or atropine (100 pg/kg),
(ml) and reinwith pro-
(Fig. 3B). Atropine plus propranolol nearly abolished heart rate increases induced by graded hemorrhage. Central venous pressure was not measurably altered from the control response shown in Fig. 1C by either propranolol or atropine. Passive Tilt Head-up passive tilting resulted in a transient decrease in carotid arterial pressure. Figure 4 shows a O-90” tilt in a Z.&kg anesthetized animal. Mean carotid arterial pressure initially fell 33 % but recovered to the pretilt level in about 2 min. Femoral arterial pressure did not decrease in this experiment, suggesting that the increased hydrostatic pressure in the femoral artery balanced the fall in pressure attributed to tilting. The base line was checked in both carotid and femoral transducers to assure that pressure changes did not represent a base-line shift. Nearly uniform increases in cardiac rate resulted from head-up tilts. Table 3 summarizes the results of 10 experiments performed on three large iguanas (2.8-3.7 kg).
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BLOOD
PRESSURE
REGULATION
IN
389
REPTILES TABLE
E#kt
3.
ofjmsivahead-up tilt on
heart rate in Iguana iguana Heart
Rate Change Angle
Anesthetized
4o 30
“X
k
l-f
2 oIO-
B. Heart
15" 25" 3Q0 45" 60" 7o" 90"
xx Propranolol -1
( 3
,
,
Y/K
1
1
1
1
1
8 Ot
Mean ps
Atropine (100 7/K)
70
Angle
IO
20 Blood
30
40 Volume
50 Deficit
60
70
4, Effect of passive blood pressure in base-line check.
tilting (o-90”) on carotid and a conscious, restrained iguana.
%
3h -5 -2
*
(1) O(2) 13 (2) 0)
11 lzt
of experiments
(1) 8
of Tilt
18 0 13 16 21 33 24
thetized
zk 18 (2) (1) zk 8 (4) II= 7 (7) zt 17 (3) szts 25 (4) =t: 23 (3)
19 *
16
in parentheses.
Treatment
on heart rate
Percent Increase in Heart Rate Over Horizontal Control
30" 60"
None
13 19
30" 60"
Atropine
11 17
30" 60"
Atropine
80
(mt)
FIG. 3. Effect of propranolol (3 &kg) and/or atropine (100 pg/kg) on heart rate changes induced by hemorrhage and reinfusion in a conscious, restrained iguana. Regression coefficients (r) and treatments are designated.
FIG.
Rate,
Unanes
TABLE 4. E$ect of atrqh’na and @@anoh/ during passive head-up tilt
Rote
t beotslmin)
arterial indicates
-3
& SD
Number
in Heart
of Tilt
femoral Asterisk
Variability in responses did not allow a quantitative study of the relationship between heart rate change, arterial pressure, and angle of tilt. Heart rate changes in relationship to tilt angle are not significantly different for anesthetized and unanesthetized animals. If the responses of the unanesthetized animals to all angles of tilt are grouped, however, and compared to the responses of anesthetized
+ propranolol
3 7
animals at all angles of tilt, a significant difference does exist (P < 0.01). In one experiment mean femoral blood flow was monitored during passive tilting. At all angles of tilt (30, 40, 60, and 90°) a decrease of femoral flow resulted that was not uniformly proportional to the angle of tilt (- 25, - 13, -25, and -38 %, respectively). In all cases the decrease in femoral blood flow lasted longer than the transient fall of arterial pressure. Central venous pressure fell proportionately with increased angle of tilt up to 60”. Central venous pressure responses to tilt were thus similar to the responses observed during graded hemorrhage. The effect of atropine and propranolol on heart rate changes induced by tilt was studied in one experiment. The results are summarized in Table 4 and are expressed increase above resting horizontal controls, as percent Atropine had a slight inhibitory effect on heart rate increases; propranolol in combination with atropine greatly reduced the heart rate increases in response to tilt,
To meet the needs and requirements of an active cellular metabolism animal transport systems have been modified progressively toward a more closed system of vessels, greater separation of oxygenated and deoxygenated blood, higher pressures, and enhanced flow rates. The most efficient and best characterized circulatory system is that of mammals but studies on lower vertebrates are increasing (10). Fn reptiles where important advances toward a double circulation are evident, pressures approaching those of
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390 mammals can be measured. It seems reasonable that appropriate controls must also exist to regulate the arterial pressure that is so fundamental to homeostasis. This study has demonstrated that the iguana can regulate arterial blood pressure during periods of graded hemorrhage and passive head-up tilting. The gradual decrease in arterial pressure in response to initial stages of hemorrhage is followed by a more marked fall in pressure after additional blood loss. This two-component response to graded hemorrhage is interpreted as an initial compensatory response followed by loss cf compensation during extreme blood loss. Thus as the compensatory reserve becomes exhausted arterial pressure falls more rapidly in response to hemorrhagic insult. If reflex adjustments did not exist arterial pressure would probably fall proportionately with blood loss without the slope change noted in Fig. 1 and less blood affecting the loss could be tolerated without adversely survival of the organism. The experiments on passive head-up tilting provide additional evidence for a controlled arterial pressure. In humans (19) and dogs (4) cardiac output and mean arterial pressure are reduced during passive tilting, an effect probably related to venous pooling of blood and thus a lowered venous return. The reduced arterial pressure in iguanas after tilt is probably related to a lowered cardiac output for the same reason it is reduced in man and dog after tilt. The increased heart rate after head-up tilts is undoubtedly one of several concerted reflex responses that act to counterbalance the fall in blood pressure+ Correlated with the fall in arterial pressure during graded hemorrhage is an increased heart rate and a decreased CVP and femoral artery blood flow. Calculated femoral resistance increased initially but fell as the arterial pressure decreased more sharply to continued hemorrhage. Heart rate increases were greatly reduced after treatment with propranolol in both hemorrhage and tilt experiments. The arterial pressure curve after propranolol (Fig. 2,4) was lower than the control curve but the jackknife estimates of CBVD and the general profile of the curve were unchanged. Atropine had a lesser effect on both heart rate changes induced by hemorrhage (Fig. 3B) or tilt (Table 4) and also had no appreciable effect on the arterial pressure changes caused by hemorrhage (Fig. ZB). These results suggest that iguanas have a low vagal tone and that heart rate increases are predominantly caused by an increased sympathetic drive on the heart. Fluorescence microscopy (Hohnke, unpublished observations) revealed relatively dense adrenergic innervation of the ventricular myocardium and provided additional evidence for the sympathetic innervation of the iguana heart. Similar observations have been reported for the adrenergic innervation of the lizard Trachysaurus rugosus (7) ; studies with TiZiqua rugosa (3) reveal that most cardiac sympathetic nerves travel independently of the vagus and derive almost exclusively from the ‘Lstellate complex.” Microscopic examination for fluorescence in cross sections of the femoral artery did not reveal unusual patterns or densities of adrenergic innervation. Specific fluorescence was confined to the adventitial-medial border and was relatively sparse (Hohnke, unpublished observations). It is interesting that arterial pressure changes induced
L. A. HOHNKE
by hemorrhage were largely unmodified by doses of blocking agents that did prevent increases in heart rate. One interpretation might be that changes in heart rate contribute little to major changes in arterial blood pressure in iguanas. A study of the reflex adjustments in regional flows and resistance in response to hemorrhage would probablv be very instructive in this regard. A dimerent interpreiation might argue that the hypothesis of neurogenic control mechanism is weakened by the failure of atropine and propranolol to affect the response to hemorrhage. A nonneurogenic mechanism of arterial blood pressure control might rely on physical factors to explain the presence of two slopes in the hemorrhage curves. One such factor might be fluid absorption to expand the circulating blood volume with subsequent failure of this mechanism during late stages of hemorrhageAnother possibility would be a sudden decrease in volume of the vascular system initiated at some critical low arterial pressure. The reduced capacity of the reservoir from which blood could be removed might result in a steer>er slope to the hemorrhage curve. The various possi ble i nterpreta tions are not necessarily independent from one another and each can be tested experimentally. The degree to which different mechanisms may be important in the control of arterial bIood pressure in reptiles might contribute to an understanding of the development of arterial pressure control mechanisms in higher vertebrates. Studies similar to the hemorrhage experiments in iguanas have also been done on birds. The anesthetized domestic hen appears quite sensitive to hemorrhagic insult. Removal of less than 10 % of the estimated initial blood volume lowered the mean AP about 22 mmHg; this effect was more than doubled if 15-25 % of the blood volume was removed (20). I n unanesthetized ducks, however, arterial pressure showed little change af‘ter removal of up to 25 Y& of the estimated blood volume (6). In both studies increased heart rate and decreased hematocrit accompanied the fall in arterial pressure. The ability of ducks and pigeons (11) to resist blood loss seems related to a great capacity for fluid mobilization from skeletal muscle (6) In the d uck this is effected by a strong reflex vasoconstriction an d a large ca pillary surface area in the skeletal muscle. These mechanisms may operate in lizards also but, as suggested, this has not been well studied. In mammals arterial pressure is maintained relatively constant by reflex adjustments initiated through changes in pressoreceptor and chemoreceptor activity. If similarly mediated reflex adjustments are operative to regulate arterial blood pressure in the iguana and lizards generally, then the location and characterization of reflexogenic zones become verv important. Information on presloreceptors and chemoreceptors in lower vertebrates is generally unavailable; this is particularly true for reptiles. If the tachycardia that accompanies hemorrhage in the iguana is initiated by pressoreceptors it may be a very helpful reflex to use in a search for reflexogenic areas in reptiles. The reflex adjustments to hemorrhage could be mediated by pressoreceptor and/or chemoreceptors, depending on their presence and/or relative importance. Responses to tilt, however, would most likely be initiated by a change in pressoreceptor activity. This is interesting
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BLOOD
PRESSURE
REGULATION
IN
391
REPTILES
since the carotid sinus reflex does not seem to exist in iguanas (Hohnke, unpublished observation). The increasedv femoral resistance, estimated for the vascular bed supplied by the femoral artery, is probably a reflex response also and could provide another useful end point in a search for receptor zones and neuronal pathways involved in controlling arterial blood pressure.
The statistical assistance of Dr. David S. Salzburg (Pfizer Research) is gratefully acknowledged. This work was supported by National Science Foundation GB 49310, Public Health Service Training Grant IIL-05696, the American I-Ieart Association-Greater Los Angeles Affiliate 4371G. Present address: Pfizer Central Research, Groton, Conn. Received
for
publication
13 February
Central Grant and by Grant 06340.
1974.
REFERENCES 1. ARVESEN,
11.
2.
12.
3.
4.
5. 6.
7.
8.
9. 10.
J. Jackknifing U-statistics. Ann. Math. Stat, 40: 20762100, 1969. BAKER, L. ,4., AND F. N. WHITE. Redistribution of cardiac output in response to heating in Iguana iguana. Gomp. Biochem. Physiul. 35 : 253-262, 1970. BERGER, P. J* The vagal and sympathetic innervation of the heart of the lizard Tiliqua rugosa. Australian J. Exptl. Biol. Med. Sci. 49: 297-304, 1971. CONSTANTINE, J. Mr., W. K. MCSHANE, AND S. C. WANG. Comparison of carotid artery occlusion and tilt responses in dogs. Am. J. Hiysiol. 221 : 1681-1685, 1971. QEBLEY, V. G. Miniature hydraulic occluder for zero blood flow determination. J. A@. P&iol. 3 1 : 138-l 39, 197 I. A. M., B. F~LKOW, AND A. G. B. KOVACH. The QOJOSUGITO, mechanisms behind the rapid blood volume restoration after hemorrhage in birds. Acta Physiol. Stand. 74 : 114-l 22, 1968. FURNESS, J. B., AND J. MOORE. The adrenergic innervation of the cardio-vascular system of the lizard Trachysaurus rugosus. 2. Zellforsch. 108 : 150-l 76, 1970. GREGERSON, M. I. A practical method for the determination of blood volume with the dye T-1824. J. Lab. Clin. Med. 29: 12661269, 1944. JOHANSEN, K. Circulation in the three-chambered snake heart. Circulation Res. 7 : 828-832, 1959. JOHANSEN, K. Comparative physiology: gas exchange and circulation in fishes. Ann. Rev. Phvsiol. 33 : 569-612. 197 1.
13.
14.
15. 16. 17. 18. 19.
20.
KOVACH, A. G. B., AND E. SZASZ. Survival of pigeon after graded haemorrhage. Acta Physiol. Acad, Sci. Hung. 34 : 301-309, 1968. MORGAREIDGE, K. R., AND F. N. WHITE. Cutaneous vascular changes during heating and cooling in the Galapagos marine iguana. Nature 223 : 587-591, 1969. MOSTELLER, F., AND J. W. TUKEY. Data analysis, including statistics. In : Handbook of Social Psychology, edited by G. Lindzey and E. Aronson. Reading, Mass.: Addison Wesley, 1968. STEGGERDA, F. R., AND II. E:. ESSEX. Circulation and blood pressure in the great vessels and heart of the turtle (Cheledra serpentina). Am. J. Physiol. 190: 310-326, 1957. WHITE, F. N. Circulation in the reptilian heart (Caiman sclerops). Anat. Record 125 : 417-431, 1956. WHITE, F. N. Functional Zool. 8: 211-219, 1968. WHITE, F. N. Redistribution gator, Copeia 3: 567-570,
anatomy of cardiac
of the
heart
output
of reptiles. in
the
diving
Am. alli-
1969.
WHITE, F. N. Central vascular shunts and their control in reptiles. Federation hoc. 29: 1149-l 153, 1970. WEISSLER, A. M., W. H. ROEHXLL, AND R. G. PEELER. Effect of posture on the cardiac response to increased peripheral demand. J. Lab. C/in. Med. 59: 1000-1007, 1962. WYSE, D. G., AND M, NICKERSON. Studies on hemorrhagic hypotension in domestic fowl. Can. J. Physiol. Pharmacol. 49 : 9 19-926, 1971.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
JOURNAL
OF PHYSIOLOGI-
Vol. 228, No. 2, February
Regulation
1975.
Printed
in U.S.A.
of arterial
in the common LYLE A. HOHNKE Defiartment of Physiology,
green
blood
pressure
iguana
Uiziuersity of California .
School of Medicine,
HOHN KE, LYLE A. Regula tiolz of arterial blood pressure in the commongreen iguana. Am. J. Physiol. 228(2) : 386-391. 1975.-Arterial blood pressure (ABP) responses to graded hemorrhage and passive head-up tilt were studied in restrained, anesthetized, and unanesthetized iguanas. The ABP fell slowly in response to hemorrhage up to a critical deficit of 35 + 19 yO of the estimated blood volume; the rate of ABP fall then increased nearly 40-fold to continued hemorrhage. Increased heart rate and decreased femoral arterial blood flow accompanied progressive hemorrhage. Propranolol (2-3 pg/kg) did not appreciably alter arterial pressure-hemorrhage curves but hemorrhage-induced increases in heart rate were diminished nearly 50%. Atropine had little effect on either the blood pressure or heart rate changes induced by hemorrhage. During passive tilts of U-90’ carotid arterial pressure
90024
the results are interpreted on the basis of reflex compensatory changes that may operate to assist systemic arterial pressure control. METHODS
AND
MATERIALS
Iguanas were obtained commercially. The animals, ranging in weight from 3 12 to 4,200 g, were kept in a partitioned room on a 12-h light-dark cycle. Heat lamps, 4 feet above the floor, and a stainless steel container, approximately 18 inches x 18 inches x 6 inches filled with water, were provided at opposite ends of the straw-covered area. Canned dog food, lettuce, and bananas were made available regularly to the animals. Animals were anesthetized for surgery with pentobarbital (Somnopental), 15 mg/kg ip, with additional anesthetic throughout the day as necessary. Other drugs administered via the indwelling venous catheter were propranolol (2-4 pg/kg), atropine (0.01-O. I mg/kg), and isoproterenol U-10 Pglkg). Direct recordings of pressures were made with indwelling saline-filled catheters connected to Statham pressure transducers. The femoral artery, femoral vein, and jugular vein were catheterized for arterial pressure, drug injection, and central venous pressure, respectiveIy. Temperatures were monitored rectally with a digital thermometer and experiments not run at room temperature were controlled by adjustable infrared heat lamps. Blood flow measurements were made with noncatheterizing electromagnetic flow probes (Micron) on the femoral artery. Flow probes were calibrated with vessel segments by gravity-induced saline flow controlled by an adjustable clamp. In vivo zero flows were obtained by occluding the vessel with forceps or a miniature hydraulic occluder (5). Flow probes were connected to a Biotronix electromagnetic flowmeter and all recordings were made with a Grass model 7 polygraph recorder. Blood volume (BV) determinations were made by dye dilution (8) with estimated values for the plasma volume (PV): BV = PV,/l - (Hct X 0.92). During both hemorrhage and tilt experiments, animals were restrained on a platform elevated about 12 inches above the surgical table. Experiments were performed at least 18-24 h after surgery on both anesthetized and unanesthetized animals. Surgery was well tolerated and good chronic preparations were used until physiological deterioration became evident.
fell 33 yO before returning to control levels (2 min). Heart rate increased and femoral arterial blood Aow and central venous pressure fell in response to head-up tilts. It is concluded that hemorrhage and passive head-up tilting can induce reflex cardiovascular changes that assist ABP regulation in iguanas. hemorrhage;
Los Angeles, Cd~urnia
passive tilt; reptiles; blood volume
THE TRANSITION OF ANIMAL LIFE from an aquatic to a terrestrial environment was associated with significant changes in circulatory requirements. In particular, lung development and dependence on aerial respiration required progressive separation of pulmonary and systemic circulations. A structurally double circulation first appears in the reptilian class Crocodilia; Squamata (lizards) possess a single ventricle that is tripartite, but confluent. The ventricular continuity of the squamates has invited numerous studies on the patterns of circulation of systemic and pulmonary venous blood (2, 10, 12, 15-l 8). As a result of such studies it has become clear that distributional patterns for systemic and pulmonary venous return may vary with the functional state of the animal, e.g., during diving and thermal stress. TFhe unique cardiac architecture of squamates fostered much of this work and has led to a considerable revision of views on reptilian circulation held earlier by comparative anatomists. Very little insight has been provided, however, on the ability of these animals to regulate cardiovascular functions such as arterial blood pressure and on reflexes, if any, that operate to maintain these functions. The present study was undertaken to partially characterize arterial blood pressure regulation in the common green iguana Iguana iguana. Pressure changes were induced by graded hemorrhage or passive tilts and
386
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BLOOD
PRESSURE
REGULATION
IN
387
REPTILES
Graded hemorrhage consisted of removing a fixed percentage (l-5 “r) of the estimated blood volume at fixed intervals (2-4 min); blood was later restored to the animal along the same volume and time course. The volume of blood removed was stored at room temperature in heparinized syringes and mixed well before reinjection. Passive head-up tilting was performed by elevating one end of a platform with a rope and pulley arrangement. Selected tilts were secured in IO-15 s and resDonses monitored several minutes before making further lchanges. All transducers were mounted on the tilt platform and b&e-line shifts were checked before making the measurements. Statistical analyses were done with a calculator (Monroe model 1766) with programs for computing standard deviations, t statistics, and correlation coefficients* Arterial blood pressure responses to hemorrhage and reinfusion were analyzed by the statistical jackknife technique (1, 13) on a PDP-10 computer.
A. Mean
70
Arterial Pressure (mm
80
6 O
Hg)
30
BHeart
l
-m
Hemorrhage
l
80-
Rote
(beotdmin)
7 ‘-
RESULTS Blood
Volume
Estimated blood volumes and their relationship to total body weight are shown in Table 1. Single determinations were run for each animal after all surgery was complete but before hemorrhage and reinfusion experiments were begun.
c. Central Pressure
(mm
Femoral
Normal responses. Responses to graded hemorrhage in seven iguanas were studied in 17 separate experiments; femoral arterial blood flow was measured in three of these experiments on two different animals. Figure 1 shows the response pattern of a hemorrhage experiment that included a determination of femoral arterial blood flow. Arterial pressure changes induced by hemorrhage and reinjection were divided arbitrarily into two regions for mathematical analysis. During early stages of hemorrhage (region I) arterial pressure fell slowly but then decreased dramatically (region II) upon additional blood loss. The point of greatest slope change was estimated mathematically by applying the statistical jackknife estimate to both hemorrhage and reinfusion data. This point, designated the critical blood volume deficit (CBVD), represents a level of blood loss beyond which continued hemorrhage will significantly enhance decreases in arterial pressure.
TABLE
1. Estimated Animal
VII VIII Mean
=t SD
No.
Hg)
D* IO
Graded Hemorrhage
~__~-
Venous
blood volumes of common green iguana iTeight,
g
Estim;$dm:Iood
,
1,188 960 315 2,815 1,400
63 18 116
1,870 4,200 3,625
105 244 191
Blood VoJ, ml x 1OO Body Wt, g
62
5.5%
1
Fiow (ml/mm) IO Blood FIG.
fusion
20
30 Volume
1. Cardiovascular responses to graded in a conscious, restrained iguana.
40
50 Deficit
hemorrhage
60
70
( ml)
and
rein-
The CBVD values are summarized in Table 2 in milliliters and as a percent of total estimated blood volume for all animals studied. The CBVD did not differ significantly during hemorrhage (H) and reinfusion (R) in any one experiment and the values were treated together to calculate the mean & SD for untreated animals. Unusually high CBVD values may reflect low estimates of circulating blood volume rather than real differences in compensatory response. The enhanced decrease in arterial pressure during the latter stages of hemorrhage is reflected in the higher absolute value of the regression coefficient for region II compared with region I. The mean regression value for region II was approximately 40 times greater than in region I (a
< .OOl).
5.2 6.6 5.7 4.1 5.4 5.6 5.8 5.3
76
Blood
*
0.7
Associated with hemorrhagic-induced lowering of blood pressure were increases in heart rate and decreases in central venous pressure and femoral arterial blood flow (Fig. 1) The leveling off of heart rate during late hemorrhage was not a consistent observation in all experiments. Femoral resistance, estimated from femoral blood flow and arterial-venous pressure differences, increased nearly twofold initially but decreased during the final stages of hemor-
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388
L. A. HQHNKE
TABLE 2. Summary of hemorrhage and reinfusion exj2eriments in Iguana iguana
A;““1
Hemor rhipienor . fusion
.
H H H H R H R
Critic al ? Iood VoI Deficit, ml
Pre~o~~lof Estimated Blood Vol
24.0* 1.9t 40*0+21.0 3.8rt
0.3
25.0* 34.ozt 28.0&
2.3 0.2 1.4
35.&k
4.4
A.
Regression
Coefficient
Region
Region
38 62
-0.003
22
+2.4
I
-0.58
11
Treatmerit*
M0Cln Arterial Pressure (mm
-2.75 -1.92
ControI
Hgl
-26.0
22 29 24 30
-0.30 -0.60 -0.40 -0.37
-1.7 -655 -1.6 -2.3
H R
20.0*
5.0
ll.Ozt
2.8
26 15
-0.80 -0.07
-2.5 -2.2
H R H R
47.OM6.0 50.01t 0.6 47.0* 5.5 57.ozt31 .o
71 76 71 86
-0.34 -0.09 -0.39 -0.42
-1.6
H R H
58.0& 56.011~ 64.0&
3.0 3.0 3.0
24 23 29
-1.6 3-0.7 +1.6
- 13.5 - 16.0 -17.5
H R H R H R H R
71.0~ 96.Ozt 41 .o* 50.0& 36.OzU3.0 52.0& 53.0+ 50.0zt19.0
1.6 9.6 7-9 9.6
41 50 21 26 19 27 28 26
-to.31 -0.22 -0.25 -0.00 -0.00 +0.15 -0.1.5 -0.24
-2.4 -2.4 -1.3 -1.3 -0.75 -1.8 -1.4 -0.75
-2.6 -2,3 -1.3
/ Propranolol (3 Y/K)
B.
P P
80
Mean Arterial A
Pressure (mm
Mean “A
7.4 9.8
j= SD$ =
atropine; P = propranolol. $ Untreated animals only.
35 *19x
- .13
-5.2
=t*77$
&6.8$
t 60 70 confidence
Hg’b
A
30
A A+P A + P
20
I o0
bounds.
rhage in two of three experiments. In a third experiment femoral resistance increased nearly fourfold without any subsequent decreases. After reinfusion femoral resistance was nearly equal to the prehemorrhage state except in one experiment in which femoral resistance returned to approximately one-half of the prehemorrhage level. The smaller femoral resistance in the latter experiment resulted in an increase in femoral arterial blood flow that was nearly 2.5 times greater (3.8 ml/ min) than the prehemorrhage rate (1.6 ml/min). Effect of atropine and propranolol on arterial PreSSwe and heart rate. Propranolol did not significantly change the jackknife estimate of the CBVD or the general profile of the arterial pressure response to hemorrhage (Fig. ZA). Although the jackknife estimate of the CBVD was not significantly changed by propranolol administration, the presence of two distinct slopes is less evident and a single regression line through all points correlated well with the data (r = 0.92). Atropine or atropine plus propranolol affected arterial responses to hemorrhage similarly (Fig. ZB). Heart rate decreased nearly 50 % in the propranololtreated animal (Fig. 3A) but the slope remained nearly unchanged. Slight increases in heart rate occurred after atropine treatment; this became more apparent as the degree of hemorrhage progressed since the slope increased
Atropine (IO0 Y/K)
+
Propranolol
I
I
I
I
I
(3 Y/K) I
I
c
IO
20
30
40
50
60
70
80
Mood
Volume
Deficit
FIG. 2. Arterial blood pressure responses to hemorrhage fusion in a conscious, restrained iguana after treatment pranolol (3 &kg) and/or atropine (100 pg/kg),
(ml) and reinwith pro-
(Fig. 3B). Atropine plus propranolol nearly abolished heart rate increases induced by graded hemorrhage. Central venous pressure was not measurably altered from the control response shown in Fig. 1C by either propranolol or atropine. Passive Tilt Head-up passive tilting resulted in a transient decrease in carotid arterial pressure. Figure 4 shows a O-90” tilt in a Z.&kg anesthetized animal. Mean carotid arterial pressure initially fell 33 % but recovered to the pretilt level in about 2 min. Femoral arterial pressure did not decrease in this experiment, suggesting that the increased hydrostatic pressure in the femoral artery balanced the fall in pressure attributed to tilting. The base line was checked in both carotid and femoral transducers to assure that pressure changes did not represent a base-line shift. Nearly uniform increases in cardiac rate resulted from head-up tilts. Table 3 summarizes the results of 10 experiments performed on three large iguanas (2.8-3.7 kg).
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BLOOD
PRESSURE
REGULATION
IN
389
REPTILES TABLE
E#kt
3.
ofjmsivahead-up tilt on
heart rate in Iguana iguana Heart
Rate Change Angle
Anesthetized
4o 30
“X
k
l-f
2 oIO-
B. Heart
15" 25" 3Q0 45" 60" 7o" 90"
xx Propranolol -1
( 3
,
,
Y/K
1
1
1
1
1
8 Ot
Mean ps
Atropine (100 7/K)
70
Angle
IO
20 Blood
30
40 Volume
50 Deficit
60
70
4, Effect of passive blood pressure in base-line check.
tilting (o-90”) on carotid and a conscious, restrained iguana.
%
3h -5 -2
*
(1) O(2) 13 (2) 0)
11 lzt
of experiments
(1) 8
of Tilt
18 0 13 16 21 33 24
thetized
zk 18 (2) (1) zk 8 (4) II= 7 (7) zt 17 (3) szts 25 (4) =t: 23 (3)
19 *
16
in parentheses.
Treatment
on heart rate
Percent Increase in Heart Rate Over Horizontal Control
30" 60"
None
13 19
30" 60"
Atropine
11 17
30" 60"
Atropine
80
(mt)
FIG. 3. Effect of propranolol (3 &kg) and/or atropine (100 pg/kg) on heart rate changes induced by hemorrhage and reinfusion in a conscious, restrained iguana. Regression coefficients (r) and treatments are designated.
FIG.
Rate,
Unanes
TABLE 4. E$ect of atrqh’na and @@anoh/ during passive head-up tilt
Rote
t beotslmin)
arterial indicates
-3
& SD
Number
in Heart
of Tilt
femoral Asterisk
Variability in responses did not allow a quantitative study of the relationship between heart rate change, arterial pressure, and angle of tilt. Heart rate changes in relationship to tilt angle are not significantly different for anesthetized and unanesthetized animals. If the responses of the unanesthetized animals to all angles of tilt are grouped, however, and compared to the responses of anesthetized
+ propranolol
3 7
animals at all angles of tilt, a significant difference does exist (P < 0.01). In one experiment mean femoral blood flow was monitored during passive tilting. At all angles of tilt (30, 40, 60, and 90°) a decrease of femoral flow resulted that was not uniformly proportional to the angle of tilt (- 25, - 13, -25, and -38 %, respectively). In all cases the decrease in femoral blood flow lasted longer than the transient fall of arterial pressure. Central venous pressure fell proportionately with increased angle of tilt up to 60”. Central venous pressure responses to tilt were thus similar to the responses observed during graded hemorrhage. The effect of atropine and propranolol on heart rate changes induced by tilt was studied in one experiment. The results are summarized in Table 4 and are expressed increase above resting horizontal controls, as percent Atropine had a slight inhibitory effect on heart rate increases; propranolol in combination with atropine greatly reduced the heart rate increases in response to tilt,
To meet the needs and requirements of an active cellular metabolism animal transport systems have been modified progressively toward a more closed system of vessels, greater separation of oxygenated and deoxygenated blood, higher pressures, and enhanced flow rates. The most efficient and best characterized circulatory system is that of mammals but studies on lower vertebrates are increasing (10). Fn reptiles where important advances toward a double circulation are evident, pressures approaching those of
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390 mammals can be measured. It seems reasonable that appropriate controls must also exist to regulate the arterial pressure that is so fundamental to homeostasis. This study has demonstrated that the iguana can regulate arterial blood pressure during periods of graded hemorrhage and passive head-up tilting. The gradual decrease in arterial pressure in response to initial stages of hemorrhage is followed by a more marked fall in pressure after additional blood loss. This two-component response to graded hemorrhage is interpreted as an initial compensatory response followed by loss cf compensation during extreme blood loss. Thus as the compensatory reserve becomes exhausted arterial pressure falls more rapidly in response to hemorrhagic insult. If reflex adjustments did not exist arterial pressure would probably fall proportionately with blood loss without the slope change noted in Fig. 1 and less blood affecting the loss could be tolerated without adversely survival of the organism. The experiments on passive head-up tilting provide additional evidence for a controlled arterial pressure. In humans (19) and dogs (4) cardiac output and mean arterial pressure are reduced during passive tilting, an effect probably related to venous pooling of blood and thus a lowered venous return. The reduced arterial pressure in iguanas after tilt is probably related to a lowered cardiac output for the same reason it is reduced in man and dog after tilt. The increased heart rate after head-up tilts is undoubtedly one of several concerted reflex responses that act to counterbalance the fall in blood pressure+ Correlated with the fall in arterial pressure during graded hemorrhage is an increased heart rate and a decreased CVP and femoral artery blood flow. Calculated femoral resistance increased initially but fell as the arterial pressure decreased more sharply to continued hemorrhage. Heart rate increases were greatly reduced after treatment with propranolol in both hemorrhage and tilt experiments. The arterial pressure curve after propranolol (Fig. 2,4) was lower than the control curve but the jackknife estimates of CBVD and the general profile of the curve were unchanged. Atropine had a lesser effect on both heart rate changes induced by hemorrhage (Fig. 3B) or tilt (Table 4) and also had no appreciable effect on the arterial pressure changes caused by hemorrhage (Fig. ZB). These results suggest that iguanas have a low vagal tone and that heart rate increases are predominantly caused by an increased sympathetic drive on the heart. Fluorescence microscopy (Hohnke, unpublished observations) revealed relatively dense adrenergic innervation of the ventricular myocardium and provided additional evidence for the sympathetic innervation of the iguana heart. Similar observations have been reported for the adrenergic innervation of the lizard Trachysaurus rugosus (7) ; studies with TiZiqua rugosa (3) reveal that most cardiac sympathetic nerves travel independently of the vagus and derive almost exclusively from the ‘Lstellate complex.” Microscopic examination for fluorescence in cross sections of the femoral artery did not reveal unusual patterns or densities of adrenergic innervation. Specific fluorescence was confined to the adventitial-medial border and was relatively sparse (Hohnke, unpublished observations). It is interesting that arterial pressure changes induced
L. A. HOHNKE
by hemorrhage were largely unmodified by doses of blocking agents that did prevent increases in heart rate. One interpretation might be that changes in heart rate contribute little to major changes in arterial blood pressure in iguanas. A study of the reflex adjustments in regional flows and resistance in response to hemorrhage would probablv be very instructive in this regard. A dimerent interpreiation might argue that the hypothesis of neurogenic control mechanism is weakened by the failure of atropine and propranolol to affect the response to hemorrhage. A nonneurogenic mechanism of arterial blood pressure control might rely on physical factors to explain the presence of two slopes in the hemorrhage curves. One such factor might be fluid absorption to expand the circulating blood volume with subsequent failure of this mechanism during late stages of hemorrhageAnother possibility would be a sudden decrease in volume of the vascular system initiated at some critical low arterial pressure. The reduced capacity of the reservoir from which blood could be removed might result in a steer>er slope to the hemorrhage curve. The various possi ble i nterpreta tions are not necessarily independent from one another and each can be tested experimentally. The degree to which different mechanisms may be important in the control of arterial bIood pressure in reptiles might contribute to an understanding of the development of arterial pressure control mechanisms in higher vertebrates. Studies similar to the hemorrhage experiments in iguanas have also been done on birds. The anesthetized domestic hen appears quite sensitive to hemorrhagic insult. Removal of less than 10 % of the estimated initial blood volume lowered the mean AP about 22 mmHg; this effect was more than doubled if 15-25 % of the blood volume was removed (20). I n unanesthetized ducks, however, arterial pressure showed little change af‘ter removal of up to 25 Y& of the estimated blood volume (6). In both studies increased heart rate and decreased hematocrit accompanied the fall in arterial pressure. The ability of ducks and pigeons (11) to resist blood loss seems related to a great capacity for fluid mobilization from skeletal muscle (6) In the d uck this is effected by a strong reflex vasoconstriction an d a large ca pillary surface area in the skeletal muscle. These mechanisms may operate in lizards also but, as suggested, this has not been well studied. In mammals arterial pressure is maintained relatively constant by reflex adjustments initiated through changes in pressoreceptor and chemoreceptor activity. If similarly mediated reflex adjustments are operative to regulate arterial blood pressure in the iguana and lizards generally, then the location and characterization of reflexogenic zones become verv important. Information on presloreceptors and chemoreceptors in lower vertebrates is generally unavailable; this is particularly true for reptiles. If the tachycardia that accompanies hemorrhage in the iguana is initiated by pressoreceptors it may be a very helpful reflex to use in a search for reflexogenic areas in reptiles. The reflex adjustments to hemorrhage could be mediated by pressoreceptor and/or chemoreceptors, depending on their presence and/or relative importance. Responses to tilt, however, would most likely be initiated by a change in pressoreceptor activity. This is interesting
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BLOOD
PRESSURE
REGULATION
IN
391
REPTILES
since the carotid sinus reflex does not seem to exist in iguanas (Hohnke, unpublished observation). The increasedv femoral resistance, estimated for the vascular bed supplied by the femoral artery, is probably a reflex response also and could provide another useful end point in a search for receptor zones and neuronal pathways involved in controlling arterial blood pressure.
The statistical assistance of Dr. David S. Salzburg (Pfizer Research) is gratefully acknowledged. This work was supported by National Science Foundation GB 49310, Public Health Service Training Grant IIL-05696, the American I-Ieart Association-Greater Los Angeles Affiliate 4371G. Present address: Pfizer Central Research, Groton, Conn. Received
for
publication
13 February
Central Grant and by Grant 06340.
1974.
REFERENCES 1. ARVESEN,
11.
2.
12.
3.
4.
5. 6.
7.
8.
9. 10.
J. Jackknifing U-statistics. Ann. Math. Stat, 40: 20762100, 1969. BAKER, L. ,4., AND F. N. WHITE. Redistribution of cardiac output in response to heating in Iguana iguana. Gomp. Biochem. Physiul. 35 : 253-262, 1970. BERGER, P. J* The vagal and sympathetic innervation of the heart of the lizard Tiliqua rugosa. Australian J. Exptl. Biol. Med. Sci. 49: 297-304, 1971. CONSTANTINE, J. Mr., W. K. MCSHANE, AND S. C. WANG. Comparison of carotid artery occlusion and tilt responses in dogs. Am. J. Hiysiol. 221 : 1681-1685, 1971. QEBLEY, V. G. Miniature hydraulic occluder for zero blood flow determination. J. A@. P&iol. 3 1 : 138-l 39, 197 I. A. M., B. F~LKOW, AND A. G. B. KOVACH. The QOJOSUGITO, mechanisms behind the rapid blood volume restoration after hemorrhage in birds. Acta Physiol. Stand. 74 : 114-l 22, 1968. FURNESS, J. B., AND J. MOORE. The adrenergic innervation of the cardio-vascular system of the lizard Trachysaurus rugosus. 2. Zellforsch. 108 : 150-l 76, 1970. GREGERSON, M. I. A practical method for the determination of blood volume with the dye T-1824. J. Lab. Clin. Med. 29: 12661269, 1944. JOHANSEN, K. Circulation in the three-chambered snake heart. Circulation Res. 7 : 828-832, 1959. JOHANSEN, K. Comparative physiology: gas exchange and circulation in fishes. Ann. Rev. Phvsiol. 33 : 569-612. 197 1.
13.
14.
15. 16. 17. 18. 19.
20.
KOVACH, A. G. B., AND E. SZASZ. Survival of pigeon after graded haemorrhage. Acta Physiol. Acad, Sci. Hung. 34 : 301-309, 1968. MORGAREIDGE, K. R., AND F. N. WHITE. Cutaneous vascular changes during heating and cooling in the Galapagos marine iguana. Nature 223 : 587-591, 1969. MOSTELLER, F., AND J. W. TUKEY. Data analysis, including statistics. In : Handbook of Social Psychology, edited by G. Lindzey and E. Aronson. Reading, Mass.: Addison Wesley, 1968. STEGGERDA, F. R., AND II. E:. ESSEX. Circulation and blood pressure in the great vessels and heart of the turtle (Cheledra serpentina). Am. J. Physiol. 190: 310-326, 1957. WHITE, F. N. Circulation in the reptilian heart (Caiman sclerops). Anat. Record 125 : 417-431, 1956. WHITE, F. N. Functional Zool. 8: 211-219, 1968. WHITE, F. N. Redistribution gator, Copeia 3: 567-570,
anatomy of cardiac
of the
heart
output
of reptiles. in
the
diving
Am. alli-
1969.
WHITE, F. N. Central vascular shunts and their control in reptiles. Federation hoc. 29: 1149-l 153, 1970. WEISSLER, A. M., W. H. ROEHXLL, AND R. G. PEELER. Effect of posture on the cardiac response to increased peripheral demand. J. Lab. C/in. Med. 59: 1000-1007, 1962. WYSE, D. G., AND M, NICKERSON. Studies on hemorrhagic hypotension in domestic fowl. Can. J. Physiol. Pharmacol. 49 : 9 19-926, 1971.
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