Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Hindlimb blood flow and oxygen consumption in the late gestation fetal lamb Knight, Kathleen Elizaabeth 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-389154.pdf [ 9.72MB ]
Metadata
JSON: 831-1.0089240.json
JSON-LD: 831-1.0089240-ld.json
RDF/XML (Pretty): 831-1.0089240-rdf.xml
RDF/JSON: 831-1.0089240-rdf.json
Turtle: 831-1.0089240-turtle.txt
N-Triples: 831-1.0089240-rdf-ntriples.txt
Original Record: 831-1.0089240-source.json
Full Text
831-1.0089240-fulltext.txt
Citation
831-1.0089240.ris

Full Text

HINDLIMB BLOOD FLOW AND OXYGEN CONSUMPTION IN THE LATE GESTATION FETAL LAMB by KATHLEEN ELIZABETH KNIGHT B.Sc, The University of Lethbridge, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Medicine Department of Obstetrics and Gynecology Reproductive and Developmental Sciences Program) We accept this thesis as conforming to the requirecLstandard THE UNIVERSITY OF BRITISH COLUMBIA November 1998 © Kathleen Elizabeth Knight, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten permission. Department of r^-teW.^ -> fivj^^ng.j The University of British Columbia Vancouver, Canada D a t e ^Qr o r - n W l A >qqft DE-6 (2/88) ABSTRACT The purpose of this research was to examine hindlimb blood flow and oxygen supply in the late-gestation felal lamb under normal and pathophysiological conditions. Although the carcass, or non-visceral tissue, accounts for a large proportion of fetal weight and oxygen demand, it has received little research attention. Because the hindlimb is composed of skin, bone, and skeletal muscle, it is representative of the carcass. We implanted vascular catheters in fetal lambs to permit sampling of blood entering and leaving the hindlimb, and a transit-time flow transducer on the external iliac artery to measure hindlimb blood flow. In late-gestation, there are specific patterns of electrocortical (ECoG) and electro-ocular activity (EoG), which are associated with episodic fetal breathing and body movements. Hindlimb blood flow tends to be higher and more variable during high voltage ECoG activity. To determine whether this is due to the increased body movements during this state, the neuromuscular blocking agents gallamine and pancuronium were administered. Mean hindlimb blood flow fell 8.3 ± 3.5% following blockade, but flow still tended to be higher during quiet sleep. As neuromuscular blockade did not appear to effect the behavioural state modulation of hindlimb blood flow, this relationship may be directly mediated by the central nervous system. The effects of hypoxemia and ischemia on hindlimb metabolism were compared. No difference was seen in the relationship between oxygen delivery and consumption or in the overall magnitude of hindlimb lactate release for the two perturbations. Thus, for similar reductions in oxygen deliver}', we found no difference in hindlimb oxygen consumption or lactate production between hypoxemia and ischemia. Therefore, there is no evidence that hypoxemia is less well tolerated than hindlimb ischemia. As arginine vasopressin (AVP) has vasoconstrictor action's in the fetus and adult, the effect of exogenous AVP was examined in fetal and non-pregnant adult sheep. Both iii groups exhibited similar degrees of hypertension, reduced hindlimb blood flow, and increased hindlimb vascular resistance. However, heart rate declined significandy more in the adults. Therefore, there is no evidence for a greater sensitivity of fetal vascular smooth muscle to vasopressin, but the fetus does appear to be less sensitive to the negative chronotropic effects of AVP. In conclusion, our studies indicate that the hindlimb, and thus the carcass, plays a major role in the dynamic nature of the fetus under normal conditions as well as in its ability to adapt to pathophysiological situations. IV TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES x ABBREVIATIONS xvii ACKNOWLEDGEMENT xx DEDICATION xxi 1. INTRODUCTION .'. 1 1.1 S T A T E M E N T O F P U R P O S E 1 1.2 B A C K G R O U N D 2 1.2.1 Sheep as a model system 2 1.2.2 Oxygen demand 4 1.2.3 Compensations for reduced oxygen supply 5 1.2.4 Hindlimb as representative of carcass tissue 6 1.3 R A T I O N A L E 8 1.4 O B J E C T I V E S 10 1.5 H Y P O T H E S E S 11 2. GENERAL EXPERIMENTAL 12 2.1 C A S T I N G 12 2.1.1 Introduction '. 12 2.1.2 Methods 12 2.1.3 Results/Discussion 13 V 2.2 ANIMAL CARE AND MAINTENANCE 15 2.3 SURGICAL PREPARATION 17 2.4 PHYSIOLOGICAL RECORDING AND MONITORING PROCEDURES 2 6 2.5 CATHETER AND ELECTRODE PREPARATION 2 8 2.6 STATISTICAL ANALYSIS 3 3 3. H I N D L I M B B L O O D F L O W M E A S U R E M E N T C O R R E L A T I O N : M I C R O S P H E R E S V E R S U S T R A N S I T - T I M E F L O W T R A N S D U C E R 34 3.1 INTRODUCTION 3 4 3.2 METHODS 3 8 3.3 RESULTS AND DISCUSSION 3 9 4. H I N D L I M B B L O O D F L O W A N D W E I G H T D I S T R I B U T I O N 44 4.1 INTRODUCTION 4 4 4 .2 METHODS 4 4 4.3 RESULTS 4 5 4 .4 DISCUSSION 4 9 5. B E H A V I O U R A L S T A T E M O D U L A T I O N O F H I N D L I M B B L O O D F L O W 53 5.1 INTRODUCTION 5 3 5.2 METHODS 5 8 5.3 RESULTS 5 9 5.4 DISCUSSION 6 9 vi 6. HYPOXEMIA AND ISCHEMIA 78 6.1 INTRODUCTION 7 8 6.2 M E T H O D S 8 0 6.3 RESULTS 8 8 6.3.1 Animal information 8 8 6.3.2 Blood gas and acid-base status 9 0 6.3.3 Hindlimb blood flow, oxygen delivery, and oxygen consumption 93 6.3.4 Lactate and glucose concentrations and fluxes 9 5 6.3.5 Cardiovascular Responses 9 9 6.4 DISCUSSION 101 6.4.1 Hypoxemia and ischemia levels 101 6.4.2 Blood gas and acid-base responses 101 6.4.3 Hindlimb blood flow 102 6.4.4 Oxygen delivery and consumption 105 6.4.5 Lactate and glucose responses 110 6.4.6 Cardiovascular responses 117 7. ARGININE VASOPRESSIN INFUSION 121 7.1 INTRODUCTION 121 7.2 M E T H O D S 122 7.2.1 Animal Preparation and Experimental Protocol 122 7.2.2 Arginine Vasopressin Analysis 125 7.2.3 Statistics 126 vu 7.3 RESULTS 127 7.3.1 Arginine vasopressin effects on the fetus 127 7.3.2 Arginine vasopressin effects on the adult 141 7.3.3 Fetal versus adult responses to arginine vasopressin concentration.. 151 7.4 DISCUSSION 158 7.4.1 AVP levels-normal and pathophysiological 158 7.4.2 Blood gas and metabolic responses 161 7.4.3 Cardiovascular responses 163 7.4.4 Fetal versus adult responses 167 8. SUMMARY AND CONCLUSIONS 173 9. REFERENCES 185 10. APPENDIX. Unsuccessful fetal surgical preparations 201 viii L I S T O F T A B L E S T A B L E 1. Fetal tissues supplied by the major branches of the terminal descending aorta and hindlimb arterial vessels, based on vascular casting results from eight fetal lambs at 118-139 days gestation 16 T A B L E 2. Successful fetal surgical preparations 18 T A B L E 3. Successful surgical preparations of non-pregnant adult ewes 21 T A B L E 4. Inner and outer diameters of silicone rubber (Silastic®) catheters used in animal preparations 30 T A B L E 5. Gestational age, fetal and hindlimb weights at autopsy for the six fetuses used in the hindlimb blood flow and weight distribution study. In the case of multiple lambs, weights are those of the operated fetus only 46 T A B L E 6. Hindlimb tissue weight and blood flow distribution on the day of experimentation for the six fetal lambs used in the hindlimb blood flow and weight distribution study 47 T A B L E 7. Effect of neuromuscular blockade on blood gas status and metabolic variables in the fetal lamb hindlimb (7 experiments) 68 T A B L E 8. Individual fetal age, weight, and study limb weight at autopsy and on day of study for the animals involved in the hypoxemia and ischemia studies. In the case of multiple lambs, weights are those of the operated fetus only 89 T A B L E 9. Arterial and venous blood gas responses to hypoxemia in the fetal lamb hindlimb (7 animals). Control data are presented as absolute values; other data are change from the control values 91 T A B L E 10. Arterial and venous blood gas responses to hindlimb ischemia in six fetal. lambs. Control data are presented as absolute values; other data are change from the control values 92 ix TABLE 11. Hindlimb metabolic variables during hypoxemia and ischemia experiments. Control data are presented as absolute values; other data are expressed as a percent of the control values 94 TABLE 12. Hindlimb lactate and glucose responses to hypoxemia and ischemia. Control data and flux values are presented as absolute values; arterial and venous concentrations are expressed as a change from control values 97 TABLE 13. Hindlimb blood flow and cardiovascular responses to hypoxemia and ischemia in the fetal lamb. Control data are presented as absolute values; other data are a percent of the control values 100 TABLE 14. Blood gas and metabolic values from fetuses given either saline vehicle only (2 experiments) or exogenous arginine vasopressin (AVP) infusions at a dose of 4.5 mU/min (8 experiments) 131 TABLE 15. Cardiovascular and metabolic data obtained from fetal lambs prior to vehicle (saline) infusion (2 experiments) or infusion of 4.5 mU/min arginine vasopressin (8 experiments). Data are presented as absolute values (mean + SEM) 132 TABLE 16. Arterial blood gas and metabolic values from non-pregnant adult ewes given arginine vasopressin infusions of 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mU/min (3 experiments). Data are presented as absolute values (mean ± SEM) 144 TABLE 17. Cardiovascular and metabolic data obtained from non-pregnant adult ewes prior to receiving arginine vasopressin infusions at doses of 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mU/min (3 experiments). Data are presented as absolute values (mean + SEM) 146 X LIST OF FIGURES FIGURE 1. Ventral view of a cast of the arterial vasculature in the hindquarters of a fetal lamb at 135 days gestation. Major blood vessels are identified 14 FIGURE 2. Line drawing of fetal lamb hindquarters (ventral view) indicating location of catheters, transit-time flow transducer, and vascular occluder 24 FIGURE 3. A typical polygraph recording obtained from a fetal lamb in late gestation. Episodes of fetal breathing, rapid eye movements, and high and low voltage electrocortical activity are indicated. Abrupt changes in amniotic, arterial, and tracheal pressures are due to position changes of the ewe. Hindlimb blood flow was obtained with a transit-time flow transducer positioned on the external iliac artery 27 FIGURE 4. Construction of silicone rubber (Silastic®) catheters implanted in the late gestation fetal lamb preparations. Diagrams are not drawn to scale 29 FIGURE 5. Construction of electrocortical and electro-ocular electrodes for the late gestation fetal lamb preparations. Diagrams are not drawn to scale 32 FIGURE 6. Transonic® transit-time flow transducer and its operation (from Hartman et al, 1985). The transducer is positioned around the external iliac artery and provides continuous measurements of hindlimb blood flow 37 FIGURE 7. Comparison of fetal lamb hindlimb blood flow as measured with a transit-time flow transducer on the external iliac artery and radionuclide-labeled microspheres for five fetal lambs 40 FIGURE 8. Individual animal comparisons of fetal lamb hindlimb blood flow as measured with transit-time flow transducers and radionuclide-labeled microspheres 42 FIGURE 9. Comparison of tissue weights and blood flows for skin, bone, and skeletal muscle in six fetal lambs 48 xi FIGURE 10. Polygraph recording obtained from a fetal lamb in late gestation prior to neuromuscular blockade. Episodes of fetal breathing and high and low voltage electrocortical activity are indicated. A uterine contracture is also labeled. Abrupt changes in amniotic, arterial, venous, and tracheal pressures are due to positional changes of the ewe. Hindlimb blood flow was obtained with a transit-time flow transducer on the external iliac artery 60 FIGURE 11. Polygraph recording obtained from a fetal lamb in late gestation following the administration of a neuromuscular blocking agent (pancuronium). High and low voltage electrocortical activity and a uterine contracture are indicated. Fetal breathing movements are absent. Abrupt changes in amniotic, arterial, venous, and tracheal pressures are due to positional changes of the ewe. Hindlimb blood flow was obtained with a transit-time flow transducer on the external iliac artery 61 FIGURE 12. Effect of neuromuscular blockade on fetal hindlimb blood flow (8 experiments). Mean hindlimb blood flow as well as that during high and low voltage ECoG cycles are presented for both before and after blockade. Blood flow was measured with a transit-time flow transducer on the external iliac artery 63 FIGURE 13. Effect of neuromuscular blockade on fetal heart rate (8 experiments). Mean heart rate as well as that during high and low voltage ECoG cycles are presented for both before and after blockade 64 FIGURE 14. Effect of neuromuscular blockade on fetal arterial pressure (8 experiments). Mean arterial pressure as well as that during high and low voltage ECoG cycles are presented for both before and after blockade 65 FIGURE 15. Effect of neuromuscular blockade on fetal venous pressure (8 experiments). Mean venous pressure as well as that during high and low voltage ECoG cycles are presented for both before and after blockade 66 xii FIGURE 16. Effect of neuromuscular blockade on fetal hindlimb vascular resistance (8 experiments). Mean resistance as well as that during high and low voltage ECoG cycles are presented for both before and after blockade 67 FIGURE 17. Plexiglas chamber used to alter maternal inspired oxygen concentrations during hypoxemia experiments (figure from Rurak et al, 1990). Ewes were able to eat, drink, and lay down normally when their heads were inside the chamber 81 FIGURE 18. Protocol for hypoxemia experiments involving Plexiglas chamber. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals) 83 FIGURE 19. Protocol for hypoxemia experiments in which hypoxic gas mixture was introduced through the maternal tracheal catheter. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals during hypoxemia and 20 minute intervals during the control and recovery phases) 84 FIGURE 20. Protocol for fetal hindlimb ischemia experiments. Ischemia was achieved by partial inflation of a vascular occluder on the fetal external iliac artery. In most experiments two episodes of ischemia were conducted separated by a recovery phase. At least 30 minutes were allowed between the end of the first recovery phase and the second ischemia episode. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals) '. 85 FIGURE 21. Polygraph recording of a hindlimb ischemia episode obtained from a late gestation fetal lamb. Ischemia was achieved by partial inflation of a vascular occluder positioned on the external iliac artery upstream to a transit-time flow transducer. In this figure, hindlimb blood flow was reduced to approximately 60% of control values. The onset of the occlusion episode is followed by abrupt alterations in blood flow as the inflation of the occluder is manually adjusted to achieve the desired level of ischemia. Release (or deflation) of the occluder results in an immediate, transient hyperemia followed by a rapid return of hindlimb blood flow to control values. Abrupt changes in pressures are due to position changes of the ewe 87 xiii FIGURE 22. Relationship between hindlimb oxygen consumption and delivery in fetal lambs exposed to hypoxemia (n=7) or ischemia (n=6) 96 FIGURE 23. Relationship between hindlimb blood flow and gestational age in the fetal lamb. The longitudinal study was conducted on 16 fetuses (each symbol represents an individual animal). Mean daily blood flow was determined with a transit-time flow transducer on the external iliac artery. Only days in which no other experiment was performed are included 104 FIGURE 24. Protocol for exogenous arginine vasopressin infusion experiments in fetal and non-pregnant adult ewes. The arrows indicate blood samples which were taken at ~15 minute intervals during the 30 minute control period and the 90 minute infusion period and at 30 minute intervals during the 120 minute recovery period 123 FIGURE 25. Plasma arginine vasopressin concentrations before, during, and after AVP infusion to fetal lambs. The lambs received either saline vehicle (2 experiments), or exogenous AVP at 0.72 mU/min (1 experiment), 4.5 mU/min (8 experiments), or 9.0 mU/min (1 experiment) 128 FIGURE 26. Plasma arginine vasopressin concentrations in fetal lambs before, during, and after either saline vehicle (n=2) or exogenous AVP infusion at 4.5 mU/min (n=8) 129 FIGURE 27. Change in fetal arterial pressure during and after arginine vasopressin administration to fetal lambs. The lambs received either saline vehicle (n=l) or exogenous AVP at 4.5 mU/min (n=8) 134 FIGURE 28. Change in fetal heart rate during and after arginine vasopressin administration to fetal lambs. The lambs received either saline vehicle (n=2) or exogenous AVP at 4.5 mU/min (n=7) 135 FIGURE 29. Change in hindlimb blood flow during and after arginine vasopressin administration to fetal lambs. The lambs received either saline vehicle (n=2) or exogenous AVP at 4.5 mU/min (n=8). Blood flow was measured with a transit-time flow transducer on the external iliac artery 136 XIV FIGURE 30. Change in hindlimb oxygen delivery during and after arginine vasopressin infusion to fetal lambs. The lambs received either saline vehicle (2 experiments), or exogenous AVP at 0.72 mU/min (1 experiment), 4.5 mU/min (8 experiments), or 9.0 mU/min (1 experiment) 137 FIGURE 31. Change in hindlimb blood flow during and after arginine vasopressin infusion to fetal lambs. The lambs received either saline vehicle (2 experiments) or exogenous AVP at 0.72 mU/min (1 experiment), 4.5 mU/min (8 experiments), or 9.0 mU/min (1 experiment). Blood flow was measured with a transit-time flow transducer on the external iliac artery 138 FIGURE 32. Change in hindlimb oxygen consumption during and after arginine vasopressin infusion to fetal lambs. The lambs received either saline vehicle (2 experiments), or exogenous AVP at 0.72 mU/min (1 experiment), 4.5 mU/min (8 experiments), or 9.0 mU/min (1 experiment) 139 FIGURE 33. Change in hindlimb oxygen extraction during and after arginine vasopressin infusion to fetal lambs. The lambs received either saline vehicle (2 experiments), or exogenous AVP at 0.72 mU/min (1 experiment), 4.5 mU/min (8 experiments), or 9.0 mU/min (1 experiment) 140 FIGURE 34. Plasma arginine vasopressin concentrations before, during, and after AVP infusion to non-pregnant adult ewes. The ewes received exogenous AVP at doses of 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mU/min (3 experiments) 142 FIGURE 35. Plasma arginine vasopressin concentrations before, during, and after AVP administration to non-pregnant adult ewes. The ewes received AVP at 25 mU/min (n=2), 45 mU/min (n=3), or 90 mU/min (n=3) 143 FIGURE 36. Change in arterial pressure during and after arginine vasopressin administration to non-pregnant adult ewes. The ewes received AVP at 25 mU/min (n=2), 45 mU/min (n=3), or 90 mU/min (n=3) 147 XV FIGURE 37. Change in heart rate during and after arginine vasopressin administration to non-pregnant adult ewes. The ewes received AVP at 25 mU/min (n=2), 45 mU/min (n=2), or 90 mU/min (n=3) 148 FIGURE 38. Change in hindlimb blood flow during and after arginine vasopressin administration to non-pregnant adult ewes. The ewes received AVP at 25 mU/min (n=2), 45 mU/min (n=3), or 90 mU/min (n=3). Blood flow was measured with a transit-time flow transducer on the external iliac artery 149 FIGURE 39. Change in hindlimb oxygen delivery during and after arginine vasopressin infusion to non-pregnant adult ewes. Ewes received AVP at 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mU/min (3 experiments) 150 FIGURE 40. Change in hindlimb blood flow during and after arginine vasopressin infusion to non-pregnant adult ewes. The ewes received AVP at a dose of 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mU/min (3 experiments). Blood flow was measured with a transit-time flow transducer on the external iliac artery 152 FIGURE 41. Relationship between percentage change in arterial pressure and plasma arginine vasopressin concentration during the infusion of vasopressin to fetal (10 experiments) and non-pregnant adult (5 experiments) sheep. The regression equations are not significantly different 153 FIGURE 42. Relationship between percentage change in heart rate and plasma arginine vasopressin concentration during the infusion of vasopressin to fetal (9 experiments) and non-pregnant adult (5 experiments) sheep. The slopes are significantly different (p < 0.01) 155 FIGURE 43. Relationship between percentage change in hindlimb blood flow and plasma arginine vasopressin concentration during the infusion of vasopressin to fetal (10 experiments) and non-pregnant adult (5 experiments) sheep. Blood flow was measured with transit -time flow transducers on the external iliac arteries. The regression equations are not significantly different 156 xvi F I G U R E 44. Relationship between percentage change in estimated hindlimb vascular resistance and plasma arginine vasopressin concentrations during the infusion of vasopressin to fetal (10 experiments) and non-pregnant adult (5 experiments) sheep. Blood flow was measured with transit-time flow transducers on the external iliac arteries. The regression equations are not significantly different 157 xvii ABBREVIATIONS A-V arterial-venous difference AMP adenosine monophosphate ANOVA analysis of variance AUC area under the concentration versus time curve AVP arginine vasopressin D P m beats per minute C/S Cesarean-section CIA circumflex iliac artery cm centimeter CNS..... central nervous system dL deciliter Do2 oxygen delivery ECoG electrocortical Eo2 oxygen extraction EoG electro-ocular FBM fetal breathing movements 8 acceleration due to gravity g ......... gram ID inner diameter HA internal iliac artery XV111 IU international units k8 kilogram L liter m3 cubic meter mEq/L milliequivalents per liter min minute(s) ml milliliter mm millimeter mM millimolar mmHg millimeters of mercury mU milli-units ng nanogram ° 2 oxygen OD outer diameter P probability factor Pp2 arterial partial pressure of oxygen Pco2 partial pressure of carbon dioxide Pg picogram pH negative logarithm of hydrogen ion concentration Po2 partial pressure of oxygen r correlation coefficient xix REM rapid eye movements rPm revolutions per minute S/L spontaneous labour SEM standard error of the mean U units v ° 2 oxygen consumption [G] glucose concentration [E] lactate concentration °C degrees Celsius Mm micrometer /*mol ••••• micromoles micro-units /*V microvolts XX A C K N O W L E D G E M E N T I would like to express my appreciation to my research supervisor Dr. Dan Rurak for his guidance. Also, thank you to the other members of my supervisory committee: Drs. Wayne Riggs, Anthony Perks, Darlene Reid, and Joseph Skala. Their advice and interest in this project have been invaluable. Also, I am grateful to our laboratory technicians, Caroline Hall and Eddie Kwan, and to my fellow students, Weiping Tan and Cindy Wu for their friendship and support. Many thanks to Becky Leopold for her friendship and secretarial skills. Financial support in the form of scholarships from the Natural Sciences and Engineering Research Council of Canada and the British Columbia Research Institute for Child and Family Health is gratefully acknowledged. This research was also supported by the Medical Research Council of Canada. xxi DEDICATION This thesis is dedicated to my family: my Mom, Elizabeth; brother, Patrick; and to the memory of my Dad, Art. Also, to my beloved husband Greg. Thank you for your love, encouragement, understanding, and enthusiasm. 1 1. INTRODUCTION 1.1 Statement of Purpose The purpose of this research was to examine hindlimb blood flow and oxygen supply in the late gestation fetal lamb under normal and pathophysiological conditions. Because the hindlimb is composed almost exclusively of skin, bone, and muscle, it is representative of the carcass (non-visceral tissues). In late gestation the carcass elements comprise 70% of fetal weight and account for 40% of oxygen demand. Thus, the response of these tissues to alterations in oxygen supply can have a profound effect on the fetus as a whole. To study the hindlimb required a surgical protocol which allowed sampling of hindlimb blood and continuous measurement of hindlimb blood flow. Vascular casting and comparison of hindlimb blood flows obtained with radionuclide-labeled microspheres and a transit-time flow transducer on the external iliac artery were used to establish a surgical protocol which minimized sources of collateral circulation and ensured accurate measurement of hindlimb blood flow. This led to an investigation of the effect of the catheterization protocol on hindlimb weight and blood flow. The normal late gestation fetal lamb experiences a complicated pattern of behavioural states involving rapid eye movements, fetal breathing and body movements, and high and low voltage electrocortical activity. These cycles are associated with alterations in blood flow, metabolism, and cardiovascular variables. Hindlimb blood flow appears to be higher and more variable during high voltage activity which we attribute to the greater prevalence of body movements during this state. Thus, the administration of neuromuscular blocking agents, which inhibit fetal breathing and body movements, should allow us to determine if skeletal muscle activity is responsible for the fluctuations in hindlimb blood flow that occur in relation to behavioural state. In addition, we sought to determine the effect of behavioural state and neuromuscular blockade on hindlimb oxygen consumption. Inhibition of skeletal activity should reduce fetal oxygen requirements. Fetal hypoxemia is associated with an inhibition of fetal breathing and rapid eye movements, and fewer incidences of low voltage electrocortical activity. Therefore, these may be compensatory mechanisms to lower oxygen consumption when the fetus faces a reduction in oxygen delivery. We compared the cardiovascular and hindlimb metabolic responses to hypoxemia, in which fetal arterial Po2 is reduced, and ischemia, where Po2 is maintained. The hypothesis to be tested was that a fall in oxygen delivery achieved by ischemia would be better tolerated by the fetal lamb hindlimb than hypoxemia, and this latter perturbation would be associated with an earlier fall in oxygen consumption or greater release of lactate by the hindlimb. Hypoxemia is associated with a rise in circulating levels of arginine vasopressin. A previous study observed a greater hypertensive effect of arginine vasopressin in the fetus compared to the adult ewe while another report found no difference in the pressor response but a greater degree of bradycardia in the adults. Thus, we examined the effect of exogenous arginine vasopressin infusion on cardiovascular and hindlimb metabolic responses in the fetus and non-pregnant ewe. 1.2 Background 1.2.1 Sheep as a model system Sheep are used extensively in laboratory work due to their availability and docility. Pregnant ewes demonstrate similar changes in physiological parameters to those observed in pregnant humans,- making ewes good models for human obstetrical research (Cissik et al, 1991). Furthermore, in late gestation the fetal lamb is similar in weight to a human fetus (~3.5 kg). Thus, as technical and ethical constraints limit study of the human fetus, the fetal lamb is a useful alternative model. A major difference between these two species is in the type of placentation: humans possess a hemochorial placenta (fetal chorionic epithelium is bathed directly by maternal blood) whereas sheep have an epitheliochorial placenta (fetal 3 chorionic epithelium is in contact with maternal endometrial epithelium). Depending on the type of experiments being conducted, the advantages of the sheep model generally outweigh the disadvantage of placental dissimilarity (Assali etal, 1974). Early studies on the fetal lamb were usually acute studies on anesthetized animals. Reports using exteriorized lambs, in which the fetus was removed from the uterus but the umbilical circulation remained intact, are also prevalent in the early literature. Although much information was revealed by such work, anesthetized and exteriorized fetuses cannot be considered physiologically representative of the in utero state (Rurak and Taylor, 1986; Jones, 1977). Meschia and colleagues (1966) first reported the use of chronically catheterized fetuses. As the sheep uterus is thinner and less muscular than that of many other species, most notably humans, surgery on fetal lambs carries a reduced risk of premature labour. Using aseptic techniques catheters can be inserted into fetal vessels, the ewe and fetus allowed to recover from surgery, and thereafter the fetus can be studied in utero in a non-anesthetized state approximating physiological normality. As chronic catheterization does not seem to adversely affect the fetus both acute and long-term studies can be conducted. In addition, advancements in radionuclide-labeled microspheres and transit-time flowprobes have allowed researchers to study blood flow and its distribution. These developments have made it possible to continuously study the fetus in utero for days or weeks at a time. Direct measurements of cardiovascular, metabolic, and behavioural state variables are possible in fetal lambs thus avoiding the subjectivity when observing the human fetus remotely i.e., by ultrasound. Although the oxygen delivery systems of the sheep and human fetus seem to differ quantitatively, oxygen consumption seems comparable in the two species (Rurak et ai, 1987). As well human and sheep fetuses exhibit similar compensatory mechanisms when faced with a reduction in oxygen supply. 4 1.2.2 Oxygen demand As the organ of gas exchange in the fetus is the placenta rather than the lungs, oxygen delivery (Do2) to the fetus is a function of umbilical blood flow and the oxygen content of the umbilical venous blood. Likewise, oxygen delivery to specific organs or tissues is the product of arterial oxygen content and blood flow to that particular tissue. By the Fick Principle the amount of oxygen taken up by fetal tissues or oxygen consumption (V o2) can be calculated as the product of blood flow and the arterial-venous oxygen content difference. The ratio of oxygen consumption to delivery constitutes oxygen extraction (Eo2). Uterine contractions, maternal or fetal hemorrhage, and constriction of uterine, umbilical, or other fetal vessels may reduce oxygen supply to fetal tissues. Hypoxemia by definition is a reduction in arterial Po2. Experimental perturbations that reduce fetal systemic oxygen delivery primarily via fetal hypoxemia include reducing uterine blood flow (Jensen etal., 1991) and decreasing maternal inspired oxygen concentration (Rurak et al., 1990a; Rurak et al, 1990b; Conn etal, 1974). Under normal conditions oxygen consumption by the placenta accounts for about half of the total uterine-umbilical venous Po2 difference (Wilkening and Meschia, 1983; Meschia etal, 1980). Thus, when oxygen availability is decreased, this high rate of oxygen consumption can significantly reduce oxygen delivery to the fetus (Groome, 1991). Hypoxia occurs when tissue oxygen supply is not sufficient to meet normal metabolic demands. Due to the high rate of blood flow to fetal organs, hypoxemia is not necessarily associated with hypoxia (Richardson, 1989). Hypoxia ranges in severity with the more severe reductions in oxygen delivery accompanied by metabolic acidosis (Peeters et al, 1979). If fetal Po2 drops below that required to maintain the diffusion gradient from the blood to the inner mitochondrial membrane (critical Po2), anaerobic glycolysis occurs producing lactic acidemia. This diffusion gradient is a particularly important factor when oxygen supply is limited (Kennedy and Jones, 1986; Jones and Kennedy, 1982). Oxygen supply can also be diminished by other means such as 5 fetal hemorrhage (Rurak et al, 1989) or reduced umbilical blood flow (Itskovitz el al, 1987; Itskovitz el al., 1983). In these cases the fall in delivery is generally caused by alteration of perfusion while fetal Po2 is generally maintained. Lactate and glucose are the major substrates of normal fetal metabolism (Singh et al, 1984). Severe hypoxemia, accompanied by hypoxia, results in a net lactate production by the fetus (Milley, 1988; Jones, 1977). Metabolic acidemia further reduces blood oxygen content probably by a rightward shift in the oxyhemoglobin dissociation curve or Bohr Shift (Rurak et al, 1990b). The shift from pH optima as a consequence of the acidemia reduces intracellular enzyme activity and may lead to tissue necrosis. Compensatory mechanisms, primarily increased blood flow, attempt to maintain oxygen delivery to the heart, brain, and adrenal glands at the expense of less vital organs, often resulting in regional acidemia. When hypoxia exceeds the capacity of these compensatory mechanisms, even the vital organs face a severe reduction in oxygen supply with death usually imminent. 1.2.3 Compensations for reduced oxygen supply The fetus exists in a state of physiological hypoxemia relative to the neonate and adult but has developed a number of adaptations which permit it to thrive in its unique environment These include: circulatory shunts to direct oxygen-rich blood to the vital organs (Rudolph, 1985; Edelstone and Rudolph, 1979), overperfusion of organs relative to their oxygen requirements (Richardson, 1989), and a higher oxygen affinity hemoglobin (Itskovitz etal, 1984). The main compensatory mechanisms which the fetus possesses to combat acute disturbances in oxygen supply are to maintain oxygen consumption by increasing extraction and to decrease total body oxygen demands. Below the critical Po2, acidemia develops which eventually limits the capacity of these mechanisms to sustain physiological well-being in the fetus. In the fetal lamb the normal range of oxygen extraction is 25 to 35% (Rurak et al, 1987; Rudolph, 1984). By increasing oxygen extraction, consumption can be maintained in 6 the face of reductions in oxygen delivery to about 50%. This compensation is termed the oxygen margin of safety or oxygen reserve (Richardson, 1989). Extraction increases in a curvilinear fashion as delivery falls (Richardson, 1989; Schumacker and Samsel, 1989), peaking at approximately 60-70% (Edelstone, 1984; Itskovitz et al, 1983). Beyond approximately a 50% fall in delivery, oxygen consumption begins to decline precipitously. As extraction rises, the Po2 of the blood perfusing tissues downstream will be reduced. Thus the need to maintain Po2 above critical values imposes a limitation on the degree of oxygen delivery that can be tolerated. In addition, when contending with reduced oxygen delivery, available oxygen is preferentially redistributed to the vital organs, such as the heart and brain, (Sheldon et al., 1979; Cohn et al, 1974) and away from peripheral tissues like the carcass (Jensen and Berger, 1991; Boyle etal, 1990; Peeters etal, 1979). This compensatory mechanism is limited by the development of systemic and tissue lactic acidemia. Arginine vasopressin (AVP) has been implicated in the increased peripheral vascular resistance (Iwamoto etal, 1979). The late gestation fetal lamb also reduces or abolishes body movements (Natale et al, 1981) and breathing activity (Hohimer and Bissonnette, 1991) in order to lower oxygen requirements when the supply is limited. 1.2.4 Hindlimb as representative of carcass tissue Carcass, defined as the non-visceral tissues, primarily includes bone, skin, and skeletal muscle. The carcass elements of the fetal lamb receives nearly 40% of combined ventricular output (Rudolph and Heymann, 1970), and these tissues constitute approximately 70% of fetal weight in late gestation (Wilkening et al, 1988; Wallace, 1948). It has been estimated that these tissues account for 40% of total fetal oxygen demand (Rudolph, 1984). As a result, any change in substrate delivery, consumption, or extraction by the carcass elements can have a profound effect on the fetus as a whole. As the fetal lamb hindlimb consists almost exclusively of skin, bone, and muscle, a number of 7 researchers consider it to be representative of the carcass (Boyle etal, 1990; Wilkening et al, 1988; Singh etal, 1984). Because the peripheral circulation in skin and muscle receives a large percentage of fetal cardiac output, vasomotor responses of these tissues can lead to a major redistribution of the fetal circulation (Rudolph and Heymann, 1970). 8 1.3 Rationale 1. In late gestation, the carcass (non-visceral tissues) comprises a large fraction of fetal body weight and receives a significant proportion of fetal cardiac output. However, there is limited information on carcass blood flow and metabolism under normal and pathophysiological conditions. 2. The hindlimb is composed of skin, bone, and skeletal muscle and is considered to be representative of the fetal carcass. Blood primarily enters and exits the hindlimb via the external iliac artery and vein. It should thus be possible to catheterize and instrument the hindlimb so as to determine its blood flow and metabolism. 3. Under normal conditions, the late gestation fetal lamb experiences cyclical variations in behavioural state, including electrocortical and electro-ocular activity, as well as breathing and body movements. Hindlimb blood flow fluctuates with behavioural state; however, the mechanisms involved in this relationship are not known. 4. When reductions in fetal oxygen delivery are achieved via hypoxemia, the rise in circulating lactate concentrations is greater than when oxygen delivery is reduced to a similar degree via decreased blood flow. This may be because hypoxemia results in greater falls in capillary and tissue Po2 compared to ischemia, thereby limiting tissue oxygen utilization to a greater extent and resulting in an increased glycolytic rate with enhanced lactate production. The carcass could be involved in this difference; however, comparisons of fetal carcass metabolic responses to hypoxemia and ischemia are lacking. 5. During severe fetal hypoxemia, carcass blood flow falls which exacerbates the fall in Po2. Arginine vasopressin has been implicated in this response as well as in the cardiovascular effects (hypertension and bradycardia) seen with hypoxemia. Previous studies suggest that this hormone may be a more potent vasoconstrictor or a less potent negative chronotrope in the fetus compared to the adult. However, a direct comparison of the cardiovascular and vasoconstrictor effects in the fetus and adult has not been conducted. 10 1.4 O b j e c t i v e s 1. To examine the vascular anatomy of the fetal lamb hindlimb through casting and dissection. These data were used to design a surgical protocol to permit measurement of solely hindlimb blood flow and metabolism. 2. To compare estimates of fetal hindlimb blood flow measured with a transit-time flow transducer on the external iliac artery to those obtained with radionuclide-labeled microspheres. 3. To compare the distribution of blood flow and weight in the study and non-study hindlimb tissues (skin, bone, and muscle) of the late gestation fetal lamb using radionuclide-labeled microspheres. 4. To measure hindlimb blood flow; blood gas status; cardiovascular responses; oxygen supply, consumption, and extraction; and glucose and lactate fluxes in the fetal lamb hindlimb before and after neuromuscular blockade. Also, to record hindlimb blood flow and oxygen consumption in relation to behavioural state. 5. To measure hindlimb blood flow; arterial and venous blood gas values and oxygen contents; oxygen consumption, delivery, and extraction; glucose and lactate fluxes, and cardiovascular variables of the fetal lamb in response to hypoxemia and ischemia. Hypoxemia was achieved by reducing maternal inspired oxygen concentrations and ischemia by partial inflation of a vascular occluder on the fetal external iliac artery. 6. To measure hindlimb blood flow; arterial and venous blood gas values and oxygen contents; oxygen consumption, delivery, and extraction; glucose and lactate fluxes; and cardiovascular variables of the fetal lamb in response to exogenous arginine vasopressin (AVP). Also, to compare the effects of vasopressin administration to fetal and non-pregnant adult sheep. 11 1.5 Hypotheses 1. Ligation of the circumflex iliac, internal iliac, and pudendoepigastric arteries will be necessary to enable a flow transducer located on the external iliac artery of the fetal hindlimb to measure solely hindlimb blood flow and metabolism. 2. With the proposed surgical protocol, a transit-time flow transducer positioned around the external iliac artery can be used to measure total hindlimb blood flow. This will be demonstrated by a good correlation with hindlimb blood flow measurements obtained with radionuclide-labeled microspheres. 3. Distribution of blood flow to and weight of hindlimb tissues will be similar between the study and non-study limbs. 4. Hindlimb blood flow and oxygen consumption will be greater during quiet sleep (high voltage ECoG). Administration of neuromuscular blocking agents to the fetus will abolish fetal breathing and body movements, including movements of the hindlimb. As a consequence, the metabolic demands of hindlimb muscles will not increase during quiet sleep so that hindlimb blood flow will not rise. 5. The hindlimb of the fetal lamb will be less able to tolerate a reduction in oxygen delivery resulting from hypoxemia than from ischemia as shown by an earlier fall in oxygen consumption and/or a greater release of lactate by the hindlimb, contributing to systemic lactic acidemia, with hypoxemia. 6. Exogenous arginine vasopressin administered to the fetal lamb will result in a rise in arterial pressure, a fall in heart rate, and a fall in hindlimb blood flow. Similar changes will be observed during vasopressin infusion to non-pregnant adult ewes. 12 2. GENERAL EXPERIMENTAL 2.1 Casting 2.1.1 Introduction Because this thesis focuses on the hindlimb and assumes that the external iliac artery serves only hindlimb tissue, it was necessary to first determine which tissues and blood vessels constitute the fetal lamb hindlimb. We employed the Batson casting technique (Polysciences Data Sheet #105), in which a partially polymerized monomer is injected into fetal blood vessels at autopsy, to help define the hindlimb tissues. Because radionuclide-labeled microspheres were used in some experiments to measure hindlimb blood flow, it was important to determine the bones, muscles, and skin which are served by the external iliac artery under our surgical protocol. By tracing the distribution of the main hindlimb vessels through the skin, bone, and muscles, it should be possible to eliminate potential sources of error in the calculation of hindlimb weight. For example, the inclusion of non-hindlimb tissue, especially from the gluteal region, would lead to an overestimate of total hindlimb weight and an underestimate of blood flow per unit weight of tissue. Furthermore, we also used a transit-time flow transducer on the external iliac artery to measure blood flow in the hindlimb. Thus, it was necessary to determine possible sites of collateral circulation. In their studies of the fetal lamb hindlimb, Wilkening (1988) and Boyle (1990) ligated the circumflex iliac artery and pudendoepigastric arterial vessels to minimize collateral circulation and ensure they were recording solely blood flow to the hindlimb. 2.1.2 Methods Vascular casting was performed on eight unoperated fetuses at 118-139 days gestation. Casting was performed at autopsy using Batson's No. 17 Replica and Corrosion Kit (Polysciences Inc., Warrington, Pennsylvania). In five animals, a midline incision was 13 made in the fetal lamb and the abdominal tissues carefully dissected to reveal the aorta. A small incision was then made in the abdominal aorta and Tygon flexible plastic tubing (R-3603, Norton Performance Plastics, Akron, Ohio; 2.38 mm ID, 3.97 mm OD) was inserted approximately 2 cm and secured. A 16 gauge needle hub was then secured at the end of the tubing and the vessels were gently flushed with a small amount of saline to clear them of any remaining blood. A coloured, partially polymerized polymer was prepared according to the manufacturer's directions and gently injected into the descending aorta In one case, after allowing the polymer to harden for several hours, the fetus was placed into macerating solution (340 g KOH: 1 L water) for several days until the tissues dissolved, leaving a coloured cast of the hindlimb vasculature (Figure 1). In three fetuses, hindlimb vessels (external iliac artery and/or internal iliac artery, femoral artery) were catheterized with appropriate diameter Silastic® medical grade tubing (Dow Corning, Midland, Michigan). The polymer was injected into the individual vessels, allowed to harden, and the fetal hindlimbs were then dissected to determine which tissues were served by the catheterized vessels. In some animals several vessels were catheterized, and casting material containing different coloured pigments was simultaneously injected via each catheter. Finally, in the four remaining fetuses, the abdominal aorta was catheterized as above and the internal iliac, pudendoepigastric, and circumflex iliac arteries were ligated. The casting polymer was then injected and allowed to harden for several hours before the tissues of the hindlimbs were carefully dissected. These final animals represented the hindlimb vasculature as it would appear after our surgical intervention. 2.1.3 Results/Discussion Figure 1 illustrates the arterial vascular cast of the hind quarters of a fetal lamb (135 days gestation); in this case the casting material was injected via the descending aorta. From our dissection of the tissues following casting, we defined the hindlimb as the muscles of the upper and lower pelvic limb as well as the gluteal and deep gluteal muscles and the skin 14 Major Blood Vessels A descending aorta B umbilical artery C external iliac artery D anterior circumflex iliac artery E posterior circumflex iliac artery internal iliac artery sagittal artery deep femoral artery pudendoepigastric artery femoral artery anterior femoral artery posterior femoral artery M saphenous artery N popliteal artery O anterior tibial artery P posterior tibial artery Q recurrent tibial artery F G H I J K L FIGURE 1. Ventral view of a cast of the arterial vasculature in the hindquarters of a fetal lamb at 135 days gestation. Major blood vessels are identified. overlying these tissues. Skin over the genitalia, rectum, and tail was excluded. Hindlimb bones included the femur, tibia, tarsals, and metatarsal, but not the iliosacral bones. The results from casting individual vessels indicate that the circumflex iliac artery, internal iliac artery, and pudendoepigastric artery serve primarily non-hindlimb tissues or are potential sources of collateral flow (Table 1). A flow transducer placed on the external iliac artery distal to the origin of the circumflex iliac artery would underestimate hindlimb blood flow unless the latter vessel was ligated. Furthermore, catheterization of the pudendoepi gastric artery (and vein) permits sampling of hindlimb arterial and venous blood without compromising limb perfusion. These finding were incorporated into our surgical protocol. Our results were consistent with the findings of other researchers (Boyle et al, 1990; Wilkeningera/., 1988). 2.2 Animal Care and Maintenance Ewes of Dorset-Suffolk breeds were housed at the University of British Columbia Farm where they underwent time-dated natural service mating. Groups of ewes (2 to 4 animals per group) were brought into synchronous estrus by implantation of vaginal pessaries (Veramix®, Tuco Products Company, Orangeville, Ontario) containing medroxyprogesterone acetate which inhibits ovulation. Two weeks later the pessaries were removed, follicle development was induced with intramuscular injections of 333 to 500IU pregnant mare's serum gonadotropin (Equinex®, Ayerst Laboratories, Montreal, Quebec), and the ewes were placed in with a ram. Pregnancy was determined by measurement of maternal serum progesterone levels at approximately 17 days post-ovulation and was confirmed 50 to 90 days later by real-time ultrasound. Each group of ewes was brought to the Research Institute approximately one week prior to surgery where they were kept in large pens in view of other sheep. The animals had free access to water, food, and hay. All studies were approved by the University of British Columbia Committee on Animal Care and conformed to the guidelines of the Canadian Council on Animal Care. 16 TABLE 1 • Fetal tissues supplied by the major branches of the terminal descending aorta and hindlimb arterial vessels, based on vascular casting results from eight fetal lambs at 118-139 days gestation. Blood Vessel Major Tissue Served Femoral and saphenous arteries Deep femoral artery Anterior femoral artery Pudendoepigastric artery anterior branch posterior branch Circumflex iliac artery anterior branch posterior branch Internal iliac artery Skin over lower leg, thigh, and lower leg muscles Skin and muscles of thigh Thigh and gluteal muscles Abdominal muscles Skin of inguinal and mammary regions Abdominal and gluteal muscles Abdominal muscles, skin of rump and thigh, thigh and gluteal muscles Skin and muscle of inguinal and mammary regions Sagittal artery Tail 17 2.3 Surgical Preparation Successful experiments were conducted on 22 fetal lambs (Table 2) and 3 adult non-pregnant ewes (Table 3). Unsuccessful preparations are presented in the Appendix. Surgery was conducted on the fetuses at 118-130 days gestation (term approximately 140 days). Food was withheld for about 18 hours prior to surgery. Ewes received 3 mg of atropine sulfate (Astra Pharma, Mississauga, Ontario) intravenously to control salivation followed by induction of anesthesia with 1 gram thiopental sodium (Pentathol®, Abbott Laboratories, Montreal, Quebec). The ewes were then placed in a supine position, intubated, and maintained on a ventilator for the duration of surgery. Anesthesia was maintained with 1.0-1.5% halothane (Fluothane®, Wyeth-Ayerst Canada Inc., St-Laurent, Quebec) and nitrous oxide (70%) in oxygen. Approximately 500 ml of 5% dextrose solution (Baxter Corporation, Mississauga, Ontario) was given at a rate of ~2 ml/min via the maternal jugular vein. Aseptic technique was observed throughout the surgical procedure. The ewe's abdomen was shaved, 1% lidocaine hydrochloride (Xylocaine®, Astra Pharma, Mississauga, Ontario) was injected subcutaneously along the midline, the surgical area was swabbed with 7.5% povidone-iodine (Betadine® surgical scrub, Purdue Frederick, Pickering, Ontario), and sterile sheets and drapes were positioned around the surgical Field. Avoiding major blood vessels and following the lidocaine injection site, a midline abdominal incision was made in the ewe to expose the uterus. An incision was then made in the uterus in a site free of cotyledons and major blood vessels and the fetal head was exteriorized. Silicone rubber catheters (Silastic®, Dow Corning, Midland, Michigan) with an outer diameter of 2.16 mm (1.02 mm ID) were placed in the trachea and amniotic cavity. The tracheal catheter was inserted through a small incision made between two rings of cartilage just below the larynx. It was advanced distally approximately 4 cm into the trachea and was anchored to the skin overlying the incision with 3-0 silk (Davis+Geck Cyanamid Canada Inc., Montreal, Quebec). A drop of surgical glue (Vetbond tissue adhesive, 3M 18 cu 1 3 cu 2 0 * WD CU O >r cu CU s •2-S cs ^—' sn 3 _> 13 Q c s •c u l o 00 GO C A G o O H O H o '§> C A 3 8 CO W - J cu E r cu a CU £l * E W 3 z s c i T3 co CN co CN -G I ca a .G T3 <U N •a a •3 3 <D CA 3 cu cu CO ON co ON CO c3 s o C A T3 3 C J . C D CO cN Ti-ro ro TI-CS CN Ti-en T3 <4 N •a ca •5 CA 3 <u <u in CN CN ro ro ON ro ro T3 T3 73 «4 <U <U N N N •a •a •a cd a CO •s $ •3 3 3  <U <U <U CA CA C A 3 3 3 ,<u Ci) <*-< t*—1 -a S T3 T3 s ca <u CU eg cu CD CU 00 00 00 CA T l i—i CN r—4 ro CN ro <-H vo co 00 ro 00 ro ro ON CN CO VD ro CN CN ro Tl-CN 00 TI-CS .G O cd E X a >  .G B D a in T f > < 6 3 IT) T ^ a* > vo ?] CN w W 13 CU •a 8 S3 CJ C A 3 s 2 3 (U C T—I § -3 3 •c CO <N .8 3 -O •c 8 00 7 3 g X I •c 2 1 c o 1 o o T3 o +s X 00 '53 7 3 & O JS 00 '53 2 -3 ^ -S ^ e J 3 o c C3 3 o 1/1 o X 00 '53 G O I O O ro ro 03 3 CJ CZ1 co X 03 <U a X CN o ro o CO G o I o 8 •3 3 X •c •a Xi 00 '53 ca cu X CJ c*> I |"e3 "ob 2 EC s 3 ea.2 > a •8'a > ^ - s 1 ca 3 a ca I M l C A " ~ ' ca co > 3 I L i i p s i s » ° ° II 3 t3 < &o 19 co 3 ^ •g M h i •<-> O -M <u DC .2 * •*-» co 4) 0 -a CD 3 1=1 '-3 C o o § a ctf CX, oo 3 c/1 00 <U O O 3 GO CN W 1 3 a •c ci (3 00 3 00 c £ a x z T3 a 3 <D cc 3 00 0 00 o 1—I CN c •a •a t & 3 O oo 3 a 2 3 <U C s <—I CI w X I <D N •a ctf r g 3 <U co s B 3 _C T ? <D T3 vo CO CO CO 00 CO CN CO B 3 C 13 00 CN CO s B 3 C T3 00 -a N 3 3 T3 <D N "1 X ! 4—» 3 <U GO 3 •a ^ T3 <D 3 O 3 00 00 CN CN CO CO CO —i CO CO CO i n CO CO o CO VO CN in CN CO CN o CO CN CO CO CO CN 00 o CN CN *r> oo CN «-< VO CN e D B CN o OH > < a C L , > < I o CN w CN w 3 13 00 •a 1 3 a i c 3 T-H C) L L ] a a in C L , > t—, CI g • f i CO & o XI 00 "53 & o CN W a a a C L , > < a 3 1 3 <U i3 O 3 o CO 3 a 2 3 a X ! O a a X a X ! 3 a a vo C L , > < a 3 •a 2 3 O u <u o 3 o oo 3 a 2 3 1) C IS "Bo •a IE 3 U . 2 I'u c^ 1 GO IX! II I I °1 CN CI X ! O oo a O '-3 | 2 ci > 3 co H i M oo U ! p i II II '> 20 CA 3 ^ • £ M "d •«-» " S 3 . 2 P la CU • 3 O * J Z r9* cu DC < s >> .2 « *S 73 CO w +•> CA CU o 2 3 i T3 _> Q o a 1 PJ CU 00 I-, 3 CO <N CO CO o CO e cu s r CU u CU cu ja ^ s W 9 CN -a 00 CU •a S3 1 3 a i CU c oo CO CN PJ CN CO § " f i GO 7 3 o 53 § 1 o 7 3 <U 7 3 en 3 .a CO CN 7 3 <U N s GO 3 CO CO CO CO 7 3 CU N 7 3 cu N XI -3 "3 3 3 CO 3 +-» .CU CO VO ON CO 3 CU CO 3 CO O CO 3 CN ON CO ' — 1 CN TJ" CO CO CO CN CN TT r-CO CN ON CO 00 CO o CN O CO CO CN 3 a in T T OH > < CN cj I X 1 CU o CU > OH > a a T £ OH > CN T l a a e o ON, > < 3 3 •a 2 3 cj cu -a X> 3 CJ Cfl 3 a 2 3 <U 3 ccj a <U -3 ccj 3 cj x 2 B 3 e 5 T3 T i i—1 00 T i CN T i CN ON 1 T3 3 cu OH > 7 3 CU 7 3 GO 3 CO 0 ON ci CN ON CO VO CO o CO s XI I XI 1 O ^•ob3 111 « OH cd «» 3 .y 1-8 11 CU CCJ g n Cd ^ I'l oo p- S P || XI II u II 00 c ^ S o c ° § s S a § i •IIS > J 5» < co U 21 TABLE 3. Successful surgical preparations of non-pregnant adult ewes. Ewe Experiment Ewe Weight Fate Number (kg) EOlOlz arginine vasopressin infusion 79 returned to UBC for rebreeding (90 mU/min) arginine vasopressin infusion (45 mU/min) E4146 arginine vasopressin infusion 60 returned to UBC for rebreeding (25 mU/min) arginine vasopressin infusion (45 mU/min) arginine vasopressin infusion (90 mU/min) E4215 arginine vasopressin infusion 52 returned to UBC for rebreeding (25 mU/min) arginine vasopressin infusion (90 mU/min) arginine vasopressin infusion (45 mU/min) Animal Care Products, St. Paul, Minnesota) was applied to the catheter insertion point, and the incision was closed. The non-occlusive nature of this catheter did not interfere with lung fluid flux through the trachea. The amniotic catheter was simply anchored to the skin of the fetal neck. While the fetal head was still exposed, electrodes for recording electrocortical (ECoG) and electro-ocular (EoG) activity were implanted in the skull. A transverse incision was made across the dorsal skull just rostral to the ears, the skin retracted, and two small holes (1.6 mm diameter) were drilled approximately 5 cm apart through the parietal region of the skull on either side of the midline. Electrodes for recording ECoG activity were positioned in the holes so as to rest on the parietal dura mater and adhered to the fetal skull with surgical glue applied to the plastic mounting disks. A grounding wire was sutured to the scalp skin. The scalp incision was then closed with 3-0 silk. Next, a 1 cm vertical incision was made over the orbital ridge of the zygomatic arch of each eye and, using a curved cutting needle, the EoG electrodes were passed through the bone. The stripped section of the wire was knotted in place and secured with tissue glue (Vetbond tissue adhesive, 3M Animal Care Products, St Paul, Minnesota). After closing the incisions, the ECoG and EoG electrode catheters were secured to the fetal skin. After completing the catheterization of the upper body, the uterine incision was closed and a second uterine incision was made through which the fetus' lower body could be exposed. Silicone rubber catheters were inserted into the femoral artery and lateral tarsal vein and advanced into the descending aorta and caudal vena cava, respectively. Both of these vascular catheters have an inner diameter of 0.63 mm (1.19 mm OD). This leg was designated the non-study or recording limb. In the contralateral leg, which serves as the sampling or study limb, smaller diameter catheters (0.51 mm ID, 0.94 mm OD) were inserted into the pudendoepigastric artery and vein and advanced 3-4 cm so that the catheter tips were in the external iliac artery and vein, respectively. Catheter placement involved making a small skin incision and then using blunt dissection to expose the vessel to be 23 catheterized. Three pieces of 3-0 silk were placed under the artery or vein. The blood vessel was tied off with the most distal piece of silk and the proximal silk was lifted up to temporarily constrict the vessel. A tiny incision was then made in the wall of the vessel between the distal and middle silks, the catheter inserted and advanced, and finally the three silk sutures were used to secure the catheter. A drop of surgical glue was also applied to the catheter entry point. Finally, the catheter was anchored to the adjacent muscle on either side of the incision with the silk tie attached to the catheter. A second amniotic catheter was also sutured to the fetal abdominal skin. Next, a 5 cm posterior flank incision was made in the fetus and, using a lateral approach, the study limb's external iliac artery was exposed. Two pieces of Silastic® tubing were placed under this vessel and gently lifted so as to expose the vessel. A 4 mm (4S or 4R series) transit-time ultrasonic blood flow transducer (Transonic Systems, Ithaca, New York) and a 4 mm inflatable perivascular occluder (In Vivo Metric, Healdsburg, California) were positioned on this vessel, with the occluder placed proximal to the origin of the circumflex iliac artery and the transducer distally. The tubing was removed and the flow transducer and occluder were anchored to the adjacent muscle. To ensure accurate measurement of hindlimb blood flow, we ligated the circumflex iliac arterial trunk with hemoclips (Hemoclip®, Edward Week and Company, Research Triangle Park, North Carolina). In addition, the internal iliac artery was ligated to minimize potential collateral circulation to the limb. The placement of an occlusive catheter in the pudendoepigastric artery effectively eliminated this vessel as a source of non-hindlimb blood flow, while permitting blood sampling without compromising limb perfusion. The location of fetal vascular catheters, transit-time flow transducer, and vascular occluder are illustrated in Figure 2. The fetus was then gently returned to the uterus and amniotic fluid lost during surgery was replaced with approximately 1500 ml of 0.9% irrigation saline (Baxter Corporation, Mississauga, Ontario). In the event that the ewe carried more than one lamb, only one fetus was instrumented (referred to as the operated fetus). FIGURE 2. Line drawing of fetal lamb hindquarters (ventral view) indicating location of catheters, transit-time flow transducer, and vascular occluder. 25 The uterine incision was closed with a continuous 2-0 gut chromic suture and then oversewn. Finally, using the same procedure as outlined above, catheters (1.02 mm ID, 2.16 mm OD) were placed in the maternal femoral artery and vein and advanced into the maternal descending aorta and caudal vena cava, respectively. Several ewes also had a transit-time flow transducer (4RB or 6RB series; Transonic Systems, Ithaca, New York) positioned on the external iliac artery for measurement of adult hindlimb blood flow. All catheters and cables were tunneled subcutaneously to emerge from an incision in the ewe's flank. The ewe's abdominal muscle layer was closed with discontinuous 3-0 Dexon (Davis+Geck Cyanamid Canada Inc., Montreal, Quebec) and Ethibond (Ethicon, Peterborough, Ontario) sutures. Finally the abdominal and flank skin incisions were closed with 3-0 Dexon. In some animals, a non-occlusive catheter (4.8 mm ID, 7.9 mm OD) made of Tygon tubing (Fisher Scientific, Vancouver, British Columbia) was also inserted into the maternal trachea for introduction of hypoxic gas mixtures. All catheters were fdled with heparinized saline (24 U/ml saline; Hepalean®, Organon Teknika, Toronto), capped, and stored in a denim pouch adhered to the ewe with medical adhesive. Elastic crepe bandages were wrapped around the ewe's midsection to keep the pouch and incision clean and to prevent the other animals from damaging the catheters. The patency of the catheters was maintained by daily flushing with heparinized saline. Prophylactic antibiotics consisting of 500 mg ampicillin sodium (Novopharm, Scarborough, Ontario) and 80 mg gentamicin (Garamycin®, Schenng Canada Inc., Pointe-Claire, Quebec) were administered to the ewe intramuscularly for 5 days postoperatively. Ampicillin (500 mg) and gentamicin (20 mg) were injected daily into the amniotic cavity for the duration of the preparation. In order to monitor the condition of the fetus, blood samples were taken daily for determination of blood gas status, oxygen saturation, and hemoglobin, glucose, and lactate concentrations. The animals were allowed to recover from surgery for a minimum of three days before being used in experiments. 26 2.4 Physiological Recording and Monitoring Procedures After several days to recover from surgery, the ewes were placed in monitoring pens and given free access to food and water. The fetal tracheal, amniotic, femoral arterial, and lateral tarsal venous catheters were connected to disposable DTX transducers (Spectramed, Oxnard, California) for continuous monitoring. Fetal heart rate was measured from the arterial pulse with a cardiotachometer (Gould Biotach amplifier model 13-4615-66, Gould, Cleveland, Ohio). The variables were recorded on either a Beckman R-711 polygraph recorder and on a computerized data acquisition system developed in our laboratory (Kwan et al., 1995a) or on a Gould TA 4000 polygraph recorder combined with Labtech Notebook for Windows data acquisition program (Version 7.2.1 Advantec, Laboratory Technologies Corporation, Wilmington, Massachusetts). Figure 3 shows a typical polygraph recording obtained from a late gestation fetal lamb in which amniotic, arterial, and tracheal pressures, fetal heart rate, hindlimb blood flow, and electrocortical and electro-ocular activity were recorded. Episodes of fetal breathing, rapid eye movements (REM activity), and high and low voltage electrocortical activity (ECoG) are clearly evident. Venous pressure was not obtained from this animal. Abrupt changes in the pressure variables are due to position changes of the ewe. For data analysis, fetal pressures were corrected for amniotic pressure by the computer (eliminating artifacts due to the mother's position) and one minute average values were stored on disk. Blood pH, Po2, and Pco2 were measured with an IL 1306 pH/Blood gas analyzer (Allied Instrumentation Laboratory, Milan, Italy) corrected to 39°C for maternal samples and 39.5°C for fetal blood. An OSM2 Hemoximeter (Radiometer, Copenhagen, Denmark) was used to measure hemoglobin and oxygen saturation in triplicate. Blood glucose and lactate concentrations, also measured in triplicate, were determined with a 2300 STAT Plus glucose/lactate analyzer (YSI Bioanalytical Products, Yellow Springs, Ohio). Oxygen content was calculated as the product of hemoglobin concentration, oxygen saturation, and a 27 .o \ I i 2 c CD 6 CD > o E . CL) CiJ 3 CD CD T3 ctj E CD 3 T3 GO £ } -j CD 00 CD "S •S He all ,«D T3 "r" o CD 3 T3 GO O -?? GO t l W o t •2 is c i « 8 B E «>'« 2 00 0 0 -S -c a J 2 - | D > i2 < T3 CS S £? g.I-8 c g O - C -1-1 oo 1 i - '> "2 -r: Q c o <J fi § i 2 ^ ° ^ GO <^ Si O T3 ' G O 28 hemoglobin oxygen binding capacity of 1.34 ml/dL, and converted to mM. Other values were calculated as follows: Oxygen delivery = Do2 = hindlimb blood flow x arterial 0 2 content Oxygen = ^ = hindlimb blood flow x (arterial - venous O, content) consumption 2 v 2 1 „ _ oxygen consumption Oxygen extraction (%) = Eo2 = — — x 100 oxygen delivery Lactate flux = hindlimb blood flow x (arterial - venous lactate concentration) Glucose flux = hindlimb blood flow x (arterial - venous glucose concentration) Hindlimb (arterial pressure - hindlimb venous pressure) vascular resistance = hindlimb blood flow Fetal weight in utero was estimated using an equation for fetal growth in sheep (Koong and Rattray, 1975) that predicts an average rate of growth of about 2% per day which is similar to the value determined in our laboratory using blood volume estimates of weight in utero (Kwan et al., 1995a). Fetal results were then normalized to hindlimb weight at the time of experimentation, with hindlimb weight calculated from the following equation: hindlimb weight , ° autopsy Hindlimb weight. t = fetal weight. ^ x b in utero b in utero fetai weight 6 autopsy 2.5 Catheter and Electrode Preparation Catheter construction is illustrated in Figure 4. Catheters were made of silicone rubber (Silastic®, Dow Corning, Midland, Michigan) with outer diameters appropriate for the vessel size (Table 4). For ease of insertion into the blood vessel, the catheter tip was cut on an angle. Ties made of 3-0 silk were securely knotted around the catheter tip for securing of the catheter in the vessel. Catheters made for insertion into the amniotic cavity Amniotic catheter Tracheal catheter H 10cm Femoral artery catheter V 10cm H Tarsal vein catheter Pudendoepigastric artery/vein catheters FIGURE 4. Construction of silicone rubber (Silastic®) catheters implanted in the gestation fetal lamb preparations. Diagrams are not drawn to scale. 30 T A B L E 4. Inner and outer diameters of silicone rubber (Silastic®) catheters used in animal preparations. Catheter Inner Diameter (mm) Outer Diameter (mm) Amniotic 1.02 2.16 Fetal trachea (non-occlusive) 1.02 2.16 Fetal femoral artery 0.63 1.19 Fetal lateral tarsal vein 0.63 1.19 Fetal pudendoepigastric artery 0.51 0.94 Fetal pudendoepigastric vein 0.51 0.94 Maternal femoral artery 1.02 2.16 Maternal femoral vein 1.02 2.16 31 were similarly prepared except that a series of small holes was cut along the length of the catheter distal to the ties. This design helped reduce the likelihood of catheter tip obstruction by the membranes lining the amniotic cavity. On the contralateral end of all catheters (i.e., end that emerges from the flank incision) bands of coloured silicone were applied to enable identification of the vessel in which each catheter was positioned. Catheters prepared for the pudendoepigastric vessels were modified slightly from the description above. In these catheters, the portion of the catheter actually inserted into the vessels was 0.94 mm outer diameter (0.51 mm ID). This was then telescoped into larger diameter tubing (1.02 mm ID; 2.16 mm OD). The small lumen section, approximately 3.75 to 4 cm in length, was appropriate for the small diameter of these vessels. However, because withdrawing blood samples through a long length of small lumen catheter is very slow, the addition of the larger lumen section was required. As illustrated in Figure 5 electrocortical and electro-ocular electrodes were prepared by threading Teflon sheathed multi-strand stainless steel wire (Cooner Wire, Chatsworth, California) through Silastic® tubing with an outer diameter of 1.95 mm (1.47 mm ID). The ECoG electrodes were composed of two lengths of wire whose ends were threaded through small plastic disks, knotted, and the stripped ends formed into a stiff oblong ball. The coiled ends were inserted through holes made in the skull for contact with the brain while the disks were used for securing the electrode to the skull with tissue glue. A third length of wire, used as a ground, was secured to the fetal scalp. Electro-ocular electrodes consisted of a total of two lengths of wire inserted through a silicone rubber catheter. Stripped sections of the end wires were secured next to each eye in the fetus. Small sections of the contralateral ends (i.e., ends emerging from ewe's flank) of the EoG and ECoG electrodes were stripped for connection to the computer cable which records their output. 32 Electrocortical (ECoG) electrode The plastic disk was 0.5cm in diamet, Electro-ocular (EoG) electrode FIGURE 5. Construction of electrocortical and electro-ocular electrodes for the late gestation fetal lamb preparations. Diagrams are not drawn to scale. 33 2.6 Statistical Analysis Data are presented as means ± 1 standard error of the mean unless otherwise indicated. Data were analyzed using StatView statistical software (Abacus Concepts Inc., Berkeley, California). Results were compared using two-tailed paired or unpaired t-tests as appropriate. For multiple comparisons, one-way (factorial) analysis of variance followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test were used. Differences were considered significant if p < 0.05. 34 3. HINDLIMB BLOOD FLOW MEASUREMENT CORRELATION: MICROSPHERES VERSUS TRANSIT-TIME FLOW TRANSDUCER 3.1 Introduction In order to study oxygen delivery and consumption in the fetal lamb hindlimb, continuous accurate measurements of hindlimb blood flow were necessary. This required the determination of total blood flow into and out of the hindlimb. Radionuclide-labeled microspheres are widely used to determine blood flows to specific tissues but do not enable continuous measurements. Thus, we elected to use a transit-time flow transducer positioned on the external iliac artery to provide these continuous measurements. By correlating the hindlimb blood flow measurements obtained with each of these techniques, we hoped to determine sources of error, including potential sources of collateral flow or blood vessels in the hindlimb that serve primarily non-hindlimb tissues. Based on these results and data from other researchers, we sought to develop a surgical protocol which would permit us to obtain continuous accurate measurements of hindlimb blood flow using a transit-time flow transducer positioned on the external iliac artery. Radionuclide-labeled microspheres are non-biodegradable, inert, polystyrene spheres with a radioactive nuclide incorporated into the plastic as opposed to simply attached to the surface. Nuclide labels are chosen for well defined energy peaks with a minimum overlap of energy spectra. This permits the injection of up to five different radionuclide labels into a single animal. Computer software integrated with a gamma counter differentiates the radioactivity emitted when multiple nuclides are present in each sample. Microspheres are available in a number of diameters and approximate the specific gravity of blood. To prevent aggregation of the spheres, they are generally suspended in isotonic saline with Tween-80 (polyoxyethylene 80 sorbital monooleate) added. Theory dictates that the microspheres are distributed in proportion to blood flow and will be trapped 35 in capillary beds on the first circulation; obviously the microspheres must remain entrapped until counting can be completed. In sheep, 15 jwm diameter microspheres are generally used to satisfy the criteria of distribution similar to erythrocytes and the absence of recirculation or non-entrapment (Heymann etal., 1977). Similar counts obtained for paired organs such as the kidneys indicate good mixing and even distribution of microspheres. Radionuclide-labeled microspheres have been widely used to measure the distribution of cardiac output and organ blood flow since the pioneering work of Rudolph and Heymann (1967) and subsequent introduction of the reference sample method (Makowski etal., 1968). For adequate mixing of the microspheres in blood, they should pass through the left side of the heart before reaching the systemic circulation. In newborns and adults this requires left atrial or ventricular injection. In the fetus it is possible to inject the microspheres into the systemic venous circulation. Because of shunts in the fetal circulation, in particular the foramen ovale and ductus arteriosus, it is possible to inject the microspheres into the systemic venous circulation; most of the spheres bypass the lungs and become entrapped in the systemic circulation. Rudolph and Heymann (1967) concluded that microspheres do not significantly recirculate, are distributed in proportion to blood flow, and do not alter circulatory physiology. Calculation of blood flow to individual organs is simplified by the reference sample method introduced by Makowski and colleagues (Makowski etal., 1968). In this technique, an arterial blood sample is collected at a known, constant rate into a glass syringe which thus acts as an artificial organ. Blood flow to a specific organ can then be calculated as the product of this reference blood flow rate and the ratio of the radioactivity in the organ to that in the reference sample. In order to keep errors ±10%, it is estimated that a minimum of 400 microspheres should be present in each sample (Buckberg et al., 1971). Archie etal. (1973) used a calibrated pump during heart bypass in lambs and dogs to create known cardiac outputs. Microspheres were found to give cardiac output measurements within 10 to 15% of the known values. The microsphere method also 36 compares favourably with blood flow measurements obtained with electromagnetic flowprobes (Buckberg et al, 1971; Rudolph and Heymann, 1967). Furthermore, a number of experimenters have used serial injections of microspheres for multiple flow measurements with little loss of precision or alteration of hemodynamics due to embolization of vascular beds (Jansson, 1991; Baer et ai, 1984). The Transonic® transit-time flow transducer, unlike other widely used in vivo flowmeters, is largely unaffected by its positioning on the vessel, is not limited by electrical interference, and, as a tight fit is not required, the flow transducer is non-constrictive. The transducer body houses upstream and downstream ultrasonic transducers with a fixed acoustic reflector positioned opposite (Figure 6). The flow transducer is placed on a blood vessel such that the transducers are on one side of the vessel and the reflector is located on the opposite side midway between the transducers. A plane wave of ultrasound emitted by each of the transducers bounces off the acoustic reflector and is received by the other transducer. The sound wave emitted by the upstream transducer travels with a vector component of the blood flow. As the downstream transducer's sound wave travels against the direction of blood flow, its transit time will be prolonged relative to the transit-receive sequence of the upstream transducer. Flowmeter circuitry calculates the difference in total transit time of the two cycles to record a signal proportional to the rate of blood flow. This signal, scaled with the predetermined calibration factor of the flow transducer, is displayed as the absolute volume rate of flow through the vessel. As the Transonic® flow transducer relies solely on the difference of the transit times it is independent of vessel diameter or alignment of the flow transducer. Transit time flowmeters have been calibrated in vivo against established methods of measuring blood flow. When measuring ascending aorta blood flow in dogs Hartman and coworkers report correlations of over 0.97 between transit time flowmeters and electromagnetic flowprobes, thermodilution, and exsanguination (Hartman et al, 1985). 37 Transonic® transit-time flow transducer UPSTREAM TRANSDUCER CABLE S I D E S E C T I O N ACOUSTIC REFLECTOR E N D V I E W PROBE BODY DOWNSTREAM TRANSDUCER BI-DIRECTIONAL ACOUSTIC PATHWAY VESSEL CONSTANT • ACOUSTIC INTENSITY THROUGHOUT THE WINDOW Schematic view of a transit-time flow transducer ACOUSTIC R E F L E C T I O N WINDOW BRACKET FIGURE 6. Transonic® transit-time flow transducer and its operation (from Hartman et al, 1985). The tranducer is positioned around the external iliac artery and provides continuous measurements of hindlimb blood flow. 38 Radionuclide-labeled microspheres allow the determination of organ blood flow in vivo with little disturbance to the animal; however, they are limited to measuring blood flow at only brief moments in time and require the animal to be sacrificed. In contrast, transit-time ultrasonic flow transducers measure blood flow continuously in vivo, but they do not give specific organ blood flows. In the present study, hindlimb blood flow in the fetal lamb was measured with a transit-time flow transducer on the external iliac artery and compared with measurements obtained with radionuclide-labeled microspheres. 3.2 Methods Five animals underwent surgery between 118-125 days gestation (mean 122 ± 2 days) using the techniques previously described (Section 2.3). Tracheal, amniotic, femoral arterial, lateral tarsal venous, and pudendoepigastric arterial and venous catheters were inserted. Hindlimb blood flow was continuously measured with a transit-time flowmeter on the external iliac artery distal to the circumflex iliac artery. Flow through this vessel was altered by partial inflation of the vascular occluder achieved by injecting saline into the occluder through a 3 cc syringe mounted on a Harvard infusion/withdrawal pump. Blood flow was maintained at each level for 10-15 minutes. A total of 19 observations were made in the 5 animals. The distribution of hindlimb blood flow was measured with 15 pirn diameter radionuclide-labeled microspheres using established technique (Rurak etal., 1990a). Briefly, a pre-weighed 10 cc glass syringe mounted on a Harvard Pump was set to withdraw blood from the femoral artery at 2.2 ml/min. After 30 seconds, approximately 1.2 x 106 sonicated microspheres (3M Health Care, St. Paul, Minnesota), labeled with one of1 4'Ce, 8SSr, 1 S 3Gd, s l Cr, and 9SNb, were injected into the tarsal vein over 1 minute. The pump continued to withdraw blood for 2 minutes after microsphere injection. The syringe was then reweighed and the blood transferred into counting tubes. The availability of five different radionuclide-labels permitted up to five observations, at different degrees of 39 external iliac artery occlusion, on each animal. After completion of the serial microsphere injections, the animals were euthanized, tissues were collected from both hindlimbs and other lower body (distal to the diaphragm) sites, weighed, and catheter placement was verified. The tissues were carbonized in a 300°C oven, packed into counting tubes, and analyzed in a gamma counter (Micrad Inc., Knoxville, Tennessee). Tissue blood flow was calculated as follows: Tissue blood flow (ml/min) = withdrawal rate (ml/min) x tissue radioactivity reference sample radioactivity 3.3 Results and Discussion In all the microsphere observations presented, similar numbers of microspheres were obtained for the right (13407 ± 882) and left kidneys (13569 ± 698), indicating good mixing and even distribution of microspheres. In addition, the blood and tissue samples contained at least 400 microspheres, the minimum number stipulated by Buckberg for blood flow errors to be ±10% in sheep (Buckberg etal., 1971). The calculated hindlimb blood flow obtained from the microspheres was compared with the average transit-time flowmeter reading during the same interval. The overall regression line for the 19 observations is presented in Figure 7. The regression data does not go through the origin (y-intercept = 3.02) indicating that the flow transducer is measuring some blood flow that is not accounted for in the microsphere calculations, probably reflecting the fact that data was collected from animals that did not undergo identical surgical procedures. The difference is within the inherent inaccuracies (±5-15%) of the two methods for determining blood flow {Burton and Gorewit, 1984; Archie et al., 1973; Buckberg etal., 1971). For the individual animals the correlation coefficients (r) ranged from 0.865 to 0.999 indicating a very good agreement between microsphere and transit-time flowmeter (ujuu/iLu) M O I J pooiq j a o n p s u e j ; M O W a u n w i s u e j i 41 measurements of hindlimb blood flow (Figure 8). The casting results indicated that the circumflex iliac (CIA) and internal iliac arteries (IIA) are potential sources of error in determining hindlimb blood flow. This was also suggested by the above results as animal E207HIN, in which no hindlimb vessels were hemoclipped, demonstrated a lower correlation coefficient compared to the animals in which the CIA and IIA were ligated. Animal E133x, in which only the CIA was hemoclipped, had a very high correlation coefficient (r = 0.998) indicating that, because the CIA serves both hindlimb and non-hindlimb tissue, it must be ligated to ensure accurate measurements of blood flow. The internal iliac artery, a potential source of collateral flow, plays a less important role. Thus, hindlimb blood flow would be overestimated by a flow transducer on the external iliac artery proximal to the origin of the CIA unless the latter vessel was ligated. Considering these data and the casting results, ligation of both the CIA and IIA was incorporated into the surgical protocol. In one animal (E2123) we attempted to catheterize the sagittal artery and advance the catheter into the descending aorta; however, the vessel was too small and had to be ligated. As the sagittal artery was found to serve only the tissues of the tail, it was not hemoclipped in subsequent animals. To further confirm the casting results, total occlusion of the external iliac artery followed by injection of microspheres was carried out on several animals to determine potential sources of collateral blood flow. However, although complete occlusion was achieved as indicated by a flowmeter reading of 0 ml/min, microspheres appeared in the hindlimb tissue in normal or above normal numbers. We speculated that microspheres in the systemic circulation accumulated in the length of the external iliac artery proximal to the occluder during total occlusion and when the occluder was subsequently released, these microspheres flowed into the hindlimb tissues as normal. Considering these results, any data points in which hindlimb blood flow was reduced to less than 20% of normal blood flow were considered inaccurate and excluded from subsequent analyses. 42 = -2.977 + 0.791 x r=0.865 no vessels clipped 0 1 0 20 30 40 50 Microsphere blood flow (ml/min) FIGURE 8. Individual animal comparisons of fetal lamb hindlimb blood flow as measured with transit-Ume flow transducers and radionuclide-labeled microspheres. The ewes' numbers are indicated on each graph (i.e., E133x). Each symbol represents a separate blood flow measurement (different radionuclide label). The regression equations and the blood vessels that were ligated are also indicated. CIA = circumflex iliac artery; IIA = internal iliac artery 43 In conclusion, the circumflex iliac, internal iliac, and pudendoepigastric arteries are potential sources of error when measuring total hindlimb blood flow with a transit-time flow transducer on the external iliac artery. When used in conjunction with a surgical protocol that ligates these vessels, blood flow measured with a flow transducer provides a good correlation to measurements obtained with radionuclide-labeled microspheres. Thus, a transit-time flow transducer can be used to provide continuous accurate determinations of hindlimb blood flow in the late gestation fetal lamb. 44 4. HINDLIMB BLOOD FLOW AND WEIGHT DISTRIBUTION 4.1 Introduction Although the use of occlusive-type catheterization is commonly used in hindlimb vessels (Rudolph and Heymann, 1984), there is limited information about its effect on hindlimb blood flow and growth. In the present study, we compared weight and blood flow distribution in the late gestation fetal lamb hindlimb under normal conditions. We implanted catheters in the femoral artery and lateral tarsal vein of one hindlimb (non-study limb) and smaller diameter, shorter catheters in the more peripheral pudendoepigastric artery and vein of the other (study) limb. The effect of these different catheterization protocols on the weight and blood flow distribution of the study and non-study hindlimbs was assessed. This research has been previously published by our laboratory (Knight etal., 1996). 4.2 Methods Surgery was performed on 6 sheep between 118 and 125 days gestation (mean = 123 ± 1 days, term ~145 days), using the techniques previously described (Section 2.3). Briefly, silicone rubber catheters (2.26 mm OD; Dow Corning, Midland, Michigan) were placed in the fetal trachea and amniotic cavity. Occlusive catheters (1.19 mm OD) were placed in the femoral artery and lateral tarsal vein of one hindlimb (non-study limb) and advanced into the descending aorta and caudal vena cava, respectively. In the other limb (study limb), smaller catheters (0.94 mm OD) were inserted into the pudendoepigastric artery and vein and advanced 3-4 cm so that the catheter tips lay in the external iliac artery and vein, respectively. For the purposes of other experiments, a transit-time ultrasonic blood flow transducer (Transonic Systems, Inc., Ithaca, New York) and a 4 mm vascular occluder (In Vivo Metric, Healdsburg, California) were placed around the external iliac artery of the study limb, with the transducer being distal to the origin of the circumflex diac artery of the study limb, and the occluder being proximal. The circumflex iliac arterial trunk 45 and the internal iliac artery were ligated. The right leg was instrumented as the study limb in 2 fetuses while the left limb was used in the remaining 4 animals. Finally, catheters (2.16 mm OD) were inserted into a maternal femoral artery and vein. The studies were conducted between 129 and 139 days gestation (mean = 134 ± 2 days), 6-14 days (mean 10 ± 1 days) following surgery. Hindlimb blood flow distribution was measured using radionuclide-labeled microspheres during the control periods of other experiments. Only the microsphere flow data were used and only that obtained prior to flow manipulation in the hindlimbs. The ewes and fetuses were euthanized with pentobarbital sodium (Euthanyl Forte, M.T.C. Pharmaceuticals, Cambridge, Ontario) 1-2 days after experiment completion (8-15 days post-surgery). Data are presented as mean ± standard error of the mean. Results were compared using two-tailed paired t-tests with differences considered significant if p < 0.05 (StatView statistical software, Abacus Concepts Inc., Berkeley, California). 4.3 Results The values for arterial Po2 (18.4 ± 1.2 mmHg), Pco2 (51.0 ± 1.0 mmHg), pH (7.284 ± 0.012), glucose (0.61 ± 0.11 mM) and lactate concentrations (2.07±0.84 mM), arterial blood pressure (46.9 ± 0.8 mmHg) and heart rate (150 ± 1 bpm) on the day of blood flow estimation were within the normal range for our laboratory. Table 5 presents the weights of the study and non-study hindlimbs for the 6 animals studied, as well as fetal weight. Although non-study limb weight was 2.8 ± 1.3% less than that of the study limb, the difference was not statistically significant. In Table 6, the hindlimb weight data are broken down into skin, bone, and muscle components, and again there was no significant weight differences between the study and non-study limb tissue components. In addition, the distribution of hindlimb blood flow to skin, bone, and muscle in the study and non-study limbs is given in Table 6. The values for hindlimb blood flow to each tissue, expressed as a percentage of total hindlimb flow, are plotted in Figure 9, along 46 TABLE 5. Gestational age, fetal and hindlimb weights at autopsy for the six fetuses used in the hindlimb blood flow and weight distribution study. In the case of multiple lambs, weights are those of the operated fetus only. Ewe Number # of Gestational Fetal Study limb fetuses age (days) weight (g) Weight % of fetal (g) weight Nonstudy limb Weight % of fetal (g) weight E207HIN 132 2218 184.2 8.30 173.4 7.82 E238 133 2604 169.0 6.49 169.5 6.51 E136 137 3158 252.5 7.99 242.9 7.69 E207HLIM 1 137 3766 261.6 6.95 263.5 7.00 E1245 139 3225 225.4 6.99 210.6 6.53 E133 131 1598 117.1 7.33 115.3 7.22 Mean + SEM 135±1 2762±319 201.6±22.6 7.34±0.28 195.9±22.1 7.13±0.23 47 Xi B 7 3 C CU J 3 oo 3 CO <U § 1 s a •c & 8 c ^ 7 3 <U Xi •4—» C o G O •a 3 X 'B CO o 7 3 8 7 3 i a 4_» GO 3 00 g GO GO S.2P 1 s H 3 E s CO s o Z E s CO 3 CO en I 8 l-'i 31 •8 xi ^-v 00 00 •53 — r> oo I 8 <L>-# > 'e o ^ c 3 ^ B 3 ^3 i s •s 53 00 ON ti CO 00 CN CO CN -H O CO CN -H l> CN T f -H ON 5 CO o CN -H T—I CN CN +1 CO iS CN T f •H 00 ON jo 00 00 •H CO 00 VD cu C O •H VD -H co 00 PS CN J O ON 00 d •H +1 -H i n 00 VO 00 -H i n ON -H VD CO 00 73 CO 3 CN +1 d CN «n +i VD CO i n CN CN -H CN T—< ON ON cd CO J O • ' ' r—I * — i 0±4. 1±3. CO -H O CN +1 CN o CO o CO CO CN o VD -H O CN T f ON CN CN -H 00 VD ON X> a > » 7 3 3 co s p CU fi e •a .2PcU co o co C a "a cs op a §3 a 3 fi g oo o '53 o & -fi ^ t3 cu CJ ^ 7 3 S .3 £ CO -H D. "6 e c j ex 3 co 00 J3 a ts cu co <U 60 T 50 + 40 + CC o Z 30 o 20 + 10 + • study l imb--% of total weight • study l imb--% of total flow H non-study l imb--% of total weight 0 non-study l imb~% of total flow i l l _X_ _L JL. _T_ SKIN BONE MUSCLE FIGURE 9. Comparison of tissue weights (open columns, study limb; stippled columns, non-study limb) and blood flows (closed columns, study limb, diagonal-hatched columns, non-study limb) for skin, bone, and skeletal muscle in six fetal lambs. Bars are mean ± SEM. * = significant difference between hindlimb blood flow to each tissue, expressed as a percentage of total limb flow, and the weight of each tissue, expressed as a percentage of total limb weight (p<0.05). 49 with the weight of the tissues expressed as a percentage of total limb weight. In every animal, total absolute flow and flow per 100 g tissue were less in the non-study limb, with the mean weight normalized flow being 13.4 ± 1.8% less than in the study limb. Blood flows to muscle, skin, and bone also tended to be lower in the non-study limb, but the differences were not statistically significant. In both limbs, skin and bone blood flows, expressed per 100 g weight, were similar, and both were significantly higher than the flow to muscle. This indicates that, compared to hindlimb muscle, the skin and bone components are more highly perfused in relation to the proportion of total limb weight that they comprise (Table 6, Figure 9). Bone received the highest fraction (45.3 ± 3.6%) of total flow, compared to skin (29.5 ± 3.3%) and muscle (25.2 ± 4.5%). 4.4 Discussion Several authors have reported that the hindlimb comprises approximately 10% of fetal weight in late gestation (Boyle et al, 1992; Boyle et al, 1990; Wilkening et al, 1988; Dawes et al, 1968), while in our animals the mean value for the study limb was 7.34 ± 0.28%. This discrepancy may be due to differences in the tissues, particularly muscle, considered as hindlimb, variations in the breed of sheep used, or differences in the number of fetuses per ewe. Previous estimates of hindlimb blood flow in the fetal lamb average ~18 mlmin '• lOOg"1, while estimates of flow to the general carcass range from 19 to 26 mlmin" l- lOOg"1 (Boyle etal, 1992; Davis and Hohimer, 1991; Jensen etal, 1991; Wilkening etal, 1988; Court et al, 1984). This compares to our estimates of 23.2 ± 2.1 and 20.7 ± 2.0 mlmin"1- lOOg"1 for the study and non-study limbs, respectively. Similarly, our estimates of study limb blood flow to the skin, muscle, and bone (Table 6) fall in the range of values reported by others: skin, 19-32 ml-min"1- lOOg"1, bone, 18-30 mlmin"1- lOOg"1, and muscle, 10-17 mlmin"1- lOOg"1 (Davis and Hohimer, 1991; Rurak etal, 1990a; Wilkening etal, 1988; Jensen et al, 1987; Jensen et al, 1986). However, we found that in relation to weight, the skin and bone of the hindlimb have higher perfusion rates compared to muscle (Figure 50 9), with bone receiving the highest fraction of total limb flow. Similar results can be extracted from data given in Wilkening et al. (1988), but this was not commented on in that paper nor apparently elsewhere. The rate of bone blood flow in the fetus (~30 ml-min"1- lOOg"1) is 2 to 3 fold higher than the values reported in the adult pig, dog, and rabbit, which range from 9-14 mlmin" lOOg"1 (Kiaer etal., 1993). This is also the case for muscle perfusion, which averages ~7 mlmin"1- lOOg"1 in adult sheep hindlimb (Oddy et al., 1981), compared to our estimates in the fetus of ~14 ml-min"1- lOOg"1. The higher tissue perfusion rates in the fetus likely reflect the need to compensate for the lower oxygen content in fetal blood compared to the adult (Rurak, 1994). In both the fetus and adult there appears to be a higher rate of blood flow to bone than to muscle in the hindlimb. In adult rat hindlimb, blood flow to marrow is higher than to bone, and marrow perfusion is increased by about 3 fold when hematopoiesis is stimulated by hemorrhage, normovolemic anemia, or erythropoietin administration (Iverson et al, 1992). The high perfusion rate that we have observed in fetal bone may also result from hematopoiesis. In the last 2 weeks of gestation, there is a rapid switch from fetal to adult hemoglobin (Bard et al, 1972). This, plus the rapid growth rate (~2%/day) in late gestation (Kwan et al, 1995a), likely results in a high rate of hematopoiesis. Skin perfusion is elevated probably due to vasodilation in response to the high ambient temperature in the amniotic cavity. When amniotic fluid and fetal temperature are decreased, skin blood flow, particularly in the lower body, decreases significantly (Van Bel et al., 1993). Skeletal muscle blood flow in the fetal hindlimb may be lower than that to skin and bone because of the limited extent of limb movement and the lack of any requirement for thermogenesis. It increases about three-fold 2 hours after Cesarean delivery probably due to shivering thermogenesis, increased limb movement, and postural muscle activity (Richardson et al., 1989). Although chronically catheterized fetal sheep have been utilized for more than 25 years (Meschia et al., 1965), there have been few studies of the effects of occlusive 51 catheterization on the organs and tissues normally perfused by the catheterized vessel. Wilkening et al. (1988) attributed a 4% difference between hindlimb weights to the effect of arterial catheterization of the non-study limb, although they found no difference in study and non-study hindlimb blood flows. In contrast, we found no significant difference in study and non-study hindlimb weights (although non-study limb weight tended to be lower), whereas blood flow was 13.4 ± 1.8% less in the non-study limb. We attribute the difference between the two studies to variations in the surgical protocols. Wilkening et al. (1988) catheterized the non-study limb pedal artery with a 1.38 mm OD polyvinyl catheter. As there are few potential sources of collateral flow in the lower hindlimb, ligation of the pedal artery could have impaired both flow and growth of the lower limb while not greatly affecting blood flow measured in the external iliac artery. We catheterized the non-study limb femoral artery with a 1.19 mm OD silicone rubber catheter. The use of a smaller catheter in a larger vessel (i.e. femoral artery versus pedal artery) may have less effect on flow in the catheterized vessel upstream from the site of occlusion; moreover, in the upper leg there are more sources for collateral flow than in the lower limb. As a result hindlimb growth was not retarded, but we did find diminished blood flow probably reflecting local interference in tissue perfusion of the upper limb. That the reduced flow did not lead to a significant effect on weight may be due to the relatively small reduction in flow and the short time (6-14 days) between fetal surgery and the study. However, it is possible that the initial reduction in flow in the non-study limb may have been greater than the ~13% observed, particularly if there were a gradual post-surgical increase in hindlimb perfusion via existing or newly developed collateral vessels. Furthermore, in both our study and that of Wilkening et al. (1988) the effect of vascular catheterization on the hindlimb may have been underestimated since the study or reference limb was also catheterized, albeit with smaller diameter catheters in peripheral branches of the hindlimb vasculature. In view of the common employment of fore and hindlimb vascular catheters in studies of chronically 52 instrumented fetal lambs, the impact of catheterization on both limb perfusion and growth seem worthy of further investigation. 53 5. BEHAVIOURAL STATE MODULATION OF HINDLIMB BLOOD FLOW 5.1 Introduction Szeto describes a behavioural state as the concurrence of a certain set of physiological measures that is recurrent over time (Szeto, 1992). Sleep cycles consisting of a quiet and an active or rapid eye movement (REM) phase have a long phylogeny, having been observed in all terrestrial placental mammals (Campbell and Tobler, 1984; Tobler, 1984) including humans (Nijhuis et al, 1982). In species born with a relatively well developed central nervous system, organized fetal behavioural states are evident in late gestation. Cyclic alterations in behavioural state are a normal component of pregnancy and are very obvious features of fetal life particularly in the last third of gestation. Prior to about 110 days gestation in the fetal lamb, these associations of electrocortical activity, eye movements, and skeletal muscle activity are random, with the transition to more organized behavioural states developing between 115 to 120 days gestation (Szeto and Hinman, 1985; Clewlow et al, 1983). In humans, this transition occurs after approximately 36 weeks gestation (Nijhuis et al, 1982). As an animal matures, less time is spent in rapid eye movement sleep. In sheep, REM sleep occurs approximately 40% of the time in late gestation, less than 20% of the time in the neonate (Ruckebusch, 1972a), and about 2.5% of the time in adults (15% of total sleep time) (Ruckebusch, 1972b). Time spent in quiet sleep (high voltage ECoG) changes from 55% of the time in the fetus, 43% in the newborn (Ruckebusch, 1972a), to 14% of the time in the adult (Ruckebusch, 1972b). Thus there is no correlation between maternal and fetal REM or ECoG activity. In the late gestation fetal lamb, skeletal muscle activity, in the form of fetal breathing and body movements, is associated with specific patterns of electrocortical (ECoG) and electro-ocular (EoG) activity (Walker, 1984). Sleep state is usually classified into three patterns. Quiet or non-REM sleep, is characterized by high voltage slow frequency electrocortical activity, no breathing or rapid eye movements, and sluggish body movements. 54 This state occurs approximately 55% of the time. Active or REM sleep, which occurs about 40% of the time, consists of low voltage fast frequency ECoG activity with breathing and rapid eye movements, but with fetal activity limited to abrupt twitches. A third category represents an awake or aroused state in which electrocortical activity is of low voltage fast frequency without REM and is associated with breathing and vigorous body movements. This state of responsiveness is of short duration, lasting a few moments at most and occupying approximately 5% of the time. In the late gestation fetal lamb, a complete sleep cycle lasts 30 to 40 minutes (Clapp et al, 1980; Ruckebusch et al, 1977). Fetal breathing movements in late gestation are episodic, involving rhythmic movement of the diaphragm and movements of small amounts of fluid in the trachea. Their incidence is about 40% (Dawes et al, 1972) with a greater prevalence during the early-morning hours (Boddy and Dawes, 1975). Body movements include twitches, isolated limb movements, and complex changes in limb and trunk orientation. They, too, are more prevalent in the early morning. As indicated, fetal breathing movements (FBM) occur during low voltage high frequency electrocortical activity with REM (Dawes etal, 1972), while body movements are most vigorous during the transitions between electrocortical states and are very infrequent during low voltage activity with REM (Natale et al, 1981). It has been suggested that breathing and body movements in utero develop the musculature in preparation for post-natal life (Boddy and Dawes, 1975). Fetal breathing movements appear to be necessary for normal lung development although the mechanism is unclear. Fetal lambs that have undergone high spinal cord transection (integrity of phrenic nerves and diaphragm retained) exhibit marked decreases in lung growth and distensibility (Harding et al., 1993; Kitterman, 1988). Long-term inhibition of body movements in fetal rats resulted in joint abnormalities, pulmonary hypoplasia, and growth retardation involving reduced bone and muscle mass (Moessinger, 1983). An association between ECoG state transition and maternal contractures has been reported (Nathanielsz et al, 1980). Uterine contractures, or non-labour contractions of the 55 uterus, occur at 30 to 60 minute intervals in the pregnant sheep (Harding et al, 1982). The initial minutes of a contracture are associated with increased fetal carcass blood flow (Llanos et al, 1986; Parer etal, 1984) and oxygen consumption (Llanos etal, 1988) suggesting that there is increased fetal muscle activity. Nathanielsz (1982) has shown that the duration, frequency, and associated rise in uterine pressure of contractures are unchanged when the fetus is paralyzed with pancuronium. Thus fetal skeletal muscle activity does not appear to be a factor in the initiation of contractures or the associated change in sleep state. Uterine contractures result in mild hypoxemia as evidenced by a fall in Pao2, rise in arterial and venous pressures, and bradycardia (Brace and Brittingham, 1986; Harding et al, 1983; Jansen et al, 1979). Cessation of fetal breathing and rapid eye movements have been observed with the onset of a contracture (Harding etal, 1981). This abolition of activity may be a result of the mild hypoxemia. Many reports emphasize the significance of behavioural state on fetal functions. Fetal micturition or voiding is more prevalent during low voltage ECoG activity; however, bladder drainage to abolish spontaneous voiding did not alter the duration or number of low and high voltage ECoG episodes (Wlodek et al, 1989). Thus voiding appears to be linked to electrocortical state but bladder contractions and voiding do not alter the normal cycling of ECoG states. Episodes of fetal swallowing are associated with low voltage ECoG and periods of arousal (Harding et al, 1984). Fetal heart rate and arterial pressure are lower during the low voltage state in comparison to observations made during high voltage activity (Clapp et al, 1980). In addition, cerebral blood flow is greater during low voltage ECoG episodes (Jensen et al, 1986; Richardson et al, 1985). Fetal animals exhibit a high proportion of low voltage ECoG with REM activity which, as this state is associated with increased cerebral metabolism, may contribute to the rapid prenatal growth and development of the brain (Richardson et al, 1985). Walker el al. (1984) did not find a relationship between ECoG state and fetal oxygen consumption, but oxygen consumption does increase significantly during fetal breathing episodes (Rurak and Gruber, 1983a). 56 Booking et al. (1985) have demonstrated a very close relationship between gross body movements and fetal heart rate accelerations in sheep. This relationship has been used clinically as an assessment of fetal well-being. The non-stress test monitors fetal movements and heart rate with Doppler ultrasound. A healthy fetus will generally exhibit at least two heart rate accelerations in a 20 minute interval, defined as a reactive non-stress test (Hacker and Moore, 1986). Some women also undergo a contraction stress test in which a low dose of oxytocin is given to induce several uterine contractions. A healthy fetus will , respond to these contractions with heart rate decelerations. Fetal breathing and body movements may also be assessed by ultrasound. The biological significance of alterations in behavioural state is unknown (Szeto, 1992). However, many forms of fetal distress are associated with reductions in skeletal muscle activity. Acute hypoxemia, for example, is associated with a reduced incidence of low voltage electrocortical activity, a virtual abolition of REM, and an inhibition of fetal breathing (Hohimer and Bissonnette, 1991) and limb movements (Natale etal., 1981). It has been suggested that this reduction in movements and breathing activity is a compensatory mechanism that lowers oxygen requirements when the fetus is faced with a reduction in oxygen supply (Richardson, 1989; Harding etal., 1983; Rurak and Gruber, 1983a). This is supported by evidence showing a reduction in oxygen consumption when gallamine triethiodide or pancuronium bromide, neuromuscular blocking agents which inhibit both breathing and body movements, are administered to the fetus (Wilkening et ai, 1988; Rurak and Gruber, 1983a). Blood flow to the umbilical and abdominal veins may be modulated by fetal breathing movements, suggesting that peripheral blood flow may be modulated by behavioural state in both sheep (Hassaart and de Haan, 1985) and humans (Marsal et ai, 1984) . However this may not be a direct relationship to behavioural state but rather to the phasic changes in intrathoracic pressure with breathing activity (Hassaart and de Haan, 1985) . We have previously shown in our laboratory that high voltage ECoG activity is 57 associated with higher fetal hindlimb blood flow and heart rate as well as decreased hindlimb vascular resistance when compared to the low voltage ECoG state (Rurak et al., 1992). The mechanism responsible for the relationship between blood flow and behavioural state may be a direct central nervous system one. This is suggested by evidence that the fall in umbilical blood flow with quiet sleep precedes the switch from low to high voltage (Slotten et al, 1989; Walker et al., 1984). A second explanation for this relationship may be that skeletal muscle activity, and thus increased oxygen demand, is modulating blood flow. As quiet sleep is associated with body movements of longer duration and more complexity than during REM sleep (Natale et al, 1981), it seems likely that quiet sleep would be associated with a greater oxygen demand by the tissues involved. This leads to the postulate that such movements would elicit an increase in blood flow in the fetal lamb hindlimb during quiet sleep. The administration of a neuromuscular blocking agent, which would abolish skeletal muscle activity, can be, used to help elucidate whether the relationship between state and blood flow is a direct one or a result of skeletal muscle activity. Previous studies have shown that neuromuscular blockade decreases total body and hindlimb oxygen consumption and hindlimb blood flow (Wilkening etal, 1989; Wilkening etal, 1988; Rurak and Gruber, 1983a). However, the effect of neuromuscular blockade on hindlimb blood flow modulation with electrocortical state does not appear to have been examined previously. One of the objectives of our study was to administer neuromuscular blocking agents to the fetus in order to abolish skeletal muscle activity. This will allow us to determine if these movements are responsible for the fluctuations in hindlimb blood flow that occur in relation to fetal behavioural state. In addition, we attempted to obtain blood samples during high and low voltage ECoG state both pre- and post-drug administration in order to determine the effect of behavioural state and neuromuscular blockade on hindlimb oxygen consumption. 58 5.2 Methods Surgery was performed on 7 fetal lambs between 120-127 days gestation (mean = 125 ± 1 days) using the techniques previously described (Section 2.3). Catheters were placed in the amniotic cavity, and in the fetal femoral artery, lateral tarsal vein, and pudendoepigastric artery and vein. Hindlimb blood flow was continuously measured with a transit-time flow transducer on the external iliac artery. Electrocortical (ECoG) and electro-ocular (EoG) electrodes were implanted for determination of fetal behavioural state. A total of 8 experiments were performed at a mean gestational age of 132 ± 1 days (range = 126-137 days). Neuromuscular blockade was achieved by a bolus injection of either 15 mg gallamine triethiodide (Flaxedil®, Rh6ne-Poulenc Rorer Canada, Ville St-Laurent, Quebec) (n=5) or 2-4 mg pancuronium bromide (Pavulon®, Organon Canada, Scarborough, Ontario) (n=3) with the drug administered around noon (range 08:30 to 16:49). Blood samples were collected from the pudendoepigastric artery and vein during both quiet and active sleep prior to and following the drug bolus. Data were analyzed from 3 complete paired high and low voltage ECoG patterns (three sleep cycles) before and after drug administration for each animal. The data used were generally collected between 6:00 a.m. and 7:00 p.m. Electrocortical state was determined by visual inspection of the polygraph record with high voltage ranging from 125 to 175 microvolts and low voltage from 25 to 50 microvolts. Arousal or awake episodes (low voltage ECoG without REM), which are most prevalent between 1:00 a.m. and 7:00 a.m., were not clearly evident on the polygraph recordings and thus were not included in the behavioural state analysis. Electro-ocular activity was classified by the presence or absence of rapid eye movements. Fetal breathing movements were detected from the tracheal pressure polygraph trace. Alterations in tracheal pressure were considered fetal breathing episodes if they were at least 1 mmHg (1.36 cm water) in amplitude and had a minimum duration of 10 seconds. As the results from gallamine and pancuronium were similar, the data from both drugs were combined. 59 Data were compared using paired t-tests with values considered significant if p < 0.05 (StatView statistical software, Abacus Concepts Inc., Berkeley, California). 5.3 Results Polygraph recordings obtained prior to (Figure 10) and following neuromuscular blockade (Figure 11) show clear episodes of high and low voltage ECoG activity. The fetus spent 46.1 + 2.0% of the sleep cycle in the high voltage state prior to neuromuscular blockade compared to 49.9 ± 1.8% following blockade; this difference was not significant. Similarly, there was no difference in cycle length (34.7 ± 2.0 minutes pre-blockade; 28.8 ± 1.5 minutes post-blockade). Thus neuromuscular blockade does not appear to alter fetal ECoG patterns. Prior to blockade, the association between fetal breathing movements, obtained from the tracheal pressure trace, and low voltage activity is evident (Figure 10). During episodes of high voltage activity hindlimb blood flow appears to be higher and more variable. A recording from the same fetus following the administration of neuromuscular blockade (pancuronium) suggests that mean hindlimb blood flow and its variability, particularly during high voltage, are reduced (Figure 11). As expected, no fetal breathing movements are evident The effectiveness of the neuromuscular blockade was demonstrated by the total lack of fetal breathing movements evident on the polygraph recordings for at least 6 hours post-administration. It should be noted that on the polygraph charts arterial, venous, and tracheal pressures have not been corrected for changes in amniotic pressure. The sudden changes in these pressures on the polygraph are due to position changes of the ewe. In addition, uterine contractures are indicated in Figures 10 and 11. The contractures are associated with a change in state from low to high voltage and hindlimb blood flow is increased in the initial moments of the uterine activity. In the pre-blockade phase, average hindlimb blood flow was 33.6 + 2.4 mlmin"1- lOOg"1 with flow tending to be higher during high voltage (34.3 ± 3.8 versus 60 62 32.8 ± 3.2 mlmin"1- lOOg"1 in REM sleep) (Figure 12). Following drug administration, mean flow fell 8.3 ± 3.5% to 30.5 ± 2.3 ml-min"1- lOOg"1, but the decrease just failed to be statistically significant (p = 0.0510). After the elimination of skeletal muscle activity, hindlimb blood flow still tended to be greater in the high voltage state, but the difference was again not significant. Variability in hindlimb blood flow, expressed as coefficient of variation, was reduced from pre-blockade (7.2 ± 0.4%) to post-blockade (7.0 ± 0.5%), but this difference was not significant. Figures 13 through 16 illustrate the change in fetal cardiovascular variables prior to and following the administration of neuromuscular blockade. The values for the high (white bars) and low voltage (solid bars) electrocortical states as well as the average of these two states (diagonal pattern) are presented for before and after blockade. The data are given as mean ± standard error of the mean. Fetal heart rate was not different in high voltage (153.2 ± 7.0 bpm) compared to the low voltage ECoG state (150.4 ± 6.3 bpm) and, although heart rate tended to rise following the administration of gallamine or pancuronium, the changes were not significant (Figure 13). No difference in arterial pressure was seen between high and low voltage activity; however, mean arterial pressure did increase significantly following administration of the drugs (Figure 14). In the pre-blockade period, fetal hindlimb venous pressure was significantly lower in low voltage (5.1 ± 0.9 mmHg) relative to the high voltage state (5.9 ± 0.7 mmHg). Venous pressure fell significantly following neuromuscular blockade (Figure 15). As shown in Figure 16, hindlimb vascular resistance averaged 1.3 ±0 .1 mmHg/mlmin'MOOg"1 in the control period, and although resistance rose to 1.5 ± 0.3 mmHg/mlmin'1- lOOg"1 with neuromuscular blockade, this change was not statistically significant. Table 7 presents the blood gas status and metabolic variables obtained from the fetal hindlimb as a baseline as well as that obtained after the administration of neuromuscular blockade. The data represent only 7 experiments due to failure of the arterial sampling catheter during one experiment. All pre-blockade values were within the normal range seen O LO O LO ^ CO CO c\J ( t 6 0 0 L . T .U!LU . |Lu) M O | i pooiq qimipujH 64 65 CD T3 CC O O XI I •4—• CO o Q. CD T3 CO O O I CD k _ Q_ (6HWUU) ejnssejd reueyv 66 CD T3 CO o O X i CO o CL CD T3 CO O O XI I CD a 3 !r? III «•> ro aj > T3 /1> O CJ w CD 6 0 GO CO £ co W 0 0 a" 3 GO GO a a, GO 3 O 3 co > 3 O CO T 3 CS ^ CD o O > x: oo - T—» X J 6 o e CO GO CL, o GO ' CD M—, ° s o c o PL) 3 d co 3 > CO "8 3 X> 0 0 CO 0 0 n > o x i —.5 cd •a O - 3 go 0 0 s 6 CO o c a 0 0 c •c 3 T3 •"1 3 0 0 ° ^ 4 3 CO co p S3 a 3 GO GO CO C3 > GO B CCJ O •a c CD •a i n o d CXX> o (6HUUUU) ajnssajd snoueA 67 CD T3 CO o o Xt t -*—' CO o CL CD "D CO o o XI I CD 3 §| •c-S <u o o,x> 00 t*-u e <u CO c CO <U '3 o 1/5 on O « >> > O -a m c •2 w X! 00 1 2 ^ o CD "2 3 C •in jO -~ CO -H 3 o CO 3 3 *-• ~ c c5 - <u -a 6 <D  CO e 3 S UJ CD c3 vo 3 o § ( t .B00 I- .^.m w. | W / 6 H w w ) 80UB1SIS9J JB|nosBA qwnpuiH 68 TABLE 7. Effect of neuromuscular blockade on blood gas status and metabolic variables in the fetal lamb hindlimb (7 experiments). Pre- Post-Neuromuscular Neuromuscular Blockade Blockade PH 7.333 ± 0.010 7.346 ± 0.009 Po2 (mmHg) 17.6 ± 1.4 20.1 ± 1.5f Pco2 (mmHg) 52.0 ± 1.6 51.0 ± 1.1 Base excess (mEq/L) 2.2 ± 0.9 2.4 ± 0.8 Hemoglobin concentration (g/dL) 10.2 ± 0.6 10.2 ± 0.6 0 2 saturation (%) 38.9 ± 4.3 45.4 ± 5.3 f 0 2 content (mM) 2.48 ± 0.36 2.92 ± 0.44f Lactate flux (|/molminlOOg"1) -2.78 ± 0.80 -0.05 ± 1.21 Glucose flux (jrniolmin1- lOOg"1) 4.35 ± 0.60 4.11 ±0 .62 0 2 delivery—absolute value (jrniolmin1- lOOg"1) —percentage of pre-blockade value 75.2 ± 6.5 72.3 ± 6.4 (96.8 ± 5.6) 0 2 consumption-absolute value (//mol-min"1- lOOg"1) —percentage of pre-blockade value 18.6 ± 1 . 6 16.4 ± 1.6 ( 8 8 . 2 ± 4 6 ) f 0 2 extraction—absolute value (%) —percentage of pre-blockade value 26.0 ± 2.9 24.0 ± 3.2 (91.1 ±2 .2) t Values are mean ± SEM. Data are presented as absolute values except those in parentheses which are expressed as a percentage of the pre-blockade values, t indicates a significant difference from pre-blockade data (p < 0.05). 69 in our laboratory. After gallamine or pancuronium injection, significant increases in fetal Po2, oxygen saturation, and oxygen content were observed. Similar changes following neuromuscular blockade have been reported by other researchers (Chestnut etal., 1989; Wilkening et al., 1989; Wilkening et al., 1988; Rurak and Gruber, 1983b). Lactate and glucose fluxes were unaltered by gallamine or pancuronium administration. Although oxygen delivery did not change following neuromuscular blockade, oxygen consumption fell 11.8 + 4.6% from pre-blockade values (Table 7). There was also a significant fall in oxygen extraction to 8.9 ± 3.2% below the baseline value. No difference was found in oxygen consumption or extraction values between high and low voltage ECoG activity either before or after neuromuscular blockade. 5.4 Discussion Gallamine triethiodide and pancuronium bromide are competitive neuromuscular blocking agents and thus abolish skeletal muscle activity including fetal breathing and body movements. During the control phase, our fetuses spent 46.1 ± 2.0% of their time in high voltage ECoG activity with an average sleep cycle length of 34.7 ± 2.0 minutes. These values are similar to values previously reported for the late gestation fetal lamb (Ruckebusch et al, 1977; Dawes et al, 1972). The drugs did not alter normal ECoG patterns as shown by the lack of effect on sleep cycle length and the amount of time spent in each electrocortical state. Gallamine generally exhibits more ganglionic blockade effects than pancuronium although the latter drug may also cause some tachycardia and hypertension (Gilman et al, 1990). At the doses used in the current study, both drugs caused an increase in arterial pressure but heart rate was not changed. The ganglionic effects of the neuromuscular blocking agents thus appear to have been similar and the results from both drugs were combined. Mean arterial pressure rose from 47.3 ±1.2 mmHg in the pre-blockade period to 48.2 ± 1.2 mmHg post-blockade (p = 0.0164) while venous pressure was significandy 70 reduced from 5.5 ± 0.5 to 4.2 ± 0.5 mmHg (p = 0.0059). Fetal heart rate and hindlimb vascular resistance were not altered following neuromuscular blockade. Our results then are similar to previous studies which have reported a slight but insignificant trend for increased fetal heart rate and arterial pressure following gallamine (Rurak and Gruber, 1983a) or pancuronium (Wilkening et al, 1988) administration. Following administration of either gallamine triethiodide or pancuronium bromide, mean hindlimb blood flow fell 8.3 ± 3.5% which just failed to reach statistical significance (p = 0.0510). Hindlimb blood flow was reduced following blockade in 6 of the 8 animals. Wilkening's group (1988) reported a significant (12%) reduction in hindlimb blood flow after pancuronium administration. These data suggest that skeletal muscle activity is associated with greater hindlimb blood flow. Clapp et al. (1980) reported high voltage ECoG activity to be associated with significandy greater fetal heart rate, arterial pressure, and umbilical blood flow relative to the low voltage state. In a previous study in our laboratory, fetal heart rate was ~7% higher, arterial pressure ~3% greater, and hindlimb blood flow ~17% higher in the high voltage ECoG state compared to low voltage (Rurak et al, 1992). In the current study, during the baseline or pre-blockade period, fetal heart rate, arterial and venous pressures, and hindlimb blood flow were 150.4 ± 6.3 bpm,47.0 ± 1.8 mmHg, 5.1 ± 0.9 mmHg, and 32.8 + 3.2 mlmin'1- lOOg"1 during low voltage electrocortical activity. During high voltage ECoG, heart rate rose 2%, arterial pressure 1%, hindlimb blood flow 4%, and hindlimb vascular resistance 4%; however, the only significant change was in venous pressure which was 21% greater in high voltage (p = 0.0472). Thus, compared to previous studies, the direction of our changes were the same but the magnitude was not as great. The degree of difference between these results may reflect the way data were obtained. In our initial work (Rurak et al, 1992), the data were quite crudely obtained i.e., by direct visual analysis of the polygraph recordings without the benefit of a computerized data acquisition system. In addition, the episodes analyzed were selected so as to avoid events like postural changes by 71 the ewe and uterine contractures. We had the benefit of computer analysis for the data currently presented which increased the precision of our measurements. However, we did not specifically avoid contractures and postural changes, and these may be possible confounding factors in our results. As stated, in a previous study in our laboratory, we found hindlimb blood flow to be significantly greater during quiet sleep (Rurak et ai, 1992), and we attributed this to a greater oxygen demand required for the increased body movements that are associated with this state. This modulation of hindlimb blood flow with sleep state is also suggested by the polygraph recording (Figure 10). In the current study, pre-blockade hindlimb blood flow tended to be greater during high voltage ECoG activity than low (34.3 ± 3.8 versus 32.8 ± 3.2 mlmin"1- lOOg"1; p = 0.2200). Because of the failure of the current data to reach statistical significance, more data is required before we can conclude that hindlimb blood flow is higher during high voltage ECoG activity. However, in the pre-blockade period, 5 of the 8 experiments demonstrated greater hindlimb blood flow during high voltage. A power analysis indicated that 13 pairs of data would be required to demonstrate a 10% difference in hindlimb blood flow between high and low voltage activity, assuming that variance remained the same. Given that the trend for hindlimb blood flow to be higher during high voltage activity has been observed in many polygraph recordings made during this and other experiments, this trend appears to be consistent. Following blockade, hindlimb blood flow still tended to be greater with quiet sleep (31.1 ± 3.4 versus 29.9 ± 3.4 ml-min"1- lOOg"1; p = 0.0.0607). Again, the difference just failed to be significant, but this relationship was seen in 6 of the 8 experiments. Thus, although the data were not statistically significant, the results suggest that neuromuscular blockade did not alter the apparent behavioural state modulation of flow in the fetal lamb hindlimb. Hindlimb blood flow demonstrates marked inter-animal variability, i.e., in these experiments pre-blockade flow ranged from -17 to 49 ml-min"1- lOOg"1. The use of paired comparisons largely overcomes this, but the conservative nature of this method also makes it 72 more difficult for differences to reach statistical significance. The observed changes in hindlimb blood flow, expressed in absolute or percentage terms, are quite small. For example, the average flow difference between electrocortical states was ~1.5 mlmin"1- lOOg"1 or -5%, and the inhibition of skeletal muscle activity with neuromuscular blockade resulted in a 3.1 mlmin"1- lOOg"1 or ~8% fall in flow. With such small differences, animal condition, measurement variability of the flow transducer, and artifacts during data collection can easily sway the results. Thus, such differences may be physiologically relevant but fail to demonstrate statistical significance. The inability to demonstrate statistically significant differences in hindlimb blood flow between high and low voltage ECoG activity may also reflect the difficulty of precisely delineating high and low voltage episodes. Another possible factor is that, although quiet sleep is associated with more body movements (Natale et al, 1981), fetal breathing movements occur during low voltage electrocortical activity (Dawes et al, 1972). Both these forms of skeletal muscle activity could contribute to blood flow changes (Wilkening et al, 1989; Wilkening et al, 1988; Hassaart and de Haan, 1985; Rurak and Gruber, 1983a), and as neuromuscular blockade abolishes both breathing and body movements, their relative effects are difficult to discern. Thus, inhibition of fetal breathing movements may also contribute to the reduction in hindlimb blood flow following neuromuscular blockade. Such modulation of peripheral blood flow with fetal breathing activity, likely due to the changes in intrathoracic pressure, has been previously reported (Hassaart and de Haan, 1985; Marsal et al, 1984; Dawes et al, 1972). In addition, behavioural state was determined by visual analysis of the polygraph recording. The subjective nature of this method is prone to error in precisely delineating the beginning and end of each behavioural state episode. As well, the awake (or arousal) state, which is characterized by low voltage ECoG activity without REM and is associated with large amounts of fetal body movements, were not clearly evident on the recordings. The difficulty in detecting the awake state reflects its very short duration and the fact that is occurs only 5% of the time; thus it was not 73 identified in the behavioural state analysis. The awake state is most prevalent between 1:00 a.m. and 7:00 a.m., so we attempted to minimize the effect of this exclusion by analyzing data from the daylight hours. However, as the awake state occurs during low voltage electrocortical activity (albeit without REM), this state may have been included with the active sleep state (low voltage with REM), and thus would have the effect of masking the difference in hindlimb blood flow between true quiet and active sleep episodes. Elimination of this potential source of error would require the development of a computer program capable of objectively identifying quiet sleep (high voltage ECoG), active sleep (low voltage ECoG with REM), and awake periods (low voltage without REM). Such a system is currently not available. The polygraph records also suggest that hindlimb blood flow is more variable during quiet sleep (Figure 10) and that the amount of variability is reduced following blockade (Figure 11). In Wilkening's study (1988), although the average coefficient of variation in hindlimb blood flow fell from 7% to 6% following blockade, this was not statistically significant. Likewise, in our study, hindlimb blood flow variability fell from 7.2 ± 0.4% pre-blockade to 7.0 ± 0.5% post-blockade, and in particular, the coefficient of variation for the high voltage ECoG episodes fell from 7.1 ± 0.6% pre-blockade to 6.1 + 0.5% after drug administration; however, none of these changes were statistically significant. The lack of significance may reflect limitations in the way data were collected. Our computer acquisition system collects data in 10 second intervals and records the one minute average. Rapid variations in flow due to limb movements thus may not be adequately captured by this system. The current study suggests that with neuromuscular blockade, hindlimb blood flow still tended to be higher in quiet sleep. Thus, because hindlimb skeletal muscle activity does not appear to be responsible for the apparent modulation in flow with behavioural state, this relationship may be an effect directly mediated by the central nervous system (CNS). This is also suggested by the abrupt fall in common umbilical arterial blood flow that occurs 74 upon the transition from low to high voltage (Walker et ai, 1984). In their study the depression in umbilical blood flow with the high voltage state was transient, with flow reduced 14.3% at two minutes into the high voltage state and then slowly returning to low voltage flow values. Slotten and colleagues (1989) also found a transient but significant fall in flow measured at the common umbilical vein during the high voltage ECoG state. The decrease in umbilical blood flow preceded the transition from low to high voltage by about one minute. These changes, as well as our own findings, are consistent with a direct CNS effect on blood flow. The rapid nature of these flow changes argues against a response to altered metabolic demands of the ECoG states. It is difficult to suggest a logical explanation as to why umbilical and hindlimb blood flow would exhibit reciprocal changes with behavioural state. Both flows derive from the descending aorta making it unlikely that vasoactive agents could be responsible for the opposing effects on the umbilical and hindlimb vascular resistances. In contrast to the findings of Walker (1984) and Slotten (1989), Clapp et al. (1980) reported a 5 to 6% increase in common umbilical arterial blood flow during high voltage activity. The reason for these contradictory reports in the direction of umbilical blood flow change are not clear. All these studies employed electromagnetic flow probes and were conducted at similar gestational ages at which well differentiated behavioural state patterns had developed. The study by Clapp (1980) was done between 4 and 10 days post-operatively while Walker (1984) and Slotten (1989) began studies at 3 to 4 days after surgery. Thus in all cases the animals should have had sufficient time to recover from surgery. Until a consensus is reached as to the direction of umbilical blood flow change with ECoG activity, it will be difficult to reconcile the hindlimb and umbilical blood flow studies. The nuclei or group of nuclei responsible for electrocortical state is still uncertain (Dawes, 1992). It has been difficult to study the particular functions that make up behavioural states because many of these activities are not independent. For example, fetal breathing and body movements can alter blood flow and heart rate patterns making it hard to 75 discern which aspects involve direct input from the central nervous system (CNS). Following transection of the brain stem above the level of the pons, fetal electrocortical activity and breathing movements become dissociated and breathing movements become continuous (Dawes etal., 1983). Pharmacological agents including atropine (Moore etal, 1989), L-5-hydroxytryptophan (Fletcher etal, 1988),and ethanol (Smith etal, 1989) have also been used to disrupt the synchrony between electrocortical state, eye movements, and breathing activity. Thus it appears that electrocortical state, REM, and breathing activity are predominantly independent and may be controlled separately by an as yet unknown "state-generator" (Moore and Hanson, 1992). Our present study also suggests that the modulation of hindlimb blood flow observed with alterations in ECoG activity may be a direct action of the central nervous system rather than linkage of ECoG and skeletal muscle activity. In the present study, neuromuscular blockade, achieved either by the administration of gallamine triethiodide or pancuronium bromide, resulted in better fetal oxygenation. We observed increases of 15.3% in arterial Po2 and 17.3% in oxygen content compared to pre-blockade values. In addition, oxygen saturation increased from an average of 38.9 ± 4.3 to 45.4 ± 5.3% (a mean change of 17.1%). In a similar experiment involving gallamine administration, Rurak and Gruber (1983a) reported increases in arterial and venous umbilical Po2 values of 14% and 18%, respectively. In studies specifically looking at the fetal lamb hindlimb, a 17% increase in hindlimb arterial oxygen content was observed following gallamine (Wilkening et al, 1988); while with pancuronium, oxygen saturation rose 11.1% and hindlimb arterial Po2 increased 9.5% (Wilkening etal, 1989). Therefore, the increase in oxygenation of the hindlimb following neuromuscular blockade that we observed is similar to that obtained in other hindlimb studies as well as in the fetus as a whole. The increase in oxygenation following neuromuscular blockade appears to be due to a reduction in total fetal oxygen demands. Rurak and Gruber (1983b) reported an average 76 increase in oxygen consumption of 30% during fetal breathing movements with the increase ranging from 13.9% to 69.0% in individual experiments. Administration of gallamine, which inhibits fetal breathing and body movements, led to a 17% reduction in total body oxygen consumption (Rurak and Gruber, 1983a). In the hindlimb, oxygen consumption was reduced 7.5% by pancuronium (Wilkening et al, 1989) and 12% with gallamine (Wilkening et al, 1988). In the present study, neuromuscular blockade was associated with an 11.8 ± 4.6% reduction in oxygen consumption. As demonstrated by Rurak and Gruber (1983a), the fall in consumption appears to be due to a reduction in oxygen extraction, which in our study fell 8.9 ± 3.2% from the pre-blockade value. No difference in hindlimb oxygen consumption was found between quiet and active sleep (16.3 + 1.4^mol-min"1- lOOg"1 versus 15.6 ± 1.4/imol-min"1- lOOg"1; p = 0.4389) in the pre-blockade period. This may reflect the small number of paired samples (n =4) that were collected due to difficulty in obtaining blood gas samples during clear episodes of high and low voltage activity. Often a fetus would appear to be in a quiet or REM sleep episode but by the time the blood gas sample could be collected, the sleep state had ended or a contracture caused a switch in state. Movements of the fetal forelimbs, including simple flexion or extension and more complex, longer duration activity, have been quantified as occurring during 14.3 ± 0.9% of high voltage activity with each movement averaging 4.55 ± 0.59 seconds (Natale et al, 1981). Bursts of activity occur every 20 to 35 minutes (Natale et al, 1981). Our observed lack of difference in hindlimb oxygen consumption between ECoG states could also mean that movements of the hindlimb during the high voltage episodes were either of insufficient duration or complexity to increase oxygen consumption. In conclusion, the late gestation fetal lamb normally exhibits cyclical alterations in behavioural state including electrocortical activity, electro-ocular activity or eye movements, and breathing and body movements. Neuromuscular blockade, achieved either with gallamine triethiodide or pancuronium bromide, abolishes fetal breathing and body 77 movements. These drugs did not alter sleep cycle length or the amount of time the fetus spent in the high voltage ECoG state. Following neuromuscular blockade, mean hindlimb blood flow was reduced by 8.3 ± 3.5% suggesting that skeletal muscle activity is associated with greater hindlimb blood flow. Although the differences were not statistically significant, blood flow to the hindlimb tended to be greater during quiet sleep both before and after the administration of gallamine or pancuronium. These data suggest that abolition of skeletal muscle activity by neuromuscular blockade did not alter the apparent behavioural state modulation of hindlimb blood flow, and thus, this may be an effect directly mediated by the central nervous system. The increased variability in flow during quiet sleep observed on the polygraph recordings appeared to be reduced following drug administration. However, this decline in variability was not found to be statistically significant, perhaps because our data acquisition system did not record data frequently enough to capture the rapid variations in blood flow associated with hindlimb activity. Neuromuscular blockade was associated with a significant increase in fetal oxygenation and an 11.8% fall in oxygen consumption. This suggests that inhibition of skeletal muscle activity, such as occurs during fetal hypoxemia, may be a compensatory mechanism that lowers fetal oxygen demands when faced with a reduction in oxygen supply. We were unable to show a statistical difference in oxygen consumption between high and low voltage electrocortical activity. This may reflect the low sample size or that the duration and complexity of the hindlimb movements during the high voltage episodes were insufficient to significantly increase oxygen consumption. 78 6. HYPOXEMIA AND ISCHEMIA 6.1 Introduction The relationship between systemic oxygen delivery (Do2) and oxygen consumption (V o2) in the fetal lamb has been examined in numerous studies in which oxygen delivery was acutely reduced by various methods, including: decreasing maternal inspired oxygen concentration (Rurak et al, 1990a; Rurak et al., 1990b; Cohn et al, 1974), reducing uterine blood flow (Jensen et al, 1991; Boyle et al., 1990; Wilkening and Meschia, 1983), and diminishing umbilical blood flow (Itskovitz et al, 1987; Itskovitz et al, 1983). Generally a curvilinear relationship is seen between the two variables with V o2 being maintained via an increase in oxygen extraction over a fairly wide range of oxygen delivery (Jensen and Berger, 1991 ; Carter, 1989). This compensation has been termed the oxygen margin of safety or oxygen reserve (Richardson, 1989). However, when oxygen delivery is reduced below ~50%, consumption begins to decline precipitously. As extraction rises, the Po2 of blood perfusing tissues downstream is reduced thereby imposing a limitation on this compensation. As well, when the fetus is compromised blood flow is redirected to the heart, brain, and adrenal glands at the expense of less vital tissues like gastrointestinal tract, skin, and muscle. When this occurs, significant fetal lactic acidemia develops which is thought to be due to anaerobic lactate production in fetal tissues and organs (Boyle et al, 1992; Iwamoto and Rudolph, 1985). In a recent study of acute fetal hemorrhage Kwan et al. (1995b) found a linear relationship between V o2 and Do2 with consumption falling significantly when oxygen supply was reduced by only ~15%. As delivery was further reduced, consumption continued to fall. However, the rise in fetal blood lactate concentration (to ~2.9 mM) was low in comparison to that seen in other studies in which oxygen delivery was reduced by lowering maternal inspired oxygen levels or uterine artery compression (lactate values of 79 -7-15 mM) (Boyle et al, 1992; Rurak et al, 1990b). They concluded that a reduction in fetal Do2 achieved via acute fetal hemorrhage is better tolerated than when caused by other means. This may be because with fetal blood loss, Do2 is reduced primarily by a fall in cardiac output and umbilical blood flow with minimal changes in vascular Po2. With the other methods of reducing Do2 the effect was primarily achieved via fetal hypoxemia. Since fetal arterial Po2 is low by postnatal standards, the oxygen gradient from blood to mitochondria may also be lower. Factors which reduce arterial Po2 nearer to that required to maintain the oxygen diffusion gradient between the blood and the inner mitochondrial membrane may jeopardize the fetus' ability to maintain normal oxygen consumption. Thus, during hypoxemia, which by definition is a reduction in arterial Po2, oxygen diffusion into fetal tissues may become a limiting factor. In contrast, with fetal hemorrhage this would not be as great a problem since the main factor reducing systemic and tissue 0 2 delivery is decreased perfusion. The greater degree of lactic acidemia that occurs with hypoxemia may be due to increased anaerobic glycolysis in those tissues in which 0 2 diffusion is impaired. As the carcass components receive ~40% of combined ventricular output (Rudolph and Heymann, 1970) and are subject to decreased perfusion with severe hypoxemia (Jensen etal, 1991; Boyle et al, 1990; Peeters et al, 1979), they could contribute to the systemic lactic acidemia Another potential site of increased lactate production is the placenta. Under normal conditions, the sheep placenta is a substantial lactate producer (Sparks et al, 1982). Although umbilical blood flow is maintained or even transiently increased during hypoxemia (Rurak et al, 1990b; Richardson, 1989; Peeters et al, 1979), the placenta increases the amount of lactate it releases into the fetal circulation (Milley, 1988). This also occurs during labour (Tan, 1997). Thus both fetal and placental tissues could be sites of increased lactate production during hypoxemia. The lesser tolerance of the fetal lamb to hypoxemia compared to hemorrhage may be due to the marked lactic acidemia that occurs 80 in the former situation. This further reduces blood oxygen content via a rightward shift in the oxyhemoglobin dissociation curve (Bohr Shift) (Rurak et al, 1990b). The objective of the present study was to measure the fetal cardiovascular and metabolic responses to hypoxemia, which reduces arterial Po2, and to hindlimb ischemia, in which Po2 is maintained. The hypothesis to be tested was that a fall in oxygen delivery achieved via hypoxemia will be tolerated less well than ischemia, and this will be reflected by an earlier fall in hindlimb oxygen consumption and/or greater release of lactate, and that the latter process will contribute to the systemic lactic acidemia. 6.2 Methods Surgery was performed on 11 sheep between 118-130 days gestation (mean = 124 ± 1 days), using the techniques previously described (Section 2.3). Briefly, catheters were placed in the amniotic cavity and in the fetal trachea, femoral artery, lateral tarsal vein, and pudendoepigastric artery and vein. A transit-time flow transducer on the external iliac artery continuously measured blood flow to the hindlimb. Periods of graded ischemia were achieved by partial inflation of the vascular occluder located proximal to the flow transducer. In some ewes a non-occlusive catheter was inserted into the maternal trachea for introduction of hypoxic gas mixtures. Hypoxemia studies were conducted on 7 animals between 124-136 days gestation (mean = 130 + 2). Hypoxemia was achieved by reducing maternal inspired oxygen content either by introducing nitrogen direcdy through a maternal tracheal catheter (n=4) using the technique of Gleed (1986) or by placing the ewe's head in a 0.25 m3 Plexiglas chamber (Figure 17) and introducing a hypoxic gas mixture of oxygen, 1.2% carbon dioxide, and nitrogen (n=3) as utilized by Rurak (1990a). The use of the Plexiglas chamber was introduced as we found it difficult to maintain low arterial Po2 values using the maternal tracheal catheter and, during one experiment, a ewe suddenly died upon introduction of the nitrogen into her tracheal catheter (see El 116 in Appendix). F I G U R E 17. Plexiglas chamber used to alter maternal inspired oxygen concentrations during hypoxemia experiments (figure from Rurak et al, 1990). Ewes were able to eat, drink, and lay down normally when their heads were inside the chamber. 82 During all experiments the behaviour of the ewes did not seem altered, and they continued to eat and drink as usual. The hypoxemia experiments were divided into a control period, a hypoxemia period of approximately 30 minutes, followed by a recovery period during which the ewe's breathed normoxic air (Figures 18 and 19). In experiments utilizing the Plexiglas chamber, the ewes first breathed room air (chamber open) for 30 minutes followed by a 30 minute period in which the chamber was closed and 40 L/min of air was circulated through it. Similarly, in these experiments, the initial recovery phase involved the ewes breathing 40 L/min air from the chamber for 30 minutes, followed by opening of the chamber and the ewes breathing room air. In all experiments, gas mixtures were allowed to equilibrate ten minutes before paired blood samples (~1 ml each) were taken from the pudendoepigastric artery and vein. For practical purposes we used change in fetal Pao2 as an estimate of the level of fetal hypoxemia, following the example of Akagi and Challis (1990b). We attempted to produce three levels of hypoxemia by reducing fetal arterial Po2 to approximately 3, 5, or 7 mmHg below control values. After each blood sample was taken, the fetus was transfused via the tarsal vein with an equivalent amount of maternal blood. The replacement blood was collected from the ewes in heparinized syringes prior to the start of the experiment. Ischemia experiments were conducted on 6 animals between 130-140 days gestation (mean = 137 ± 1 days). Following a control period, hindlimb ischemia was achieved by injecting saline into the vascular occluder through a 3 cc syringe mounted on a Harvard infusion/withdrawal pump. By continuously monitoring hindlimb blood flow with the downstream flow transducer, the infusion rate into the occluder could be adjusted to achieve the desired reductions in hindlimb blood flow. We attempted to achieve three groupings of ischemia by reducing hindlimb blood flow by 25, 50, or 75%. Once the desired level of occlusion was achieved, blood samples were taken at 10 minute intervals for approximately 30 minutes (Figure 20). This was followed by a 20 minute recovery period with the first recovery sample taken 10 minutes after release of the occluder. In 5 of the 6 83 CONTROL (room air) CONTROL (40 L/min air) HYPOXEMIA RECOVERY (40 L/min air) RECOVERY (room air) Chamber open to room air Chamber closed; 40 L/min air started Hypoxic gas mixture started Hypoxic gas mixture off; 40 L/min air started Chamber opened to room air FIGURE 18. Protocol for hypoxemia experiments involving Plexiglas chamber. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals). 84 CONTROL (room air) HYPOXEMIA RECOVERY (room air) Ewe breathing room air Hypoxic gas mixture started through ewe's tracheal catheter Hypoxic gas mixture off; ewe breathing room air FIGURE 19. Protocol for hypoxemia experiments in which hypoxic gas mixture was introduced through the maternal tracheal catheter. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals during hypoxemia and 20 minute intervals during the control and recovery phases). CONTROL ISCHEMIA (first level) Vascular occluder inflated RECOVERY Vascular occluder released ISCHEMIA (second level) Vascular occluder inflated RECOVERY Vascular occluder released FIGURE 20. Protocol for fetal hindlimb ischemia experiments. Ischemia was achieved by partial inflation of a vascular occluder on the fetal external iliac artery. In most experiments two episodes of ischemia were conducted separated by a recovery phase. At least 30 minutes was allowed between the end of the first recovery phase and the second ischemia episode. Arrows indicate fetal blood samples (taken at approximately 10 minute intervals). 86 expenments, this recovery phase was followed by a second 30 minute episode of ischemia (at a different level of occlusion) followed by another recovery phase. Thus a total of 11 ischemia episodes were studied. At least 30 minutes were allowed between the end of the first recovery phase and the beginning of the second ischemia trial. As with the hypoxemia experiments, an equivalent amount of maternal blood was used to replace fetal blood lost to sampling. Figure 21 illustrates a polygraph recording of a typical ischemia episode in which hindlimb blood flow was reduced to ~60% of the control value. Initially, blood flow is quite variable as the occluder is manually adjusted to achieve the desired degree of compression. Release of the occluder results in a transient hyperemia followed by a rapid return to normal flow values. Data from both the hypoxemia and ischemia experiments were analyzed by grouping the results based on change in hindlimb oxygen delivery (Do2), expressed as a percentage of control values. The interventions were classified as mild if oxygen delivery fell to 67-100% of control values; moderate when Do2 was between 34-66% of baseline; and severe as a Do2 below 34% of control values. For the hypoxemia study, 26 samples were collected during the control and recovery phases, 14 during mild (from 4 different fetuses), and 10 during both moderate (6 fetuses) and severe hypoxemia (3 fetuses). The hypoxemia levels were somewhat difficult to maintain; thus, in 2 of the 7 experiments samples corresponding to 1 level of hypoxemia were obtained, in 4 experiments samples were from 2 different levels of hypoxemia (the levels were adjacent i.e., either mild and moderate or moderate and severe), and from 1 fetus mild, moderate, and severe hypoxemia samples were collected during a single episode. From the ischemia group, a total of 12 control samples were obtained, 3 during mild ischemia (from 2 different fetuses), 9 during moderate (from 5 fetuses), 6 during severe (from 4 fetuses), and 18 during the recovery phase. The ischemia levels were easier to sustain as in only 1 episode were the samples collected equivalent to 2 different ischemia levels (moderate and severe). 87 3 3 <u _ 3 o L,J3 c 2 a 2 * »i u S; a a §•§ I a cx c C u s 23 3 ' 3 — m w cd c-C a l l ? * 5 ^ 5i 00 • CO 3 oo.~ -S.J5 - i= .2 _g 2 GO «J s? c «'S ^ c CO S . V Hi §11 •g § 0 & 2 .2 o -a _o 2 3* fi OO' GO > > 3 =5 <U 3 X JU <L) . > 3 O 2 c E . i « 2 j e a f 88 Data collected prior to the introduction of the hypoxic gas mixture or inflation of the vascular occluder were considered baseline or control data with each fetus serving as its own control. Control data are generally presented as absolute values, with other data calculated as either a change from or a percentage of the individual animal's control values. For analysis of the cardiovascular variables, the fetal cardiovascular data from 3 minutes before to 3 minutes after each blood sample (total of 7 minutes of data) were used. Data are generally presented as mean ± standard error of the mean. Data were analyzed with StatView statistical software (Abacus Concepts Inc., Berkeley, California). Changes in the variables during experiments were analyzed with one-way (factorial) analysis of variance (ANOVA) and Fisher's Protected Least Significant Difference (PLSD) post-hoc test. Repeated measures ANOVA could not be used as not all animals were exposed to the three levels of intervention. Differences between the hypoxemia and ischemia groups were compared using two-tailed unpaired t-tests. Differences in regression equations were compared by applying unpaired t-tests to the slopes and y-intercepts. In all cases, differences were considered significant if p < 0.05. 6.3 Results 6.3.1 Animal information Table 8 presents individual animal gestational age, fetal weight, and study limb weight at the time of experiment and at autopsy for the animals involved in the hypoxemia and ischemia studies. The hypoxemia experiments were conducted at a significantly earlier gestational age compared to the ischemia studies (130 ± 2 days versus 137 + 1 days). The number of fetuses carried by each ewe was the same in both groups. Furthermore, no difference was found between the two groups in terms of fetal weight or hindlimb weight at autopsy and on the day of study. 89 TABLE 8. Individual fetal age, weight, and study limb weight at autopsy and on day of study for the animals involved in the hypoxemia and ischemia studies. In the case of multiple lambs, weights are those of the operated fetus only. Ewe No. of Experiment Autopsy Number Fetuses gestational estimated estimated gestational fetal hindlimb age fetal hindlimb age weight weight (days) weight (g) weight (g) (days) (g) (g) HYPOXEMIA E3210 1 128 3708 273 142 5294 389 E2224 1 132 3466 255 137 3944 290 E1223 1 124 2444 180 136 3471 255 E1245 3 136 3005 210 139 3225 225 E967 2 136 2776 204 139 2979 219 E207HIN 2 124 1734 144 132 2218 184 E1154 1 132 3409 251 132 3409 251 Mean±SEM 1.6±0.3 130±2 2935±259 217±17 137±1 3506±359 259±25 S C H E M IA E1245 3 138 3152 220 139 3225 225 E722 2 138 3026 222 139 3096 228 El 16 2 139 3206 236 139 3206 236 E540 2 140 3798 279 142 3960 291 E136 1 136 3082 246 137 3158 252 E207HIN 2 130 2095 174 132 2218 184 Mean±SEM 2.0±0.3 137-hlt 3060±224 230±14 138±1 3144±226 236±14 t = significandy different from hypoxemia value (p < 0.05). 90 6.3.2 Blood gas and acid-base status Prior to the introduction of the hypoxic gas mixture for the 3 experiments using the Plexiglas chamber, blood samples were collected with the chamber open and the ewe breathing room air as well as with the box closed and the ewe breathing air administered at 40 L/min into the chamber. As there were no differences in the blood gas values between these two situations, the data were pooled as control samples. Similarly, we grouped together as recovery samples the data collected after the completion of the hypoxemia phase when the ewe breathed 40 L/min of air circulated through the Plexiglas chamber and when she breathed room air with the chamber open. The blood gas values and acid-base status for the 7 animals studied in the hypoxemia experiments and the 6 animals involved in the ischemia studies are presented in Tables 9 and 10. The control data (presented as absolute values) indicated no significant blood gas or acid base status differences between the animals which were to be subjected to hypoxemia or ischemia except for higher baseline arterial (3.27 ± 0.17 versus 2.70 ± 0.16 mM; p = 0.0384) and venous (2.47 ± 0.16 versus 1.94 ± 0.14 mM; p = 0.0310) oxygen contents in the ischemia study group. During hypoxemia, fetal arterial Po2 fell by 3.4 ± 0.5 mmHg during mild, 5.3 ± 0.5 mmHg with moderate, and 6.8 ± 0.3 mmHg during severe hypoxemia. During severe hypoxemia fetal arterial Po2 averaged 13.0 ±1.0 mmHg compared to the control value of 20.2 ± 0.6 mmHg. In addition, arterial oxygen saturation and content were reduced at each level of hypoxemia, falling by 19.0 ± 0.9% and 1.00 ± 0.04 mM, respectively, during severe hypoxemia. Fetal arterial Pco2 also fell with each level of hypoxemia. Arterial pH and base excess were maintained until severe hypoxemia when they fell significantly with pH falling by 0.094 ± 0.016 and base excess decreasing by 7.5 ± 1.0 mEq/L. As with the arterial values, with each level of hypoxemia venous Po2 was significantly reduced reaching values 2.8 ± 0.5, 5.5 ± 0.6, and 8.3 ± 0.4 mmHg below control (to 14.9 ± 0.4, 12.6 ± 0.9, and 7.4 ±1.2 mmHg). Venous oxygen saturation and 91 5* tt O U W tt ti tt ti ti cc d d -H +| O —, d ^ CO d +i 00 vd in d -H 00 CO VO o d -H I/O o VO T—( o d -H OS o d T q T OS i — i d d +i +l +i o r-H 00 d I in -H WO o +) o OS d +i o OS IT) o d +i r - H d o +i o o H— O d d d d •H +l +i +i -H VO 00 CO CN d • • o 1 d I d • 4— CN CN _l 00 O <N *—I d d d t—H d -H +) +i -H CO CN CO T—I 00 1 "St , — i 00 1 d 00 d +l 00 -H o vd s d +i in CO d b d +i OS d ti H ti Q 0 in d d -H +) co in r-3 Os o o d +i 00 o d vo d Os CN d +1 wo d d +i -H vo vd CO Os vo d •H in +l CN d Os o o d +l 8 o d vo d -H CN CN CN d -H in d d +i •H vd Os Os Q -J 0 tt H Z o u CO in CO o o CN o H— CO d d d d d -H +1 -H •H -H "fr co CO O CO co' • 1 o o d d d vo d +i CN d CN in d -H O os o o o d -H O CO CO d +i in d o +) o d "fr CN -H in 9 o +l vo vo d -H O r-CN in d +i oo CN in d •H "fr vd CO d -H d vo d -H 00 in o o o d +l o o o d o o d -H CN CO s CN n— CN d d -H -H +1 VO- T - H VO d d Os 00 o d +i 3 d "fr d d CN d -H -H +1 +l CO o "fr d Os r — 1 CO tt ti H tt oo S 8 00 e e CL, s GO <U o X CQ ~ob "5' o e u o c 8 c o *ob o 6 <u X a o 1 00 CO 0 z ti 00 X B S 00 X X c x B GO (D CJ X o o PQ ~ob cT" o c <u o § o e X) o "ob o S <u c o I GO c § 92 CO £3 > o u w w CG H w Q O Q O H Z o u o CO i n o CO 00 d d d d d d +i -H -H +l -H •H -H CN i n CN CO CO 00 d d o o O d "* CO d > d ON VO q 8 i n vcT d r - H d d d CN d -H -H -H -H •H +i CX) CN i n CN O o T — l O d d v i d i d • o CO CO q o CN ON o d d d d d d -H -H •H -H +l -H +1 CN CN r~- i n CN O N d i d o o d i d d • d d i i r -VD o CO CN o O N CN d d d d >-< d +1 -H -H +i +i +i +l 1—( q CO i > i - H 1— I 1 d O o d I i CS O d • i T—1 o vo o i n O N 1— I d d d d CN d •H -H +i -H +l -H +1 CN CO O N 00 CO i > i — i <M 3 CO d i d ON CN CO CN d +l vo i n d CN VO d +i 00 i n o ON o CO d d d •H +i -H +i i n vo O CN d I o o d d d i +1 o CO 00 d +i i n o o d +i ON o d o -H i n o d i CO CN d -H CO d CO d -H co d co i n i — i -H 00 i n 4— O vo q O i n CN d d d d CO +i -H -H -H +i +1 1 . — < CO vo CN i n (N i o o d d i VD O N O O +1 d < 4— CN o d CN d -H +i i n CN d • 00 1 vo d .—( CN d d -H •H +i i 1 ON o 1 s 1 d CN d -H d O N 00 Y—1 o 00 i n q vo d d d d d CO d -H +l -H -H -H -H -H vo o O r—i O N CO CN ON i — ( d l > >n CN i i — i CO CN < a: w H oo 00 X B B CN I X cx >-J 6, 00 Vi <D CJ X u <u ~oo o a <u o § o a -S o o E CD o 1 co a c o CJ o co O z w 00 E 6 00 E CN I C X & Ei CO oo 8 <u S3 OQ J 33 ~ob "c' o c CJ § CJ c IS o •5b O E <u X a o c o o O O 93 0 2 content also fell with each level of hypoxemia reaching values of 16.0 ± 1.4% and 0.91 ± 0.07 mM below the control values, respectively, during severe hypoxemia. The severe perturbation was also associated with a significant rise in venous Pco2 (by 1.7 ± 0.8 mmHg), and reductions in pH (by 0.142 ± 0.022) and base excess (by 8.3 ± 1.2 mEq/L). During the recovery phase mean arterial and venous Po2 values returned to baseline levels; however, the other variables remained significantly different from the control levels. In contrast to hypoxemia, fetal arterial Po2, pH, base excess, and O z saturation declined with increasing levels of ischemia but none of the changes were significant. Venous pH and base excess were also not statistically affected by reducing hindlimb blood flow. Arterial oxygen content did fall significantly with severe ischemia reaching 0.41 ± 0.16 mM below the control value and remained low during recovery. Venous Poz was reduced by 2.1 ± 0.6 mmHg during mild, 1.8 ± 0.6 mmHg during moderate, and 2.1 ± 0.5 mmHg during severe ischemia (to 16.7 ± 1.3, 16.2 ± 0.7, and 14.3 ± 1.2 mmHg, respectively). Similarly, drops in venous 0 2 saturation and 0 2 content were observed, with average reductions of 8.5 ± 2.0% and 0.62 ± 0.12 mM, respectively, during severe ischemia. During the recovery phase, these latter 3 variables remained below baseline levels. 6.3.3 Hindlimb blood flow, oxygen delivery, and oxygen consumption The data on hindlimb blood flow, and oxygen delivery and consumption are given in Table 11. Data collected during the control periods are expressed as absolute values. In order to facilitate comparisons, the remaining data (mild, moderate, severe, and recovery phases) are expressed as mean (± SEM) percentages of the individual animal control values. In the control period, blood flow in the ischemia animals (13.8 ± 0.9 mlmin"1- lOOg"1) was significantly lower than in the hypoxemia group (28.9 ± 1.8 mlmin"1- lOOg"1). This difference was accompanied by significandy lower values for 0 2 delivery (44.3 ±3.1 versus 71.4 ± 1.7 /^ molmin"1- lOOg'1) and consumption (10.7 ± 1.3 94 >< tt ti > O u ti tt ti tt ti >• ti ti H tt ti Q O Q -J O tt H Z o u o 4— VO o o C O CO vd vi -H -H +1 +l O N vo q d <—i ci 0\ CO o T—t f-H T - H vo CO in •< f r CN "t -H -H +i in «n CO 00 d d C O CN co O T 3? o x e x> '3 s ? ""3 S c w 3 00 o o I ' s § 1 X o l d ° § e co g MS, >^ •—' X O ON CO 3 4— 00 4— CN 4 — CO CN <n vd -H -H -H -H CN 00 o d wi T - H vo r-« CN in CN CO CN vd vd -H -H +i +i vo \r C O in ON ON ON T—1 ON 2; %—( 00 "fr T - H T - H +) -H -H +1 O N T - H CO 00 i - H , - H O N CN CN CN {£ c o X co c co 00 >, X o CN "fr -H 00 ot o C/2 o - r 3? o s "fr -H C O 00 in i> +l ON CO o ON T 1 CO d CO T - H -H +1 -H 00 C O CO 5 d T — « 00 o o | . ' s To £ § 1 £ 3 x O x O ON vd +i 00 "fr CN 00 00 4— ON T—| <—I <-< C N +1 •H -H -H in O vo VD ON* C N vd C N C N "fr d vo T 1 vo" 4— C O 4— VO 4— C N C N 00 -H -H +1 -H "fr o CO co d in ? vi in 4— CO d -H "fr 4— T - H co •H .1 ± 11.0 4— O N vi T - H +1 rS r> C O i—1 O N 2; vo CN -H "fr "fr. CN o '+3 1 X co E i CO 00 X o w &o -H ! 73 > 95 versus 21.4 ± 1.4//mol-min"1, lOOg"1). However, oxygen extraction was similar in both groups (24.4 ± 2.6 versus 29.3 ± 1.7%). During the ischemia experiments, hindlimb blood flow fell as expected to 77.4 + 0.3% of control values for mild, 50.4 + 2.6% for moderate, and 29.5 + 1.8% for severe ischemia. In most cases, there was a transient hyperemia following release of the occluder (Figure 21). The hyperemia was too brief to be reflected in the mean flow values during the recovery period which did not significantly differ from the control data. With mild hypoxemia, hindlimb blood flow was not altered. However, with moderate and severe levels, flow was reduced to 71.2 ± 4.1 and 38.5 ± 4.6% of the control level respectively. With mild hypoxemia and ischemia, the ~25% reduction in Do2 was not associated with a fall in 0 2 consumption. However, with moderate levels of perturbation, the reduction in delivery to 50.8 ± 2.8 and 48.0 ± 2.3% of control values for hypoxemia and ischemia, respectively, was accompanied by V o2 values that were 75.4 ± 5.2 and 74.3 ± 8.6% of control. With severe hypoxemia and ischemia, there were further reductions in both oxygen delivery and consumption. Although both variables were reduced to lower values with hypoxemia (20.5 ± 2.3 and 30.3 ± 4.5% for Do2 and V o2, respectively), compared to ischemia (26.0 +1.8 and 42.6 ± 7.9%), the differences between these 2 groups was not statistically significant. In both groups, oxygen extraction rose progressively as 0 2 delivery was reduced. Although the rise tended to be greater with ischemia (to 160.6 ± 21.3% of control) than with hypoxemia (143.3 ± 9.1%), again the difference was not statistically significant. Figure 22 plots the relationship between oxygen delivery and consumption, with both the individual measurements and the mean values for mild, moderate, and severe reductions presented. There was no difference in this relationship for the two perturbations. 6.3.4 Lactate and glucose concentrations and fluxes Arterial and venous glucose and lactate concentrations as well as hindlimb glucose and lactate fluxes are presented in Table 12. The control values and the flux data are 96 97 « W o u w tt w tt w t> w CO H tt ti Q O Q O tt H Z o u o & a CO d -H ON VO CO 3 d +i o l> CO vo CO d +i vo d -H uo d a, a 7 3 •c s i n CO d +l o 00 co o vo d -H 00 00 C O "fr CO d -H 5 3 < N d +i o i i (Z> 3 O 3 CD > i n O N +1 O o ci C N ON d < N C N i n o d -H 00 o o -H C O co d oo -H 00 O O -H 00 o 00 o o 3 2 B o B 3 7 3 s CO 3 •c < i n o d -H s d 2 ~ d -H o d < N CO d -H 00 CO "fr ON i n d -H VO vo "fr 3 d -H O N O 8 T—4 00 vo O 8 CO CO d d d d d d +1 -H -H -H -H -H 00 CO i n T - H 00 q i n ON O i n i—i T - H 1—1 T - H d CO o o . CO 3 o c CD > 00 o o 3 73 8 a U co vo T - H d -H ON C N "fr CN d +1 T - H d 00 T - H d -H 3 d CO d -H i n s 0 00 vo T - H d d d -H +1 +1 +1 00 VO 00 CN O 0 q "fr t d d C N VO -H i n < N vo O d +i o C O O N 0 00 O 0 VO C N d d d O +1 -H +1 •H -H i n , _ ( C N 1—1 O VO 0 O T - H i n C N 1 d d f CO d CN CN d -H 3 d CN T - H d -H CN O CN* O T - H d -H "fr CN -H CO B 3 7 3 •c T - " ^ 00 *p 7 5 a 3 , 3 d -H J so 3 O 3 CD > Cj 73 •c -8 < m o d -H VO o m o d i n vo O O CO 3 O 3 CD > CN C O 0 -H O N T - H vo T - H CN co 0 T - H T - H d d d +1 +1 +1 +1 CO i n r> 0 0 i> d d d d CN "fr CN CN 00 T - H wo CO 0 VO O d d d T - H d d d +i -H +1 -H +1 +1 +1 CN t> i n CN vo CN O O N O P CN d 1 d CO* 1 d d 0 CN r-d +1 b 00 o o S '3 S B o a a, I << 8 s CD • co Tib 8-75 11 , ~ 3 o a 8 ^ a !^ 0 H 2 w I 8 s> * 9 y Is XJ g-O CD CD *~* CD c3 c3 • 3 I S G <g - H 00 00 3 II ' t o ' 5 o 7 3 - 7 II II 98 presented as absolute values while the remaining data are expressed as the mean (± SEM) change from the individual animal control values. During the control period, arterial glucose concentrations were significantly higher in the hypoxemia experiments than in the ischemia trials (1.08 ± 0.06 versus 0.74 ± 0.04 mM; p = 0.0005), while lactate levels were lower (1.42 ± 0.09 versus 2.02 ± 0.12 mM; p = 0.0005). With increasing severity of hypoxemia, both arterial and venous lactate levels rose progressively to values of 3.70 + 0.54 and 3.88 ± 0.60 mM above control levels, respectively, during severe hypoxemia. There was also a rise in arterial glucose concentration during severe hypoxemia (to 0.33 ± 0.10 mM above control), but venous levels were not significantly altered. With ischemia, the arterial lactate concentration was minimally altered, reaching 0.71 ± 0.24 above control values, while hindlimb venous lactate concentrations peaked at 1.01 ± 0.37 mM above control levels during severe ischemia. In both groups there was a net uptake of lactate and glucose during the control period. Control glucose uptake was higher in the hypoxemia group compared to the ischemia animals (3.50 + 0.33 versus 1.07 ± 0.72 /miol-min"1- lOOg"1), but control lactate uptake was not different between the groups (1.15 + 0.81 for hypoxemia versus 3.37 ± 1.24 for ischemia). Glucose continued to be taken up by the hindlimb during both hypoxemia and ischemia at rates that were not different from the control values. Glucose flux was higher during moderate and severe hypoxemia than the corresponding levels of ischemia. In contrast, lactate tended to be released from the hindlimb during and following both hypoxemia and ischemia. This net release of lactate from the hindlimb was observed at all levels of hypoxemia, although the lactate efflux did not increase with the severity of hypoxemia. Moreover, none of the changes were significantly different from the control value. However, when the pooled mean lactate flux during hypoxemia (-3.12 ± 0.87 /miol-min"1- lOOg"1) was compared to the control value (1.15 ± 0.81 ^mol-min"1- lOOg"1), the difference was statistically significant. During ischemia, there was not consistent lactate 99 release from the hindlimb, and none of the changes were different from control. As with hypoxemia, the pooled mean lactate flux during ischemia (-0.25 + 0.41 ^molmin"1- lOOg"1) was significantly different from the control value (3.37 ± 1.24 //molmin"1- lOOg"1). At each level of perturbation (mild, moderate, and severe) there was no significant difference in lactate flux between the hypoxemia and ischemia experiments. For example, lactate efflux during severe hypoxemia (-2.24 ± 0.92 ^mol-min"1- lOOg"1) was not different from that seen with severe ischemia (-0.25 + 1.62 ^molmin"1- lOOg"1). However, when the pooled mean lactate fluxes were compared, lactate efflux was significantly greater with hypoxemia than with ischemia. During the ischemia recovery period, there was significant lactate release from the hindlimb (-4.28 ± 1.75 /^ molmin"1- lOOg"1), whereas in the hypoxemia recovery phase, although there was net lactate efflux (-2.00 ± 1.95 ^molmin"1- lOOg"1), it was not different from the control value. 6.3.5 Cardiovascular Responses The cardiovascular responses to hypoxemia and ischemia are presented in Table 13. Once again, the control data are expressed as absolute values while the remaining data are presented as mean percentages (± SEM) of the individual control values. During the control period, hindlimb vascular resistance was greater in the ischemia animals compared to the hypoxemia group (3.6 ± 0.5 versus 1.7 ± 0.1 mmHg/mlmin"1- lOOg"1). In contrast, hindlimb blood flow and heart rate in the ischemia animals (13.8 ± 0.9 mlmin"1- lOOg"1 and 147.3 + 3.7 bpm, respectively) were lower than in the hypoxemia group (28.9 + 1.8 ml-min"1- lOOg"1 and 162.8 ± 2.5 bpm). Fetal heart rate fell slightly with moderate hypoxemia (to 91.6 + 3.8% of control). Bradycardia (77.0 ± 2.4% of control) was also observed with severe hypoxemia along with increases in vascular pressures (119.9 + 3.4 and 153.3 ± 6.5% above control for arterial and venous, respectively) and hindlimb vascular resistance (to 347.6 ± 49.6% above control). No change in fetal heart rate or arterial pressure occurred with ischemia, but 100 OS u l> o o w OS w OS CO H OS w Q O Q J O OS H Z o u WO H— 00 4— o vq CO ci co •H -H -H -H ON Tf 00 d § § ol VO +1 in 00 co T f •H CN i—i oq -H ON 00 cN 00 o o o OQ ci -H O oq CO •H vq »—i ON in ci -H 00 ci VD B 3 1 CO -H ON ON 00 ci +1 s OO X B B IT 3 co co a CX -a •c in VD +1 CO in in oo -H O o oo X B B 3 co Vi CX CO 3 s <U > vo in +i Cl CO $ -H VO CO ON ci -H in CJ CN —1 r -co ci i n vd -H +1 •H -H -H VO CO ON Tf 00 ON ON ci o d O i—i ON 00 £ 00 o o 8 a i B on 3 3 * 1 a 3 a C) r—1 00 CJ T f ci Tt -H "H -H -H 00 CO o VO 8 ci o i—i vi i—i ci <—i .—i oq +) in ON o Tf ON ci ci ON -H -H M ON 00 q 00 ON ci o CO C4 i—1 VD ci -H •<* d in d ^ £ 2 ON i n ci d .—i +1 -H -H q CO C l CO CO CO o o i—1 5 o CO 00 3 a a, o CQ a 3 3 a s e r-+1 VO 8 1—1 CN ON ( N 1 d d d CO CN d -H -H -H +t +) -H -H * " H oq CO ON O in Tf i—i TC 00 X a B IT 3 co QO CX •n fi < oo a a 3 co CO 3 O 3 <U > S co -H r -in oo CO in CO -H ci CN in d -H VO CO oo 8 S I a •22 c co 3 3 s t d & CO OJ 3 ><n c<J O Ii o 3 a o 5 ° £ a CD P • 3 IS H 00 Ci I co c2 S 3 6 .1 J S SMI CJ CO CO t j h a ! 101 hindlimb resistance increased markedly, reaching levels 385.7 ± 37.7% above control values during severe ischemia. The increase in resistance seen with hypoxemia and ischemia appears to be mainly a reflection of decreased hindlimb blood flow. 6.4 Discussion 6.4.1 Hypoxemia and ischemia levels In 10 of the 11 ischemia episodes only 1 level of ischemia was tested while during the remaining episode samples from both moderate and severe ischemia were collected. However, in only 2 of the 7 hypoxemia experiments were samples from a single level of hypoxemia obtained (in 4 experiments samples were from 2 levels of hypoxemia while in 1 experiment all 3 levels of hypoxemia were examined). Thus, fetal hindlimb ischemia, achieved by partial inflation of the external iliac artery occluder, was easier to sustain than hypoxemia, whether achieved via a maternal tracheal catheter or the ewe breathing a hypoxic gas mixture in a Plexiglas chamber. This difference reflects the local nature of the ischemia i.e., it is limited to one hindlimb of the fetus. In contrast, hypoxemia affects the whole body and, furthermore, both the ewe and fetus experience reduced levels of oxygenation. Thus, alterations in uterine blood flow, umbilical blood flow, skeletal muscle activity, fetal or maternal cardiac output or oxygen content are all variables which could influence the level of fetal hypoxemia achieved at each sampling time. 6.4.2 Blood gas and acid-base responses The control blood gas results obtained prior to hypoxemia (Table 9) or ischemia (Table 10) are typical of those obtained in our laboratory and in others. Although arterial oxygen content was significantly lower in the hypoxemia group (2.7 ± 0.2 mM) compared to the ischemia animals (3.3 + 0.2 mM), both of these values fall in the range of values previously reported for the descending aorta/femoral artery (Kwan et al, 1995b; Boyle et al, 1992; Boyle et al, 1990; Rurak et al, 1990b; Rurak et al, 1990a; Wilkening et al, 102 1988). As expected from our experimental protocol, fetal arterial Po2 fell significantly with each level of hypoxemia reaching values of 6.8 + 0.3 mmHg below the control level during severe hypoxemia. These changes were associated with reductions in fetal arterial and venous oxygen saturation and content. Severe hypoxemia was also associated with reductions in arterial pH and base excess values, the latter falling 7.5 ± 1.0 mEq/L below the control values. As arterial Pco2 levels were unchanged or fell slightly there was no evidence of fetal asphyxia. Blood gases collected following hypoxemia indicate that Po2 quickly returned to control values and the other variables were returning more slowly to the pre-insult levels. In contrast to the hypoxemia animals, arterial Po2 did not change with ischemia. Furthermore, arterial and venous Pco2, pH, and base excess levels were unaltered even with severe ischemia A minor fall in arterial oxygen content was observed during severe ischemia (to 0.4 ± 0.2 mM below the control value). This change is likely a reflection of the slight, but not significantly different, reductions in hemoglobin concentration and oxygen saturation from which 0 2 content is derived. The total amount of blood taken from the fetus during each experiment was about 20 ml or, assuming a fetal blood volume of 110 ml/kg in late gestation (Brace, 1983), approximately 6% of fetal blood volume. However, the fetal volume lost during each sampling (2 ml or ~0.7% of total fetal blood volume) was replaced with maternal blood after each withdrawal. Fetal hemoglobin concentrations were unchanged during the ischemia experiments and rose only slightly as the hypoxemia experiments progressed. Thus, there is no evidence that transfusion of small amounts of maternal blood into the fetus had an overall effect on the experimental outcomes. 6.4.3 Hindlimb blood flow We found that hindlimb blood flow was less in the ischemia control group compared to the hypoxemia animals (Table 11). Previous studies have reported estimates of fetal lamb hindlimb blood flow in late gestation (127 to 142 days) ranging from 9 to 30 103 mlmin"1- lOOg'1 with average values of approximately 18 mlmin"1- lOOg"1 (Knight et al, 1997; Boyle et al, 1990; Wilkening et al, 1988). Thus, the control hindlimb blood Hows observed in both the hypoxemia (28.9 ± 1.8 ml-min"1- lOOg"1) and ischemia (13.8 ± 0.9 mlmin"1- lOOg"1) groups fall within this range of values. That our mean blood flows were on either end of this range may explain why the difference was significant. The mean gestational age of the animals subjected to ischemia was approximately a week greater than those in the hypoxemia experiments (137 ± 1 versus 130 ± 2 days). Two of these animals were involved in both hypoxemia and ischemia experiments. As part of the hindlimb weight and blood flow distribution study (Section 4), 3 of the fetuses involved in the ischemia experiments received radionuclide-labelled microspheres. As this required the fetuses to then be euthanized, these experiments were conducted later in gestation so as to permit the fetuses to also be used for other experiments (such as hypoxemia). Thus, the order in which the fetuses were exposed to the hypoxemia and ischemia interventions could not be randomized. This is reflected in the difference in the average gestational ages of the hypoxemia and ischemia fetuses. To further investigate whether this difference could be a factor in the discrepancy between the two groups in terms of blood flow, we did a retrospective, longitudinal study of the relationship between hindlimb blood flow (measured with a transit-time flow transducer on the external iliac artery) and gestational age. The results, collected from 16 fetuses in our laboratory between 119 and 141 days gestation, demonstrated a significant fall in blood flow with increasing gestational age (Figure 23). Previous studies in which hindlimb or carcass blood flows and gestational ages have been reported have not found a significant relationship between these variables (Boyle et al, 1990; Wilkening et al, 1988; Rudolph and Heymann, 1970). In contrast, Bendeck and Langille (1992), who measured organ blood flows at 120 and 140 days gestation, found a significant (50%) reduction in hindlimb bone blood flow and, although skeletal muscle flow fell from 17.2 to 12.8 mlmin"1- lOOg"1, neither this nor blood flow to the skin was significantly different. Similar trends for fetal skeletal muscle and skin blood flow were 104 FIGURE 23. Relationship between hindlimb blood flow and gestational age in the fetal lamb. The longitudinal study was conducted on 16 fetuses (each symbol represents an individual animal). Mean daily blood flow was determined with a transit-time flow transducer on the external iliac artery. Only days in which no other experiment was performed are included. The relationship is statistically significant: y = 118.8 - 0.69x r = 0.30 105 reported by Rosenfeld (1977) when comparing sheep at 60-100 versus 130-140 days gestation. Using the regression equation from our data (hindlimb blood flow = 118.8 -0.69 x gestational age), the gestational age difference between the hypoxemia and ischemia groups would only account for approximately a 17% difference in flow. The presence of the vascular occluder on the external iliac artery of the fetuses in the ischemia group is unlikely to be responsible for the reduced control hindlimb blood flow in this group as 3 of the 7 hypoxemia fetuses also had vascular occluders implanted. Furthermore, the occluders were only connected to the infusion pumps during the experiments, and the correct positioning of the occluders and flow transducers were verified at autopsy. Thus, it is unlikely that these devices were responsible for the lower control blood flow observed in the ischemia group. Thus, although the hindlimb blood flow values for both the hypoxemia and ischemia control groups fall within the normal range reported in the literature, a complete explanation for the significant difference between these two groups is not clear. 6.4.4 Oxygen delivery and consumption It is often difficult to compare studies in which Do2 is reduced because, depending on the goal of the experiment, different variables are used to define the decline in oxygen supply. For example, oxygen supply levels may be based on the percent reduction in umbilical blood flow in experiments of umbilical cord compression, change in maternal or fetal Po2 or oxygen contents when maternal inspired oxygen levels are altered, etcetera However, it is important to consider both oxygen content and blood flow as it is both of these variables that constitute tissue oxygen supply. Thus, in the current study, we defined levels of hypoxemia and ischemia based on oxygen delivery rather than on arterial Po2 or hindlimb blood flow. In other studies of the fetal lamb hindlimb in late gestation, oxygen delivery has ranged from 50 to 86 //molmin"1- 100g'! and oxygen consumption from 12 to 28 ^molmin"1 lOOg"1 with approximate averages of 60 and 17 /^ molmin lOOg"1, respectively 106 (Boyle etal., 1992; Boyle etal., 1990; Wilkening etal., 1988). Our control Do2 and Vo 2 in the hypoxemia group (71.4 ± 1.7 and 21.1 ± 1.4 ^molmin"1- lOOg"1, respectively) fall within these ranges but those of the ischemia group (44.3 ± 3.1 and 10.7 ± 1.3 /fmol-min"1- lOOg1), appear to be low. These differences between the hypoxemia and ischemia groups appear to be consistent i.e., no one or two animals contributed unusually high or low delivery and consumption values. Fetal arterial oxygen content was actually greater in the ischemia control group, and the arterial-venous oxygen content difference was similar for the hypoxemia (0.76 ± 0.05 mM) and ischemia (0.79 ± 0.09 mM) control groups. Thus the difference in control hindlimb blood flows between the hypoxemia and ischemia groups was responsible for the significant difference seen in the baseline oxygen delivery and consumption values. To facilitate comparison of the results, oxygen delivery and consumption values for the interventions are expressed as a percentage of their respective control values. Several researchers have pointed out a problem in relating values that are calculated from a common set of direcdy measured variables: coupled errors in these measured variables may introduce a bias that leads to incorrect conclusions (Stratton et al, 1987; Moreno et al, 1986). In our case, oxygen delivery and consumption are calculated using hindlimb blood flow and arterial oxygen content values and thus, experimental errors in either of these values could create artifactual correlations between oxygen consumption and delivery. For example, an erroneous arterial content value would lead to the calculation of delivery and consumption values that were both high or both low, leading to a positive correlation between consumption and delivery that may be unrelated to any physiological phenomenon (Stratton etal., 1987). Stratton and colleagues (1987) specifically investigated the problem of coupling errors in oxygen consumption and delivery studies. They concluded that, unless the measurement errors are very large or the range of deliveries used is very small, the influence of coupled error on oxygen consumption and delivery relationships is quantitatively small enough to be ignored. As the oxygen content values are 107 similar to those reported in the literature and oxygen delivery values ranged from approximately 13% to 100% of control values, in the current study the influence of coupled errors was considered to be negligible. In both our hypoxemia and ischemia experiments, hindlimb oxygen consumption was maintained when delivery was reduced to approximately 75% of control and fell when delivery was reduced by more than ~50%. At these levels of oxygen supply, our hypoxemia animals demonstrated reductions in Po2 and oxygen content while no such changes occurred with ischemia. In our ischemia experiments, the fall in hindlimb oxygen delivery mirrored the reduction in blood flow. During hypoxemia, blood flow did not change with mild hypoxemia but fell with moderate and severe reductions in oxygen delivery. With severe levels of hypoxemia and ischemia, both arterial oxygen content and hindlimb blood flow were significantly reduced exacerbating the reduction in Do2. Jensen et al. (1991) have studied the effect of altering fetal oxygen delivery by reducing uterine blood flow (thereby reducing fetal arterial oxygen saturation and content) and compared their results with previous work done in their laboratory in which Do2 was diminished by umbilical cord compression (Itskovitz etal, 1987). Radionuclide-labeled microspheres were used to determine blood flow distribution. Oxygen delivery was reduced to 67% and 50% of control values with uterine artery compression and to 77% and 46% of control with umbilical cord occlusion. Graded reductions in uterine blood flow were associated with a fall in fetal descending aorta Po2 and oxygen content whereas these variables did not change until oxygen delivery fell by -50% during cord compression. Uterine blood flow reduction also resulted in a rise in Pco2 in the descending aorta which was not evident with cord compression. Blood flow to the fetal brain and heart greatly increased with each level of uterine artery occlusion but with umbilical cord compression, the only significant increase was in brain blood flow at a 50% reduction in Do2. In both studies, total body V o2 was maintained, primarily by an increase in oxygen extraction, until delivery was reduced by ~50%. With the more severe reduction in Do2, oxygen 108 consumption by the lower carcass fell in the uterine artery experiment but did not change when faced with umbilical cord compression. With reduced maternal inspired oxygen levels and uterine artery occlusion, fetal Po2 values fall whereas during fetal hemorrhage, hindlimb ischemia, and umbilical vein compression fetal Po2 values are generally maintained. In the latter cases, oxygen delivery is altered primarily by decreases in blood flow. When we reduced Do2 by approximately 50% (moderate intervention), 0 2 consumption fell to 75.4 ± 5.2% of control during hypoxemia versus 74.3 ± 8.6% with hindlimb ischemia. These data compare favourably with the reductions in oxygen consumption seen with uterine artery occlusion (to ~70% of control) (Jensen etal, 1991) and in studies in which fetal Po2 is maintained (to ~75% of control) (Kwan etal, 1995b; Itskovitz etal, 1987; Itskovitz etal, 1983). Furthermore, when other researchers have reduced oxygen delivery to 20-25% of control values by altering fetal Po2 or blood flow, consumption was approximately 33% (Boyle etal, 1990) and 50% of control values (Itskovitz etal, 1983), respectively. These results are similar to those seen in the current study when the reductions in maternal inspired oxygen level or hindlimb blood flow were categorized as severe (30.3 ± 43.5% versus 42.6 ± 7.9% of control, respectively). Thus, although our work as well as that of Boyle et al (1990) focused on the hindlimb, the results aligned well with those of other researchers who measured whole body responses to reductions in oxygen supply. A curvilinear relationship between oxygen consumption and delivery has been reported in many other studies (Carter, 1989; Edelstone et al, 1989; Itskovitz et al, 1983; Wilkening and Meschia, 1983). We also found a curvilinear relationship between these two variables with V o2 declining gradually until Do2 fell by ~50% when the decline in consumption fell significantly (Figure 22). Although hindlimb V o2 fell with a lesser fall in delivery during hypoxemia compared to ischemia, the difference was not statistically significant. As acute hypoxemia is associated with an inhibition of fetal breathing (Hohimer and Bissonnette, 1991; Koos et al, 1987b) and limb movements (Natale et al, 1981), the 109 earlier, though not significant, fall in oxygen consumption may reflect hypoxemia-elicited inhibition of muscle activity. Koos et al (1987b) observed significant reductions in fetal breathing movements and REM activity when fetal brachial artery Po2 was lowered beyond ~17.8 mmHg by decreasing the inspired 0 2 concentration of the ewe. In a subsequent study the same researchers (Koos et al., 1987a) noted reductions in fetal breathing and rapid eye movements in response to fetal anemia, a condition in which fetal Pao2 is generally maintained. They concluded that eye and breathing movements were inhibited not by fetal arterial Po2 but by tissue Po2, particularly in the pons region of the brain. This inhibition of fetal activity occurred with reductions in jugular venous Po2 from 20.8 mmHg to 15.0 mmHg during hypoxemia (Koos etal, 1987b) and from 19.5 mmHg to 17.8 mmHg with fetal anemia (Koos et al, 1987a). In our study, fetal venous Po2 (measured in the external iliac vein) ranged from 14.9 ± 0.4 mmHg with mild hypoxemia to 7.4 ± 1.2 mmHg during severe hypoxemia (control = 16.4 ± 0.5 mmHg). Thus, even allowing for Po2 values to be slighdy greater in the upper body, the venous Po2 values we observed with each of our hypoxemia levels (mild, moderate, and severe) should have been sufficient to reduce the incidence of fetal breathing movements and REM activity and thus reduce oxygen consumption. This suggestion is supported by the observation that during mild hypoxemia V o2 was reduced by 8.7%. This is similar to our previous results (Section 5) in which neuromuscular blockade, which abolishes fetal breathing and body movements, was associated with an 11.8 ± 4.6% reduction in hindlimb oxygen consumption. During ischemia, arterial Po2 was unchanged but venous Po2 declined significantiy (falling from 17.6 ± 0.9 mmHg at control to 16.2 ± 0.7 mmHg with mild ischemia and 14.3 ±1.2 mmHg during severe ischemia). Thus hindlimb tissue Po2 would also have been reduced. However, inhibition of skeletal muscle activity would not be expected with ischemia as this perturbation causes only local reductions in oxygen delivery rather than the whole body effects of hypoxemia. 110 For comparable reductions in oxygen delivery, oxygen consumption fell to a similar degree with both hypoxemia and ischemia. We had hypothesized that hypoxemia would result in greater reductions in oxygen consumption as this intervention reduces an already low fetal arterial Po2 which could then diminish oxygen diffusion into the tissues. In the current experiments, compared to ischemia, hypoxemia did result in significantly greater reductions in Po2of the arterial blood and in the hindlimb tissues (assuming venous Po2 is reflective of tissue Po2). However, no difference was seen in the arterial-venous Po2 gradients for the two perturbations. For example, with severe hypoxemia the arterial-venous Po2 difference was 5.6 + 0.5 mmHg compared to 4.5 ± 0.6 mmHg with severe ischemia (p = 0.1800). Thus, hypoxemia was associated with lower arterial and tissue Po2 values, but the oxygen diffusion gradient from blood to hindlimb tissue was not different for the two interventions.. Although hypoxemia and ischemia reduce oxygen delivery by two different ways, oxygen diffusion into the tissues of the hindlimb was similar and thus hindlimb oxygen consumption did not differ for the two perturbations. As a result, hypoxemia and ischemia were similarly tolerated by the hindlimb and our hypothesis was rejected. 6.4.5 Lactate and glucose responses Glucose and lactate are important substrates for oxidative metabolism in the late gestation ovine fetus with amino acids providing an alternate fuel source. Under normal conditions, approximately 61% of glucose and 72% of lactate is oxidized accounting for about 50% of fetal oxygen consumption (Hay et al, 1983). Lactate does not diffuse from the mother to fetus as fetal lactate concentrations are higher than those in the maternal circulation (Burd et al, 1975). In contrast, maternal glucose concentrations are much higher than in the fetal lamb largely due to substantial glucose consumption by the uteroplacental tissues (Meschia et al, 1980). During the control period, arterial and venous glucose concentrations and glucose uptake were significantly lower in the ischemia fetuses while lactate concentrations were 111 higher. As the ewes were allowed free access to grain and hay, these differences likely reflect the recent nutritional intake of the mothers. No significant change in glucose concentrations was seen with either hypoxemia or ischemia with the exception of a rise in arterial glucose levels during severe hypoxemia. Net hindlimb uptake of glucose was seen during the control and experimental conditions, and there were no significant changes in glucose uptake during or after either intervention. In contrast, arterial lactate levels rose during moderate and severe hypoxemia reaching 5.07 ± 0.48 mM. Severe hypoxemia was also associated with a reduction in pH (to 7.234 ± 0.031) and a marked fall in base excess (to -6.8 +1.8 mEq/L). Arterial lactate levels remained high during the recovery phase. Similar pH, base excess, and arterial lactate levels (to ~7 mM) have been previously reported in response to reductions in maternal inspired oxygen concentration (Rurak etal, 1990b) and uterine artery compression (Boyle etal, 1992; Jensen etal, 1991). Because hypoxemia affects the whole fetus and the placenta, these higher lactate levels probably reflect the larger amount of tissue responding to the reduction in oxygen delivery. Unfortunately Itskovitz and colleagues (1987; 1983) did not report lactate values in their studies of umbilical cord compression; however, only modest reductions in descending aorta pH (from 7.39 during control to -7.35) were seen. Because blood flow is maintained or even increased to the carcass and peripheral tissues during cord occlusion, regional lactic acidemia is not likely. In the current study, no change in fetal pH, base excess, or lactate flux were seen and, even with severe ischemia, only a small increase in arterial lactate concentration was observed. This implies that this intervention had little adverse effect on the placental or systemic circulation. Ischemia, unlike hypoxemia, is primarily a local phenomenon and thus is unlikely to generate whole body responses. During hypoxemia there was release of lactate from the hindlimb in contrast to net lactate uptake during the control period. Although the changes during mild, moderate, and severe hypoxemia were not different from the control level (1.15 ± 0.81 ^molmin"1- lOOg"1), the pooled mean lactate flux (-3.12 ± 0.87 /miolmin1- lOOg ') was statistically different. In 112 a study of prolonged (~4 hours) severe, nonlethal hypoxia, achieved by reducing uterine blood (low, Boyle etal. (1992) also observed net hindlimb lactate output (6.7 /miol-min"1- lOOg"1) although the systemic lactate concentrations (~15 mM) were more markedly elevated than in our study. However, in their study, lactate was also released by the hindlimb during the control or normoxemic period (~3.1 /miol-min"1- lOOg"1). Singh et al. (1984) have also reported hindlimb lactate efflux from both maternal and fetal sheep under normoxemic conditions, whereas Yeleswaram (1993), as in the current study, observed net lactate uptake by the fetal hindlimb. The reason for these differing results is not clear; however, it may relate to differences in fetal endocrine status in the various studies, particularly in terms of catecholamine concentrations. Another possible explanation relates to analytical variability in lactate measurements. As indicated in Table 12, the hindlimb arterio-venous (A-V) difference in lactate concentration is small; thus, relatively small measurement errors could result in an artifactual reversal of the A-V difference. This would be most problematic with elevated arterial and venous lactate concentrations. During ischemia, there was also a tendency for lactate efflux. As with hypoxemia, at each level of ischemia lactate flux was not statistically different from the control value (3.37 ± 1.24 /miol-min"1- lOOg"1), but the pooled mean lactate flux (-0.25 + 0.41 /miol-min"1- lOOg"1) did reach statistical significance. The pooled mean lactate flux during hypoxemia was statistically greater than during ischemia In contrast, the situation was reversed during the recovery phase, with hindlimb lactate efflux following ischemia (4.28 ± 1.75 /miol-min"1- lOOg"1) over twice as great as following hypoxemia (2.00 ± 1.95 /miol-min"1- lOOg"1). As a result, the overall lactate efflux was not different between the two perturbations. The apparent difference in the time course of the lactate flux in the two sets of experiments may relate to the difference in blood flow responses. During ischemia blood flow was reduced to a greater extend than during hypoxemia This could have limited lactate efflux so that a greater proportion of lactate was released during the recovery period when blood flow was restored. 113 The hindlimb lactate release that occurred with both hypoxemia and ischemia was not associated with any obvious increase in hindlimb glucose uptake. This suggests that the released lactate was derived from a source of glucose within the hindlimb. One likely source is skeletal muscle glycogen which accumulates in the fetus during late gestation (Shelley, 1961). Increased glycogenolysis in the hindlimb could be elicited by the rise in circulating catecholamines that occurs with hypoxemia (Cohen et al, 1982; Jones and Robinson, 1975). Circulating catecholamines increase the liberation of glucose-1-phosphate from skeletal muscle glycogen, which can then be converted to lactate. Infusion of adrenaline to fetal lambs results in hyperglycemia and lactic acidemia, actions that can be attenuated by f3-adrenergic antagonists (Jones and Ritchie, 1978), as can the rise in plasma lactate concentrations during hypoxemia (Jones and Ritchie, 1983). Yeleswaram et al. (1993) found that labetalol, a combined a and (3 adrenergic blocker with significant p\ agonist activity, elicits prolonged lactic acidemia in the fetal lamb associated with sustained hindlimb lactate release. A similar mechanism could have operated during hypoxemia. However, as such systemic effects are unlikely to have been stimulated by hindlimb ischemia, local factors may have acted in this situation to cause the lactate efflux. During hypoxemia, there was both systemic lactic acidemia and lactate release from the hindlimb. An obvious question is the extent to which lactate efflux from the hindlimb and the overall carcass contributed to the rise in circulating lactate concentrations. Yeleswaram et al. (1993) addressed this question in their study of labetalol by extrapolating hindlimb lactate release to the entire carcass and comparing this to an estimate of systemic lactate production. The latter value was obtained by multiplying the incremental increase in the area under the arterial lactate concentration versus time curve (lactate AUC) times a published value for lactate clearance in the fetal lamb. A similar approach can be used with our hypoxemia data. Overall carcass lactate efflux can be estimated by multiplying weight normalized hindlimb lactate efflux by 70% of fetal body weight to yield 1.92 mmol. Arterial lactate AUC is estimated as the average of the rise in arterial lactate concentration 114 during hypoxemia (1.72 /miol/ml) times the duration of the hypoxemia period (30 minutes), with the resulting value being 51.6 ^mol-ml'-min. This value is then multiplied by fetal lactate clearance; however, it is not clear which lactate clearance value should be used. Boyle et al. (1992) obtained a normoxemia lactate clearance value of 43.5 ml-min"1-kg"1, which is similar to the estimate of 35.5 ml-min -^kg'1 published by Sparks (1982). Using the former value and the average fetal weight of our hypoxemia animals (2.935 kg) yields an estimate of 127.7 ml-min"1. This value times the lactate AUC produced a value of 6.59 mmol for total body lactate production. The carcass contribution to the total body lactate production would then be 1.92 mmol/6.59 mmol or 29%. However, Boyle et al. (1992) found that during ~4 hours of severe hypoxemia, fetal lactate clearance fell dramatically although it could not be precisely calculated because arterial lactate specific activity was not at steady state as it had been during the normoxia portion of their protocol. The clearance value they calculated by neglecting the accumulation of tracer lactate was 10.5 ml-min"1-kg"1 or 30.8 ml-min"1 when calculated for the average body weight of our animals. This value multiplied by the arterial lactate AUC gives an estimate of 1.59 mmol for total body lactate production. The carcass contribution in this situation is thus 1.92mmol/1.59 mmol or 121%. As the level of hypoxemia we obtained was not as severe nor as prolonged as that of Boyle, fetal lactate clearance by our animals was likely greater than 10.5 ml-min"1-kg"1. Regardless of the exact values, it appears that lactate release from the carcass does contribute to the systemic lactic acidemia seen during hypoxemia, with the contribution ranging between approximately 29 and 100%. The precise contribution depends on the magnitude of the fetal lactate clearance after ~30 minutes of hypoxemia which is currently unknown. If the carcass is not the sole source for the rise in fetal circulating lactate concentrations during hypoxemia, other sites must be considered. One such potential site is the placenta. As stated earlier, under normal conditions the placenta is a net producer of lactate, and the fetus is a net consumer of this substance (Sparks et ai, 1982). This placental release of lactate is illustrated by the higher lactate concentrations found in the 115 umbilical and uterine veins compared to the corresponding arteries (Burd et al, 1975). The lactate produced by the placenta appears to be asymmetrically distributed with greater amounts entering the umbilical compared to the uterine circulation (Sparks et al, 1982). The amount of lactate released into the fetal circulation increases during hypoxemia (Milley, 1988) and labour (Tan, 1997). Under normal conditions, fetal lactate utilization exceeds the umbilical uptake of lactate from the placenta indicating that fetal tissues must produce lactate endogenously (Hay et ai, 1983). Even with increased placental lactate release during hypoxemia, other fetal tissues likely must also contribute to the elevated plasma lactate concentrations. Fisher etal. (1982) have examined the response of the fetal myocardium to hypoxemia, achieved by reducing maternal inspired oxygen levels. The fetal heart normally consumes lactate. In response to a 50% reduction in ascending aorta oxygen content, myocardial blood flow rose markedly to 160% above control values and cardiac oxygen consumption was unchanged. Arterial lactate levels were significandy increased but myocardial lactate consumption was unchanged. Thus, the fetal heart does not appear to be responsible for the increased systemic lactate observed during hypoxemia Lactate flux across the fetal liver has also been examined. Lactate appears to be an important substrate for the liver, as there is a net hepatic uptake of lactate under normal conditions in fetal, newborn, and adult sheep (Apatu and Barnes, 1991; Gleason et al, 1985a; Gleason etal, 1985b). This is supported by the observation that there is no net fetal hepatic uptake or release of glucose indicating that other carbon sources, such as lactate and amino acids, are used for oxidative metabolism by the liver (Gleason and Rudolph, 1985; Bristow et al, 1983). Abnormal conditions, such as hypoxemia, could alter glucose and lactate production, extraction, utilization, and flux. Reducing maternal inspired oxygen levels (Bristow et al, 1983) or severe umbilical cord compression (Rudolph et al, 1989) decreases blood flow to the liver leading to lower hepatic Do2 and either maintained or slightly reduced V o2. However, even under such conditions, hepatic oxygen content, Do2, 1 16 and Po2 exceed that of other organs due to receiving well-oxygenated umbilical venous blood. Thus, it is unlikely that the liver experiences tissue hypoxia. Although lactate continues to be consumed by the liver during cord compression, net hepatic lactate uptake declines significantly (Rudolph etal., 1989). This likely contributes to the observed rise in arterial lactate concentrations and may be in part responsible for the fall in lactate clearance during severe hypoxemia reported by Boyle et al. (1992). However, other factors such as changes in lactate consumption by other organs or increased fetal lactate production may also contribute to the increase in circulating lactate. Unlike during control conditions, glucose is released from the liver during hypoxemia (Rudolph et al., 1989) and cord compression (Bristow et al., 1983). Lactate is unlikely to be the source of this glucose as gluconeogenesis, in which non-carbohydrate substrates like lactate and amino acids are converted to pyruvate and then to glucose, does not appear to occur to an appreciable amount in the fetal liver under normal conditions (Townsend etal., 1989; Gleason and Rudolph, 1985) or with acute reductions in umbilical blood flow (Rudolph et al., 1989). Hepatic glycogen content is reduced during hypoxemia suggesting that glycogenolysis may be responsible (Stratford and Hooper, 1997). In the current study, hindlimb glucose consumption tended to increase, particularly during hypoxemia, but did not reach statistical significance. Hepatic glucose release may have contributed to the increased arterial glucose concentration seen with severe hypoxemia. Carbohydrate metabolism has also been examined in the fetal kidney (Iwamoto and Rudolph, 1985) and lungs (Simmons and Charlton, 1988). During normoxia, the fetal lamb kidney consumes lactate but releases glucose into the systemic circulation. In response to reduced maternal inspired oxygen levels, the situation is reversed: there is net glucose uptake and net release of lactate from the renal circulation. This resulted in significant increases in lactate levels in the descending aorta (from 1.53 + 0.32 mM during control to 4.92 ± 1.66 during hypoxemia). This rise in arterial lactate values is similar to what we observed with severe hypoxemia (from 1.42 + 0.09 mM during control to 5.07 ± 0.48 117 mM). Furthermore, under normal conditions, measurements of lactate concentrations in the pulmonary arteries and veins of fetal lambs (119-141 days) indicate that the lung releases lactate into the circulation and that this does not change over the gestational ages examined (Simmons and Charlton, 1988). Thus, the fetal lung is another potential source of plasma lactate, although whether this production continues during conditions of reduced fetal oxygen delivery does not appear to have been examined. In conclusion, there was lactate efflux from the fetal hindlimb during both hypoxemia and ischemia. With hypoxemia, most of the lactate was released during the intervention, while in the ischemia experiments, the majority was released during the recovery or post-ischemia period. However, there was no difference in the overall magnitude of the response between the two perturbations. During hypoxemia, lactate release from the carcass may contribute to the systemic lactic acidemia, with the estimated contribution ranging from approximately 29—100%. The increase in circulating lactate concentrations may involve other sources as well. The fetal liver, for example, although a net consumer of lactate, reduces its lactate uptake during hypoxemia, thereby contributing to the systemic levels. In addition, the placenta and fetal kidney appear to release lactate into the circulation in response to hypoxemia Lactate flux across the fetal lung is another potential source of systemic lactate, although its metabolism has not been examined during hypoxemia 6.4.6 Cardiovascular responses As stated earlier, control hindlimb blood flow was lower in the ischemia group of animals. As there was no difference in control arterial and venous pressures between the two groups, this difference in blood flow was responsible for the greater hindlimb vascular resistance seen in the ischemia animals. Control fetal heart rate was also significantly lower in the ischemia group (147.3 ± 3.7 bpm) compared to the hypoxemia animals (162.8 ± 2.5 118 bpm). This may reflect the greater gestational age of the ischemia animals as previous work has shown this variable to decline with increasing gestational age (Tan, 1997; Dawes, 1985). Bradycardia, hypertension, and redistribution of blood flow away from peripheral tissue have been described by many previous researchers in response to hypoxemia achieved either by decreasing maternal inspired oxygen levels (Reuss et al, 1982; Parer, 1980; Cohn et al., 1974) or reducing uterine blood flow (Boyle et al., 1992; Jensen et al., 1991). We observed similar responses in hindlimb tissue particularly with severe hypoxemia Hanson (1988) reviewed the complex interaction of factors affecting the fetal cardiovascular system during hypoxemia Vascular responses to reductions in oxygen delivery may result from chemoreceptor stimulation and/or circulating hormones including catecholamines and arginine vasopressin. Hypoxemia-related bradycardia is attributed to a vagally mediated chemoreceptor reflex. Stimulation of the sympathetic nervous system in response to hypoxemia results in a redistribution of blood flow with vasoconstriction in peripheral tissues including the carcass and gut combined with vasodilation in the vital organs like the heart and brain (Iwamoto etal, 1983; Reuss etal, 1982). The hypertensive response to hypoxemia is probably the net effect of these changes in vascular resistance (Hanson, 1988). In our ischemia animals, no change in fetal heart rate or vascular pressures were observed. Hindlimb blood flow and vascular resistance underwent reciprocal changes. For example, with severe ischemia blood flow fell to nearly a quarter of the control value while vascular resistance across the hindlimb increased approximately four fold. Local release of endothelium-derived contracting factors, such as endothelin-1, may have contributed to hindlimb vasoconstriction (Luscher and Wenzel, 1995) but was insufficient to cause a hypertensive response measurable in the descending aorta. Thus, partial occlusion of the external iliac artery resulted in mainly local effects such as vasoconstriction without the systemic responses evident with hypoxemia. 1 19 In summary, a curvilinear relationship was seen between oxygen delivery and consumption in the hindlimb with no significant difference between the curves for hypoxemia or ischemia. An earlier, but not significant, fall in consumption seen with hypoxemia may reflect an inhibition of muscle activity. Arterial and venous Po2 values were significantly lower during hypoxemia than during ischemia. Thus hindlimb tissue Po2 would be lower with hypoxemia. Hypoxemia affects the whole body, so that reduced tissue Po2 in the brain would inhibit skeletal muscle activity and thus lower oxygen consumption. Although venous, and therefore, hindlimb tissue Po2 were also reduced with ischemia, only local tissues would be affected and thus muscle activity is unlikely to be altered. Despite the significantly greater reductions in arterial and venous Po2 with hypoxemia compared to ischemia, no significant difference was seen in the arterial-venous Po2 gradient between the two interventions. Thus, although oxygen delivery is reduced by two different ways with hypoxemia and ischemia, oxygen diffusion into the hindlimb tissues was similar and thus hindlimb oxygen consumption did not differ for the two perturbations. The change in hindlimb lactate flux from net uptake during control to net release with both perturbations indicates that tissue Po2, in both cases, was sufficiently reduced to stimulate anaerobic metabolism or reduce iactate oxidation in the hindlimb tissues. The overall magnitude of hindlimb lactate efflux was not significantly different between the hypoxemia and ischemia experiments. A greater rise in plasma lactate levels was seen with hypoxemia, and carcass lactate release may contribute to this. Other organs, including the placenta, liver, kidney, and lung may also have contributed to this nse in circulating lactate. Although there was apparent lactate efflux from the fetal hindlimb during hypoxemia, the magnitude of the lactate flux was small compared to the decrement in oxygen consumption. For example, during severe hypoxemia, lactate efflux was 2.24 /miol-min"1- lOOg"1 compared to a decrease in V o2 of -14.2 /miol-min"1 lOOg"1. This is similar to the results of Boyle et al. (1992). They suggested that the marked reduction in hindlimb (and overall carcass) oxygen consumption during severe hypoxemia limits the fall in systemic 0 2 content thereby maintaining the supply of oxygen to the heart, brain, and adrenal glands, while the relatively small increase in lactate efflux is a protective mechanism to limit the rate of glucose breakdown and systemic lactic acid accumulation. This implies a suppression of hindlimb metabolic rate during hypoxemia, in that the fall in oxidative metabolic rate is not linked to an increase in glycolytic rate. In the current study, the lack of any obvious increase in lactate efflux with increasing severity of hypoxemia (and increasing decrement in V o2) is consistent with this view. The role of the various hindlimb tissue components (skin, bone, and skeletal muscle) in the response to hypoxemia and hindlimb ischemia is not known. As indicated in Section 4, muscle comprises the largest proportion of hindlimb weight. Because of its glycogen content, skeletal muscle is likely important in terms of lactate production. However, bone receives the highest fraction of hindlimb blood flow, and, compared to muscle, both skin and bone are more highly perfused in relation to the total hindlimb weight that they comprise. As a result, when oxygen delivery is reduced, bone and skin may be more greatly affected in terms of oxygen consumption. These potential differences in the response of the hindlimb components to a reduction in oxygen supply may contribute to the lack of relationship between the decrement in oxygen consumption and the magnitude of hindlimb lactate efflux. Our results illustrate that whether oxygen delivery is reduced by hypoxemia or by reducing blood flow, V o2 can generally be maintained until oxygen delivery is reduced by ~50%. Fetal hypoxemia did result in greater plasma lactate concentrations reflecting the whole body effects of this perturbation versus the local nature of the ischemia. However, there was no difference between hypoxemia and ischemia in terms of the overall magnitude of hindlimb lactate efflux. Thus, for similar reductions in oxygen delivery, there is no evidence that fetal hypoxemia is less well tolerated than hindlimb ischemia. Our hypothesis is not supported by the results obtained and must, therefore, be rejected. 121 7. ARGININE VASOPRESSIN INFUSION 7.1 Introduction Arginine vasopressin (AVP) is a cyclic nine amino acid peptide produced in the supraoptic and paraventricular nuclei of the hypothalamus and released into the circulation from the posterior pituitary. Vasopressin, one of the first hormones to be identified, was first noted in 1895 for its pressor actions although most research has focused on its later-established antidiuretic action (Share, 1988). Vasopressin acts on vascular smooth muscle receptors classified as V1 receptors, with calcium as a second messenger, to elicit vasoconstriction; V 2 receptors in the kidney, employing the adenylate cyclase-cyclic AMP system as the second messenger, are responsible for the antidiuretic action of this hormone (Share, 1988). In the adult, the main role of vasopressin appears to be the regulation of renal water excretion. It causes increased water resorption in the distal tubule resulting in increased urine osmolality. The antidiuretic action of AVP in the fetus occurs over a very low range of hormone values. Under normal circumstances vasopressin levels are very low (<5 pg/ml) and fetal urine is hypotonic to plasma. At plasma AVP levels of ~13 pg/ml maximal urine concentrating occurs and urine osmolalities at or only slightly hypertonic to plasma are achieved (Woods et al, 1986; Robillard and Weitzman, 1980). This indicates that fetal urine concentrating ability is only 20-30% of the adult potential (Ervin, 1988; Woods et al., 1986; Robillard and Weitzman, 1980). In the ovine fetus, AVP is released in response to stresses such as fetal hypoxemia (Rurak, 1978),hemorrhage (Ross etal., 1986b; Drummond etal., 1980; Robillard etal., 1979), hypotension (Ross et al, 1986a), and vena caval obstruction (Wood et al, 1982). The administration of AVP results in a fall in fetal heart rate and a rise in arterial pressure (Wiriyathian etal., 1983; Iwamoto etal., 1979; Rurak, 1978),as well as maintained (Iwamoto et al, 1979) or increased umbilical blood flow (Rurak and Gruber, 1984). In addition, dramatic falls in blood flow to the peripheral tissues, particularly skin and skeletal 122 muscle, have been reported in dogs (Liard et al, 1982) and fetal lambs (Iwamoto et al, 1979) while perfusion of the heart and brain increases (Iwamoto et al, 1979). This redistribution of cardiac output is similar to the pattern seen during fetal hypoxemia (Peeters et al, 1979; Cohn et al, 1974). These finding suggest that the release of arginine vasopressin in response to hypoxemia may play a role in the cardiovascular changes associated with this condition. Few reports in the literature have compared the effect of arginine vasopressin infusion in fetal and adult animals. Rurak (1978) demonstrated a greater increase in arterial pressure in fetal lambs given exogenous arginine vasopressin compared to adult sheep. This was speculated to be due to a greater vasoconstrictor effect of AVP on the fetal systemic circulation. Conversely, Harper and Rose (1987) observed similar pressor responses to exogenous arginine vasopressin in fetal and adult sheep but the degree of bradycardia was greater in the ewes. The purpose of the current study was to examine the effect of exogenous arginine vasopressin infusion on blood gas status, hindlimb blood flow, and cardiovascular variables in fetal lambs and non-pregnant ewes. 7.2 Methods 7.2.1 Animal Preparation and Experimental Protocol Surgery was performed on eight fetal lambs between 118-127 days gestation (mean = 122 ± 1 days) using the techniques previously described (Section 2.3). Catheters were placed in the amniotic cavity, fetal trachea, femoral artery, lateral tarsal vein, and pudendoepigastric artery and vein. A transit-time flow transducer positioned on the external iliac artery provided measurements of hindlimb blood flow. Experiments took place at a mean gestational age of 130 ± 1 day (range = 125-135 days). A total of 12 experiments were conducted consisting of a 30 minute control period, a 90 minute infusion of arginine vasopressin (Pitressin, Parke-Davis, Scarborough, Ontario) or saline vehicle, followed by a 2 hour recovery phase (Figure 24). All experiments 123 CONTROL AVP INFUSION RECOVERY -30 minutes -15 minutes -5 minutes +15 minutes +30 minutes +45 minutes +60 minutes +75 minutes +90 minutes +120 minutes +150 minutes +180 minutes +210 minutes AVP infusion started AVP infusion stopped FIGURE 24. Protocol for exogenous arginine vasopressin infusion experiments in fetal and non-pregnant adult ewes. The arrows indicate blood samples which were taken at ~15 minute intervals during the 30 minute control period and the 90 minute infusion period and at 30 minute intervals during the 120 minute recovery period. 124 conducted on the same animal were separated by at least 24 hours. Paired fetal pudendoepigastric arterial and venous samples (~1 ml each) were taken at 15 minute intervals during the control and infusion phases and at 30 minute intervals during the recovery phase. Each sample was immediately followed by the collection of approximately 3 ml of blood from the pudendoepigastric artery for subsequent analysis of plasma AVP concentrations. After each set of samples, the fetal blood lost was replaced with an equivalent amount (~5 ml) of maternal blood that had been collected from the mother prior to the experiment. Two fetuses served as controls receiving vehicle (0.9% saline) only, while the others received arginine vasopressin at 0.72 mU/min (1.8 ng/min) (n=l), 4.5 mU/min (11.25 ng/min) (n=8), or 9.0 mU/min (22.5 ng/min) (n=l) yielding a range of 0.2 to 3.2 mU-nun"1, kg"1 fetal weight (0.5-8 ngmin'kg"1). The AVP doses were made up with non-heparinized saline in 60 ml sterile syringes and administered intravenously using a Harvard infusion pump at an infusion rate of 0.16 ml/minute. Over the 90 minute infusion period, fetuses thus received approximately 14.4 ml of infusate. In addition, three non-pregnant adult ewes underwent surgery to implant catheters in the femoral artery and vein and to position a transit-time flow transducer on the external iliac artery. A total of 8 adult experiments were conducted following the same protocol outlined for the fetus (Figure 24). The animals were randomly assigned to receive arginine vasopressin administered at 25 mU/min (62.5 ng/min) (n=2), 45 mU/min (112.5 ng/min) (n=3), or 90 mU/min (225 ng/min) (n=3) yielding a range of 0.3 to 1.7 mUmin'kg"1 adult weight (0.75-4.25 ngmin'kg"1). The Harvard pump infusion rate was the same as used in the fetal experiments; thus, the adult sheep also received approximately 14.4 ml of infusate. As with the fetal lambs, at least 24 hours elapsed between experiments conducted on the same animal. 125 7.2.2 Arginine Vasopressin Analysis Arterial blood samples for AVP analysis were collected in sterile syringes then transferred to EDTA Vacutainer® tubes (Becton Dickinson, Franklin Lakes, New Jersey) and kept on ice. The samples were centrifuged at 4°C for 20 minutes at 760xg and the plasma was transferred to plastic vials for storage at -70°C. Samples were analyzed using a commercial vasopressin 1 2 SI radioimmunoassay kit (Incstar Corporation, Stillwater, Minnesota) that has been used previously in our laboratory. The kit's sensitivity or minimum detectable amount for arginine vasopressin is 2.5 pg/ml. The vasopressin antibody demonstrates <0.01 % cross-reactivity with oxytocin, 0.14% with vasotocin, and 600% with lysine vasopressin (found in pigs and related species). The blood samples were assayed according to the guidelines of the Incstar kit. The plasma samples (1 ml) were first extracted using methanol and ODS-silica (octadecasilyl-silica) Sep-Pak columns. Briefly, this involved attaching each Sep-Pak column to a polypropylene syringe and then washing it with 5 ml methanol followed by 20 ml deionized water. The plasma was then acidified with 0.1 ml 1M HC1 and pushed slowly, over a period of 1 minute, through the column. The column was then rinsed with 20 ml of 4% (v/v) acetic acid. Finally, 3 ml of methanol was passed through the column very slowly, such that it was left in contact with the ODS-silica for at least 3 minutes, and the eluate collected in a test tube. A final 1 ml of methanol was passed through the column to ensure complete elution of the peptide. After being evaporated to dryness under nitrogen in a 37°C waterbath, the samples were reconstituted with 0.5 ml 1% BSA (bovine serum albumin)-borate buffer (a greater volume of buffer was used for reconstitution if the plasma sample was expected to read greater than the highest standard). The samples were vortexed, incubated in a 37°C waterbath for 10 minutes, and then revortexed to ensure reconstitution of the sample. Aliquots of 0.2 ml of each sample were then assayed in duplicate. To each sample, 0.1 ml of the primary antibody, raised in guinea pigs against vasopressin (guinea pig anti-vasopressin), was added and the samples incubated for 18-24 hours in a shaker at 4°C. The 12SI vasopressin tracer (0.1 ml) was then added to each sample and again incubated in the shaker overnight at 4°C. On the final day, phase separation was achieved in 20 minutes at room temperature with the addition of 0.5 ml goat anti-guinea pig precipitating complex or GAGP-PPT (pre-precipitated complex of second antibody, raised in goats against guinea pig serum, plus polyethylene glycol). After a 30 minute centrifugation at 4000 rpm, the supernatant was aspirated and the tubes were counted in a gamma-counter (Micrad Inc., Knoxville, Tennessee). A calibration curve was prepared by plotting the natural logarithm of extent of binding (Logit B/B0) against stated log concentrations of the calibration standards included in the Incstar kit. This resulted in a linear response curve. The levels of AVP in the plasma samples (in pg/ml) were then interpolated from this plot. Following the example of Rurak (1978), samples whose arginine vasopressin levels registered as undetectable by the assay were assigned a value of one half the kit's sensitivity (1.25 pg/ml). 7.2.3 Statistics Data are generally presented as mean ± standard error of the mean. Data were analyzed with StatView statistical software (Abacus Concepts Inc., Berkeley, California). Arginine vasopressin and blood gas levels are presented as absolute values. Mean hindlimb blood flow, oxygen delivery, consumption, and extraction during the infusion and recovery periods are presented as a percentage change from the control values. For analysis of the cardiovascular variables, data from 3 minutes before to 3 minutes after each blood sample (total of 7 minutes of data) were used. Fetal hindlimb venous pressure was not available as the pudendoepigastric venous catheter was utilized for blood sampling and the infusate was administered via the lateral tarsal vein catheter. Likewise, because only one venous catheter was implanted and this was used for the drug infusion, no venous data were collected from the adult animals. The cardiovascular data, presented at each blood sampling point, are shown as a percentage change from the control values. In all cases, multiple comparisons 127 were analyzed with one-way (factorial) analysis of variance (ANOVA) and Fisher's Protected Least Significant Difference (PLSD) post-hoc test. Repeated measures ANOVA could not be used due to incomplete data sets (i.e., inability to draw some blood samples or record cardiovascular variables due to catheter failure, etc.). Differences were considered significant if p < 0.05. Fetal and adult hindlimb blood flow and cardiovascular variables were compared by applying lines of best fit to the two groups of data. Differences in the regression equations were compared by applying unpaired t-tests to the slopes and y-intercepts. As with the previous data, differences were considered statistically significant if p < 0.05. 7.3 Results 7.3.1 Arginine vasopressin effects on the fetus Figure 25 shows the mean plasma arginine vasopressin levels for fetuses that received either vehicle (saline) or exogenous arginine vasopressin infusions. The overall control AVP concentration for the 12 experiments was 1.8 ± 0.2 pg/ml. No change in AVP levels was observed for the two animals that received vehicle (saline) only. Infusion of arginine vasopressin resulted in mean plasma concentrations of 12.7 + 1.6, 48.4 + 3.2, and 54.4 ± 7.5 pg/ml for the animals receiving doses of 0.72 mU/min (1.8 ng/min), 4.5 mU/min (11.25 ng/min), and 9.0 mU/min (22.5 ng/min), respectively, all of which were significantly different from the control values. The mean AVP concentrations were not different for the 4.5 and 9.0 mU/min doses but this may reflect that the higher dose was only administered to one animal. As seen in Figure 26 plasma AVP levels rose rapidly during administration of the 4.5 mU/min dose, with a significant difference from control seen at 15 minutes into the infusion. Similarly, cessation of the infusion was associated with a dramatic decrease in plasma arginine vasopressin concentrations with levels returning to the control level at 30 minutes post-infusion (+120 sample). Plasma clearance of arginine vasopressin in the fetus was determined for the 4.5 mU/min (11.25 ng/min) infusion rate. Clearance rate was 128 (|Ui/6d) uojiBJiueouoo dAV ewse|d l e j s j 129 (|lU/6d) U0IIBJJU90U00 dAV BLUSB|d |BJ9J 130 calculated as the rate of hormone infusion (per kg body weight) divided by the average of the 75 and 90 minute arterial plasma AVP concentrations. The 75 and 90 minutes samples were chosen as the hormone levels at these points appeared to be at steady state. Fetal clearance of the hormone was 85.3 ± 11.4 mlmin'kg"1. Table 14 presents the blood gas and metabolic values for the fetuses given either vehicle or 4.5 mU/min (11.25 ng/min) of exogenous arginine vasopressin. As the 0.72 and 9.0 mU/min doses were only administered to 1 animal, the blood gas data for these infusion rates are not presented. All control values are within the normal range for our laboratory. Infusion of saline did not alter the blood gas or metabolic values, but the recovery phase was associated with significant reductions in arterial pH (to 7.328 ± 0.004) and base excess (to -0.1 ± 0.2 mEq/L). However, there were no alterations in lactate concentration or flux. Likewise, glucose parameters were unchanged during infusion of the vehicle. During infusion of AVP at 4.5 mU/min, no changes in fetal blood gases or metabolic values were observed. Arterial lactate values rose significantly during the recovery phase reaching a mean value of 1.82 ± 0.17 mM. Although hindlimb venous lactate concentrations tended to rise during and following AVP infusion, the changes were not significant and there was no significant change in lactate flux. No changes in glucose levels or flux were observed. Cardiovascular and metabolic data obtained prior to the infusions of either vehicle (saline) or 4.5 mU/min AVP to fetal lambs are given in Table 15. The results are presented as absolute values (mean ± SEM). There were no significant differences between the two sets of data with the exception of hindlimb blood flow which was significantly higher in the AVP group of animals (23.7 ± 2.5 versus 38.3 + 4.0 mlmin"1- lOOg"1). Results from the 0.72 and 9.0 mU/min AVP doses are not shown in this table or the subsequent cardiovascular figures as only one experiment was performed at each of these doses and thus there were insufficient samples to determine if any changes observed were statistically significant. 131 co co 2H CL, O % > CD fi ' f i '5b & co fi § CD oo o X CD c CD £ •c 8. X CD CN >> •3 o J D 73 S CD > CD 73 CO t-c CD •s CD fi CD > '5b CO CD CO a .<D 3 C CD s •c 2 el C3 X co <D fi oo 73 c % B CD WO o l ^ "a 2 ~ c3 8 PQ S o 'co 2 fi CQ OH > fr CD > 03 CA CD fc. a, o CO c 60 « e S B V i c g 'io . 3 3 CD fe-l l C *3 CD 3 _o 'S3 . 3 C 0 0 o o d -H C N i—i CO IS I d +i CO o d CO o o d +t 0 0 C N co rS CN O o d +l CN "fr CO 0 0 o o d +i o\ "fr CO rS vo 0 0 "fr d d d +i -H -H T-H CO 0 0 vi T-H T ( vo wo vo CO d d d +i -H -H o i—1 ON d CN T-H O vo vo VO "fr d d d +i +i •H CN T 1 ON •—1 CN VO wo ON "fr d d d -H +t +i i > "fr ON CN vd T 1 "fr vo vo d d d -H +i +l q CO ON CO CN r-' T-H vo d -H d -H r--d CN d "fr "fr CN T I d d d d -H -H -H +i vp ON O "fr CO rS i > / \ CN T-H <•—•> VO d +i oo d CO d +i ON ON CO d +i CO d V 0 d -H O _ "fr d -H 0 0 d vo d -H O vo o d vo CN VO d d o -H d -H O d i d 0.79 T—I d -H CN 0 0 CO 0 0 ON CN o O d d d -H +t -H VO •fr CO ON q q «—1 T-H i—H ON d +i i > co d Q. 73 'C s oo 6 B 73 •c 00 B B 8. CO fi O C CD > 00 73 •a fi < fi <D s, CD CD C CD CO CO 8 X CD CD m CD "fi 2 C 3 c o CD o > t=L t=L £ £ X) o Tib o S CD PG 73 •C CD CO 3 O 3 CD > 73 'C <D 3 O C CD > 00 o o fi B 73 B a, x 3 c •ON vo d +l "t o CN vo vo CO VO "fr T-H T-H o o CO d d d d d d +i -H +i -H +i +1 vo vq 0 0 ON ON 0 0 VO CN ON —< T-H d d d i "fr vo o o r-H CO T-H ON o CN ON 0 0 vo d d d d d d d -H -H + -H -H +i -H OO vo CN CO l > 0 0 0 0 l> W0 co d T-H d d d "fr VO o vo O ON O "fr T 1 co T-H vo CN d d d d d ,—i d -H +i +1 +i -H +1 -H ON ON ON CN vo co 0 0 CO CN & d T-H ~* d d i—i co vo o ON O 0 0 o o o $ o co d d d d d d d +l +l +l +l +i + +i 0 0 q vo q o q CN 0 0 CN "fr o ON 1 1 T 1 d d CN i—i , T-H O Y T ( r -T-H ON T 1 o CN ON ON co 0 0 d d d d d d d +i -H -H +i +i -H -H VO T 1 CN T ( q ON 0 0 wo r - "fr CO —' 1 1 i—i d d T-H CO 00 o o c "e 73 B a, x 3 o CD 3 5 8 I c CD CD C O CD CD co O CD 3 Tib II O c x ; § 1 ^ fi (33 O •§ ° in S P i fi "H -S > s ' i i i <D CD y S & II i> s.§> 3 co . 73 II 132 TABLE IS. Cardiovascular and metabolic data obtained from fetal lambs prior to vehicle (saline) infusion (2 experiments) or infusion of 4.5 mU/min arginine vasopressin (8 experiments). Data are presented as absolute values (mean + SEM). Vehicle (saline only) 4.5 mU/min AVP Arterial pressure (mmHg) 55.2 * 51.2 ± 1.4 Heart rate (beats/min) 144.9 ± 17.8 172.3 + 5.0 Hindlimb blood flow (ml/min1- lOOg"1) 23.7 ± 2.5 38.3 + 4.0| Oxygen delivery (/miol/min"1- lOOg"1) 87.2 ± 13.3 96.9+ 16.1 Oxygen consumption (^mol/min1- lOOg"1) 27.8 + 4.5 20.0 + 2.3 Oxygen extraction (%) 32.2 ± 2.4 27.1 +2.0 * only 1 data point available t = significant difference from vehicle value (p<0.05) 133 Fetal arterial pressure was unaltered by the administration of the saline vehicle (Figure 27). During the infusion of 4.5 mU/min AVP, arterial pressure rose rapidly and remained elevated for the duration of the infusion. The mean increase during infusion was 16.5 ± 1.6% above control levels. During the recovery phase arterial pressure rapidly returned to pre-infusion values. Figure 28 presents fetal heart rate responses to saline or arginine vasopressin at 4.5 mU/min. No change in heart rate was observed with the saline infusion. AVP was associated with rapid reductions in fetal heart rate, averaging 17.2 ± 1.5% below the control values. The bradycardia persisted for the duration of the infusion with heart rate quickly returning to control values when the infusion was stopped. The changes in fetal hindlimb blood flow that occurred during and following infusion of either vehicle (saline) or arginine vasopressin at 4.5 mU/min are illustrated in Figure 29. Infusion of saline alone did not alter hindlimb blood flow. AVP at 4.5 mU/min resulted in an average 28.2 ± 1.6% reduction in flow. This fall in flow was statistically significant after 15 minutes of AVP infusion and remained low for the duration of the infusion. Flow had returned to control values 30 minutes after cessation of infusion. Fetal hindlimb oxygen delivery (Do2) fell significantly with arginine vasopressin infusions of 4.5 and 9.0 mU/min, but was unchanged during the 0.72 mU/min dose (Figure 30). All Do2 values returned to control levels during the recovery period. The fall in hindlimb oxygen delivery at the medium and high doses (to 72.5 ± 2.0 % and 69.9 + 4.2% of control, respectively) mirrors the reductions in mean hindlimb blood flow seen with these infusions (to 71.8 ± 1.9% and 67.7 ± 3.2% of control) (Figure 31). During the AVP infusions oxygen consumption (V o2) was maintained (Figure 32) due to a rise in oxygen extraction (to 126.9 + 5.4% and 168.0 ± 8.5% of the control values for the medium and high doses, respectively) (Figure 33). Mean V o2 was significantly reduced during the recovery phase following the 4.5 mU/min AVP infusions. This is the effect of the small, but not significant, reductions in both hindlimb oxygen delivery (95.9 ± 3.5% of control) 134 CO CM CM t-( |OJUJO0 UJOJJ S B U B L j O %) 9jnss9Jd |BU9^B |Biei ui 96uBqo 135 £ <D E in o CM ~1 LO ~1 LO "1 o o m eg oo o in o CM o CD + in + o CD + in + co in o o CO CD C o 3 O ) 'c c O ) CD J D E o CD E i -co O co -_ CO .s -° tl) x; p3 CQ O H . CO o CM m CM (|0JJU00 IU0JJ a6uBL)0 %) ejBJ JJB9L| |Bie} Uj 96UBL|0 136 (|OJJUOO LUOJ{ 9BUBLJ0 % ) MOIJ pooiq quijipinu. |Biej U; eBuaqo 137 co > o o CD o o o o o o o CO o o co CCJ O CX co cd co O O > § CO fi O • i B B co fi .E J> r l C B co QO '8.-B > 5 -H co •—' r* S E | . CO l-i CO > j2 c? d ^ T3 00.> CX Jd * Q * CO ._, O g « CO P -D co S-2 T3 B cd CO CO 3 . CN C \ C co C B 2Pxi co co § E 2 ^ -X ! cd cd CO fi co C co B O ( I 0JJU00 JO %) AJOAI|8P ue6Axo qunipuju, |ejej UJ O B U B I J Q 138 (lOJJUOO jo %) MO|J pooiq qunipuRj \e\&i u\ o6ueu,o 139 tz c E E. => CD E E E c sali CM o sali ai CD > o o CD o co c o o LO CM c o 1— 3 S <u x ° Ei CJ 3 o cl > CJ C ^ B •a a i ? a g e I «i X fi B CN ^-9, ecu B 3 • E a . B « 3 T3 'C cd c i * x a 'cx'cS oo T3 fi 1/1 "—' B S -3 -6 GO 3 "8 D •§ fi "S 6 to & > ^ < 0 g c>2 C X I « w 1 s I +i B H u S 1 00 CJ (/j ^ I l l ' s <-> ~3 ^ • 1 s CN B 2 O cd oo CL 3 H io o LO io co LO o fe .fi < > oo CU <U O <u d 00 w (|0j}U00 JO o/o) uoudwnsuoo ueBAxo quinpinu, lejej u; e6ueu,o 140 CD > O o CD c o 'co 3 c o o CO CA CO o «> co c § ' C ° s 5? ja | ° . S <u "a J C C D § e S 73 CM eg Iw co - -8 I T 3 CO cj c ca 'C > •3. .S .11 § | oo. £ o a II 53 § •§ « X ! JS c o  c co 00 6 '53 'C eg 00 "3 -fc; co 6 CO I* cx X CO ' co p a ti CO co n, .3 CO CO £ CO O I t CO w CO 3 o 0 0 o CO o o CM O O O CO o CD 2 £ > 7 3 ^ fa.a 0 > (I0JJU00 JO %) uojpBJixe ueBAxo qoijipuju |ejej w e6ueuQ 141 and extraction (84.9 ±7.1% of control) which were observed during recovery from the 4.5 mU/min dose. 7.3.2 Arginine vasopressin effects on the adult Significant increases in plasma arginine vasopressin levels were also found in adult sheep given exogenous AVP. From an overall control value of 1.3 + 0.2 pg/ml, plasma AVP levels reached mean concentrations of 47.9 ± 5.3, 71.1+ 7.6, and 159.7 ± 16.2 pg/ml for infusion dosages of 25 mU/min (62.5 ng/min), 45 mU/min (112.5 ng/min), and 90 mU/min (225 ng/min) doses, respectively (Figure 34). Plasma AVP levels increased rapidly, achieving a significant difference from the control values after 30 minutes and remaining elevated throughout the infusion period (Figure 35). Arginine vasopressin levels returned to pre-infusion levels within 30 minutes after the infusion was ended. Clearance rates were determined in the adult ewes using the same methods as were used with the fetal animals: rate of hormone infusion (per kg body weight) divided by the average of the 75 and 90 minute arterial plasma AVP concentrations. Clearance values for the 25 mU/min (62.5 ng/min ), 45 mU/min (112.5 ng/min), and 90 mU/min (225 ng/min) doses were 23.9 + 6.6, 21.6 ± 2.4, and 19.0 + 2.4 ml-min"1-kg"1, respectively. Thus fetal clearance of AVP was 3.6 to 4.5 times greater than in the adult. No changes in maternal pH, Po2, or Pco2 were observed during arginine vasopressin infusion (Table 16). Blood base excess tended to rise during the recovery phase, achieving a significant difference from the control values for the 25 and 90 mU/min doses (to 6.1 ± 0.4 and 5.9 + 0.4 mFiq/L, respectively). Similarly, hemoglobin concentrations were lower during vasopressin infusion with changes significant at the 25 and 90 mU/min doses. Arterial lactate concentrations were significantly elevated during the medium (45 mU/min) and high (90 mU/min) dose AVP infusions and, although lactate values decreased during the recovery phase, they remained high relative to the control values. Little change in arterial 142 .3 CO CD *J CD KJ CD fa.S 13 > 5 (|Lu/Bd) UO! ;BJ }U90UOO dAV euiSB|d j |npv 143 (|uu/6d) u o | } B J i u 9 o u o o dAV e w s B | d unpv CA CA a a o CA 03 > CD C DC C '55 .3 00 O O d -H ON "fr ON O O ? in oo "fr 00 CN* -H cn cn (N O CN CN 00 d -H 00 NO* cn d +i NO cn "fr d -H ON in o -H CN d -H VO ON CN d •H ON 00 o d NO d o d d cn d +i "fr i > cn CN T-H d +l o o "fr* E o as fi 3 8 o d +i ON NO "fr >n CN +l cn +1 CN (S cn in d +i in "fr cn d -H NO ON 8 d CN CN d ON cn cn - H cn cc 03 a o CA CS > C C s. e 1 P E " T fi O '53 .3 o d -H ? "fr CN in ON CN •H ON in o CN in d -H ON in cn -H oo NO* cn -H o "fr* cn «n d •H cn d -H in CN* NO d +i 00 CN* CN d +i in ON* CN d -H O ON* cn d -H VO ON cn o d in d in o d -H ON in d CN O in cn d ON o d +i NO 00 cn o o o CN d +i "fr NO cn* 03 CA u a © CA 05 <U C S CN e o • H CA .3 fi 3 NO O O d +! ON ON o o d -H o d +l P "fr in CN* -H ON O CN in CN* +1 l > "fr CN -H CN ON 4— cn d "fr • d •H d -H in -H T - H d cn NO* T - H T—( +1 in in* cn NO d -H "fr NO* cn "fr d -H m "fr* CN d +l cn "fr CN d +l "fr ON* cn d -H NO d cn o in "fr 9 o 8 d -H NO "fr CN d +i T-H •> cn* T-H d -H CN 00 cn 00 CN d -H 00 "fr cn X 00 X £ £ 00 X £ £ CO CO CD CD c S 3 PQ c ^ ob &b o ^ £ g 4J co 73 S 732 *c S < 145 glucose values was observed except for a significant increase during the 90 mU/min infusion period. Table 17 presents the cardiovascular and metabolic data obtained from the non-pregnant adult ewes prior to infusing arginine vasopressin. These data are presented as absolute values (mean ± SEM). No significant differences were found between the control data of the three groups of animals. The low dose arginine vasopressin did not alter arterial pressure in the adult animals but at the medium and high dosages arterial pressure rose an average of 17.9 ± 2.6 and 35.9 ± 3.8% above control values, respectively (Figure 36). The hypertension was evident by 30 to 45 minutes into the infusion period. Arterial pressure had returned to pre-infusion values by 30 minutes into the recovery phase. At all three doses of the drug, arginine vasopressin infusion to the adult ewes caused a very rapid bradycardia with heart rate remaining low for the duration of the infusion period (Figure 37). Mean heart rate fell by 28.5 ± 1.9,39.9 ± 1.6, and 38.0 ± 3.4% for the 25,45, and 90 mU/min AVP doses, respectively. The recovery phase was marked by a rapid return of heart rate to the pre-infusion values. Figure 38 shows the changes in adult hindlimb blood flow at each sampling point during the infusion and recovery phases. Although blood flow tended to be less during all three doses of AVP, no significant changes were seen at the 25 mU/min level. The 45 and 90 mU/min doses were associated with reductions in flow that were statistically significant after 45 to 60 minutes of infusion. Regardless of the AVP regimen, during the recovery phase blood flow quickly returned to pre-infusion values. Mean hindlimb oxygen delivery was unchanged for the animals given 25 mU/min of arginine vasopressin whereas this variable fell to 72.5 ± 4.5 and 68.6 ± 7.6% of control values during the 45 and 90 mU/min AVP infusions (Figure 39). During the recovery phase, Do2 returned to control values for the low and medium doses but was significantly elevated (111.5 ± 8.4% of control) for the ewes that had received the 45 mU/min dose. As 146 TABLE 17. Cardiovascular and metabolic data obtained from non-pregnant adult ewes prior to receiving arginine vasopressin infusions at doses of 25 mU/min (2 experiments), 45 mU/min (3 experiments), or 90 mLVmin (3 experiments). Data are presented as absolute values (mean ± SEM). ARGININE VASOPRESSIN DOSE 25 mU/min 45 mU/min 90 mU/min Arterial pressure (mmHg) 76.3 ± 0.4 76.2 + 6.5 69.3 + 10.0 Heart rate (beats/minute) 91.3 ± 3.7 104.2 ± 15.8 102.0 ± 10.8 Hindlimb blood flow (ml-min"1) 98.0 + 3.4 76.6 + 3.9 65.0 + 6.0 Oxygen delivery (^molmin1) 632.9 + 29.3 578.2 + 47.8 545.2 ± 71.7 147 (IOJJUOO UJOJJ 96UBL)O % ) ejnssejd |Bu9jjB ;inpB u\ 96UBHO 148 ( | O J U J O O I U O J J 8 B U B L | 0 % ) 9 J B J i J B e q u n p B i n 9 6 U B U , O 149 £ E E I £ I => z> r> E E S in in o CM o> CM + O CO o m o CM o o + in + o CD in o co + m 1— + co CD C 3 o 'co 3 c "c c CD .Q E o CD E ^ O <-> ii co a >o S 6 co (3 co . 3 50 O H-> S '55 X o oo y CO O H «» CO co CO jo 3 fi 2 CO . 3 8 3 E 3 . t . * > ~ •a d £ . 8 s < ^ s « •a i | H 8 a 3 CO co CO 3 co c cd ya QJ »T3 -^H > x5 X -a 'S 3 -S a •a 8 3 *d ~ 3 <D ra co « 9-ON g g 00a t, j i g 5 § ° * 6 2^X5 co <; 00 II 3 3 5 3 W O CO o o <M O CM O O CO O 00 pjj co -i; g 3 5 '§ 5 -a co O E s g 8 (lojjuoo L U O J J eBueqo %) M O J J pooiq qainpujq unpe in eBueno 150 E E E I I I o o o o o o o o o " W ' ' - c > a > c o r ~ - < O L O (I0JJU00 JO %) A j e A | | 9 p u e B A x o q i u j i p u i u j i n p e ui e B u e u o 151 in the fetus, the change in hindlimb oxygen delivery in the adult animals reflected the alterations in hindlimb blood flow during AVP infusion. Specifically, for the low, medium, and high arginine vasopressin doses, hindlimb blood flow during infusion was reduced to 89.3 ± 7.0, 77.8 ± 4.5, and 73.6 ± 7.8% of control values (Figure 40) while Do2 fell to 80.9 ± 6.9, 72.5 ± 4.5, and 68.6 ± 7.6% of control (Figure 39). 7.3.3 Fetal versus adult responses to arginine vasopressin concentration In order to analyze fetal and adult responses to arginine vasopressin infusion, we wanted to compare similar levels of plasma AVP. This was achieved by combining the data obtained from the 25 and 45 mU/min (62.5 ng/min and 112.5 ng/min) doses given to the adults (5 experiments) with the fetal 0.72,4.5 and 9.0 mU/min (1.8, 11.25, and 22.5 ng/min) doses (10 experiments). The baseline or control value for the fetuses was 2.1 ± 0.4 pg/ml and 1.4 ± 0.2 pg/ml in the adults. These values were not significantly different. The total AVP levels (average of control and infusion values) were also not statistically different (39.1 + 3.2 pg/ml in the fetus; 50.4 ± 5.3 pg/ml in the adult), but the average AVP concentrations during infusion were significantly higher in the adult than fetus (58.9 ± 4.7 versus 45.0 ± 3.0 pg/ml). Recovery samples were not included in the comparisons so as to avoid any latent effect from the drug infusion. Thus the responses to arginine vasopressin infusion in the fetus and adult were compared over a range of approximately 1 to 112 pg/ml. The mean plasma clearance of arginine vasopressin from the fetal animals (0.72,4.5 and 9.0 mU/min doses) was 83.6 ± 11.2 ml-min"1, kg"1. In the adults, AVP clearance (25 and 45 mU/min doses) averaged 22.5 + 2.5 mlmin'kg"1. Increasing plasma AVP levels were associated with a rise in arterial pressure with the hypertension for a given arginine vasopressin level tending to be greater in the fetal sheep (Figure 41). The data were best described by polynomial regression equations. However, no significant difference in the fetal and adult regression equations was found. Over the plasma arginine vasopressin concentrations studied, both fetal and adult sheep 152 CD > o o 0 c o "co 3 c o o co o c •G co M * v 8 CD -t-» -o CO o CM o o O CD O CO o 1^- o CD ( IOJUJOO 10 o/ 0) M O | i pooiq qujjipuju ;|npe u; erjueiio 153 CO ~ Q. c o CO k— •+—• c CD CO c o o 00 00 CD k— Q. o oo CO > CD c 'c D) i— CO co E « jo Q_ CO c e PO^ T3 ca <3 CO CO S J CO'tS C1* fi . f i fi:H T3 3 00 d.S* CO CA co X O ^ CO c 55 O co « H b CO - CO 73 ,<u .2 co 1 e C CA ._ CA CO CO C CO 3 cr CO c o o o o © i! fi O CO _ C CO CO O > fO 3 § H cx cx " t OA CN m r- vo d d ll II S .2 CO CA _- t—i O 3 CX JH cd CO U - . -H . 3 W-\ 3 CO < » c7\ CO J H '—v r—H co =2 -£ £0 fi £ 1 = 1 u l e g • ° 3 .5 CXT3 C O + X ON CA & 3 3 . , =5 O X ^ C -Z3 CO O 2 g<n + X in d + C *i CO CO " 3 * c | o rt ^ ° c TT fi cO W '55 S. . co 00 CO CO CX CX § C CO O > 3 <N CN X x C N C N © © 5 © © d II II 3 (%) ejnssejd iBuejjB u| GBUBUQ 154 exhibited bradycardia as AVP levels increased (Figure 42). Although there was no difference between the y-intercepts, the logarithmic decline was significantly greater in the adult animals compared to the fetal results. For example, at a plasma arginine vasopressin concentration of 75 pg/ml, fetal heart rate had decreased approximately 18% from the control values compared to 36% in the adult sheep. Figure 43 illustrates the relationship between plasma arginine vasopressin concentration and the percentage change in hindlimb blood flow in both adult and fetal sheep. As AVP levels increased, hindlimb blood flow, as measured with a transit-time flow transducer on the external iliac artery, fell dramatically. The data were best described by logarithmic regression equations. Although the change in hindlimb blood flow was greater in the fetus compared to the adult, the regression equations were not significantly different. Hindlimb vascular resistance is normally calculated as follows: Hindlimb (arterial pressure - hindlimb venous pressure) vascular resistance = hindlimb blood flow However, in the current study, venous pressures were not available. As venous pressure is generally low (~l-3 mmHg) in both adults and fetuses, we considered it to be negligible and estimated hindlimb resistance as arterial pressure over hindlimb blood flow. Thus, we likely slightly overestimated our calculation of vascular resistance in the hindlimb. Figure 44 shows the relationship between plasma arginine vasopressin levels and the percentage change in hindlimb resistance from control for fetal and non-pregnant ewes. Over the range of AVP concentrations examined (1-112 pg/ml), hindlimb resistance rose with increasing AVP levels with no difference in the regression equations seen between fetal and adult sheep. 155 (%) 9JBJ peel) uj arJueqo 156 (%) MO|j pooiq quijipiuq in e6ueqo 157 to 3 (0 0 cj H O ) Q . o c CD O c o o CO CO CD C L O CO co > CD c 'c O ) u. CO co E CO J O D_ 3 O > S.2 a X ! 22 3 I s 6 - 3 •-; "C CO 0 0 <u S 3 -s CX <U O 0 0 ' - 3 3 £ cd is •8 g oog cd i I T ) O O d d CX CX 3 oo*.: <u s ta <u CX O fiC S ° ohs g 3 S T 3 y '55 X J S.1 s i ~* —. cd gs xt-CN O vo d d II II g o 2 <R co g C 3 0 0 0 0 + o 1—I > 5 v '3 •8 .5 '§ '5 CX 3 CX O IS go g « co W cj e « o « O cd >-i \ 3 3 w w - 1"3 o <U Jd .u U<£ td .13 Q 3 a-a ^ ^ 55 23 oo 3 ^ cj cd CJ cj T J w > I-I <*_ cd X X as - H as as •«t ON + + cs CM o o ' d d M CJ 'tog Q CO (%) eouBiSjsej jB|nosBA quijipmu, ui. 96UBU.O 158 7.4 Discussion 7.4.1 AVP levels-normal and pathophysiological Under normal conditions arginine vasopressin levels in the fetal and adult sheep are very low: <10 pg/ml as measured by bioassay (Rurak, 1978; Alexander et al, 1974) or <5 pg/ml when determined by radioimmunoassay (Akagi and Challis, 1990b; Akagi and Challis, 1990a; Iwamoto etal, 1979). In the current study, control AVP levels were 1.8 ± 0.2 and 1.3 ± 0.2 pg/ml in the fetal and adult animals, respectively. Exogenous AVP administration led to plasma vasopressin levels of 7-106 pg/ml in the fetus. As there were no significant changes in fetal hemoglobin concentration and the blood lost to sampling was replaced with an equal volume of maternal blood after every sample, we do not believe that changes in blood volume contributed to the rise in plasma AVP levels. The hemorrhage-induced threshold for arginine vasopressin release occurs at withdrawal of ~13% of fetal blood volume (Ross et al, 1986b). In the current study <2% of fetal blood volume was removed at each sample. Furthermore, in fetuses receiving saline vehicle infusions only, the blood sampling protocol was identical to animals receiving the hormone and the saline animals did not exhibit a change in AVP concentrations. Rurak (1978) administered exogenous arginine vasopressin to fetal lambs at doses of 2.25, 4.5, and 9.0 mU/min (0.5 to 4.1 mUmin'kg"1). This resulted in fetal plasma AVP levels of 15-505 pg/ml (6-202 /^ U/ml). Despite administering similar doses of exogenous arginine vasopressin (0.17,4.5, and 9.0 mU/min), when expressed per kg fetal weight our doses were lower (0.2-3.2 mUmin'kg"1) which is reflected in our lower plasma AVP concentrations (7-106 pg/ml or 3-42 /^ U/ml). Our hormone levels were more similar to those of Iwamoto (1979) who achieved plasma AVP levels of 17-91 pg/ml (6.8-36.4 |<U/ml) by administering vasopressin to fetuses at 0.9 to 2.3 mUmin'kg"1. The fetal arginine vasopressin levels achieved in this study appear to be physiologically relevant. Akagi (1990b) examined the effect of graded hypoxemia, achieved 159 by reducing maternal inspired oxygen, on AVP levels in fetal lambs of 125 to 147 days gestation. No change in plasma AVP levels was observed when carotid arterial Po2 was reduced by 4.0 to 5.3 mmHg; however, a reduction in Po2 of 8.3 mmHg was associated with a significant rise in plasma arginine vasopressin levels to 16.3-24.2 pg/ml. In addition, Daniel et al. (1983) examined the effect of various types of hypoxemia on fetal arginine vasopressin levels. Reduced maternal inspired oxygen (10% oxygen), partial occlusion of the umbilical cord for 20 minutes (blood flow values were not given), and complete occlusion of the umbilical cord for 2 minutes resulted in plasma AVP levels of 31.9 ± 0.91, 50.1 ± 11.45, and 160.4 ± 12.45 pg/ml, respectively. In a hemorrhage study, Drummond (1980) observed that a 20% reduction in fetal blood volume resulted in plasma AVP levels of 31.7 + 14.7 pg/ml in late gestation. With more severe hemorrhage (Robillard etal., 1979) or hypoxemia (Rurak, 1978) vasopressin levels rise to >200 pg/ml. Plasma arginine vasopressin concentrations of >450 pg/ml have been reported in fetuses during spontaneous (Stark etal., 1979) and induced parturition (Stark etal., 1981). No rise in vasopressin levels was seen during the preparturient period suggesting that the elevated vasopressin levels are a result of intrapartum stress and are not related to the initiation of parturition (Stark et al., 1979). A recent study has demonstrated that the antepartum and intrapartum arginine vasopressin concentrations in normal and growth-restricted late gestation fetal lambs appear to be quite different (Tan, 1997). In the normal fetuses, AVP rose from 4.2 ± 0.8 pg/ml pre-labour to a maximum of 98.3 ± 30.2 pg/ml at delivery. In contrast, the growth-restricted fetal lambs had a pre-labour AVP concentration approximately 2.7 times higher (11.7 ± 1.4 pg/ml). At delivery, AVP levels in these fetuses averaged 480.1 ± 102.7 pg/ml. Thus, the growth-restricted fetus has an elevated basal AVP level compared to appropriate-for-gestational-age fetal lambs. Furthermore, the stress of labour is associated with increased arginine vasopressin levels, with poor fetal condition, such as growth-restriction, significantly augmenting this pattern. 160 Rurak (1978) also administered arginine vasopressin to non-pregnant ewes at doses of 23.1, 46.3, 92.7, and 185.3 mU/min (0.4 to 2.9 mU-min" 'kg"1) resulting in plasma concentrations of 100-1170 pg/ml. With the exception of the highest dose, we administered similar amounts of AVP (25, 45, and 90 mU/min or 0.4 to 1.7 mUmin'kg"1). Plasma vasopressin levels between 1 and 256 pg/ml resulted. We observed a fall in hemoglobin concentration during AVP infusion to the non-pregnant ewes. Adult animals did not receive blood to replace that lost to sampling; however, only 4 ml of blood was removed during each sample for a total loss of 52 ml over the course of an experiment. Assuming an adult sheep blood volume of 53.5 ml/kg (Giles et al, 1977), this represents a total blood loss of <2% which is unlikely to have contributed significantly to the rise in arginine vasopressin levels. Rurak (1978) also reported a fall in hematocrit in adult sheep given exogenous arginine vasopressin. This may be due to a rise in plasma volume, which is an AVP effect reported in adult dogs (Szczepanska-Sadowska, 1973). Elevated plasma arginine vasopressin levels, similar to the range achieved by this study, are also seen in newborn and adult sheep in response to stress. Like the fetus, hemorrhage in adult sheep is associated with increases in plasma AVP levels (Wintour et al, 1995; Hjelmqvist etal, 1991; Cameron etal, 1985) and lambs subjected to phlebotomy of 10 to 20% of blood volume experience 37- to 44-fold increases in AVP (Leake et al, 1979). Uterine contractions, particularly at delivery, and vaginocervical stimulation increase vasopressin concentrations in the ewe (Kendrick et al, 1991). Unlike fetal lambs, plasma arginine vasopressin levels do not increase significantly in adult ewes breathing 9-10% oxygen (Curran-Everett et al, 1988; Stegner etal, 1984b; Rurak, 1978). However, vasopressin levels of >300 pg/ml are seen with severe hypoxemia when maternal inspired air is reduced to 5% oxygen (Stark et al, 1984b). Thus, hypoxemia is a potent stimulus for vasopressin release in fetal lambs, but a more profound hypoxemia is required for a similar AVP secretion in adult ewes (Stark et al, 1982). 161 7.4.2 Blood gas and metabolic responses The blood gas and metabolic variables obtained during the control periods are within the normal range seen for fetal and adult sheep in our laboratory. The fall in pH and base excess seen in the recovery phase following the infusion of vehicle (saline) is puzzling (Table 14). When these data were calculated as a change from the individual animal's control, no significant change in these variables was found suggesting that inter-animal variation contributed to this observation. Rurak and Gruber (1984) examined the effect of vasopressin on fetal lamb oxygenation using an AVP infusion rate similar to ours (4.5 mU/min for 60 minutes or 1.4 to 3.5 mUmin^kg"1). They observed a significant increase in fetal arterial Po2 following 60 minutes of AVP infusion (from 20.0 ± 1.0 to 23.1 ± 1.0 mmHg) but not at the 30 minute sample; significant declines in pH and base excess were observed at both points. Iwamoto (1979), employing lower doses of AVP (0.9 to 2.3 mUmin'kg"1), also reported a rise in Po2 after 30 minutes of arginine vasopressin infusion (from 21.9 ± 0.7 to 24.1 + 0.8 mmHg) but no change in pH. In the current study, fetal arterial Po2 rose from 19.2 + 0.6 to 20.0 ± 0.5 mmHg and pH fell from 7.316 ± 0.011 to 7.311 ± 0.007 but the changes did not reach statistical significance (Table 14). Rurak and Gruber (1984) attributed the rise in vascular Po2 seen during vasopressin infusion to an increase in umbilical blood flow and hence increased oxygen delivery to the fems. As oxygen consumption was unaltered, the lower extraction of oxygen from the blood led to a rise in Po2. Iwamoto (1979) did not see a significant rise in umbilical blood flow but peripheral blood flow was reduced 45%. In our study, umbilical blood flow was not measured but fetal hindlimb blood flow fell to 71.8 + 1.9% and 67.7 ± 3.2% when AVP was administered at 4.5 and 9.0 mU/min, respectively. This correlated with significant decreases in hindlimb oxygen delivery to 72.5 ± 2.0% and 69.9 ± 4.2% of control values. Increased fractional oxygen extraction meant that hindlimb consumption was unchanged during AVP infusion. The rise in oxygen extraction led to a reduction in hindlimb venous 162 Po2 from 14.9 ± 0.6 to 14.1 ± 0.5 mmHg at the 4.5 mU/min dose. Although this drop was not significant, the steepness of the relationship between fetal Po2 and oxygen saturation (oxyhemoglobin dissociation curve) at this value was reflected in a significant increase in hindlimb arterial-venous oxygen content difference during AVP infusion (from 0.58 ± 0.05 to 0.79 ± 0.06 mM). The observation that Po2 in the descending aorta did not fall suggests that greater umbilical blood flow and/or reduced placental oxygen extraction occurred and were able to compensate for the changes in oxygenation seen across the hindlimb. As with the fetus, no change in arterial Po2 or pH was observed in the non-pregnant ewe. The observation that exogenous AVP does not have an effect on adult blood gases has been previously reported (Nuyt etal., 1996; Harper and Rose, 1987; Rurak, 1978). Arterial lactate concentrations were elevated during AVP infusion, reaching values of 1.10 ± 0.43, 0.59 ± 0.05, and 0.76 ± 0.07 mM at the 25,45, and 90 mU/min doses, respectively. As venous values were not available, we could not determine hindlimb lactate flux for the adults. Thus, it is unclear whether lactate release from the hindlimb contributed to the rise in circulating lactate levels or whether other peripheral tissues are responsible. Lactate values began to decline during the recovery phase but were still above control levels. This lactic acidemia likely contributed to the elevated base excess during the recovery phase, but was insufficient to alter arterial pH. Hindlimb oxygen delivery was reduced to 80.9 ± 6.9%, 72.6 ± 4.5%, and 68.6 ± 7.6% of control values during the low, medium, and high AVP doses. This reflects vasoconstriction in the hindlimb tissues as illustrated by the similar reduction in mean hindlimb blood flow (89.3 ± 7.0%, 77.8 ± 4.5%, and 73.6 ± 7.8% of control values). Peripheral vasoconstriction has been reported in adult sheep (Vizsolyi and Perks, 1976) and dogs (Liard etal., 1982; Heyndrickx etal., 1976) in response to AVP administration. Adult hindlimb blood flow was quite variable over the course of the sampling (Figure 38). The variability in these data reflects the difficulty of obtaining hindlimb blood flow from semi-mobile adult animals. For example, when the animals lie 163 down their legs are positioned beneath the body resulting in a reduction in arterial blood flow whde a switch to a standing position often causes a transient hyperemia. 7.4.3 Cardiovascular responses Since 1895 it has been recognized that vasopressin causes an increase in arterial pressure and vasoconstriction in most vascular beds (Share, 1988; Liard et al, 1982; Iwamoto et al, 1979). In the current study, infusion of AVP at 4.5 mU/min was associated with a reduction in hindlimb blood flow of 28.2 ± 1.6%. Fetal arterial pressure rose 16.5 ± 1.6%, and heart rate fell 17.2 ± 1.5%. Previous reports in which AVP was administered at ~4 mU/min observed fetal arterial pressure increases of 5-29% and 20-25% reductions in heart rate (Irion et al, 1990; Tomita et al, 1985; Wiriyathian et al, 1983; Rurak, 1978). Iwamoto (1979), whose range of infusion doses (0.9 to 2.3 mUmin'kg 1; mean = 1.4 ± 0.1 mUmin^kg"1) is most similar to ours, reported a 19% rise in arterial pressures and 17% fall in heart rate in the fetus. Thus, the degree of hypertension and bradycardia noted in our fetuses in response to arginine vasopressin infusion is similar to the values observed in other studies. Over the range of plasma AVP concentrations we achieved (1-112 pg/ml) by administering vasopressin at a maximum dose of 9.0 mU/min to the fetus, arterial pressure rose steadily (Figure 41) while heart rate fell dramatically and then appeared to plateau (Figure 42). Wiriyathian (1983) found that the rise in arterial pressure is vasopressin dose-dependent but the fall in heart rate is not. These data are in contrast to Rurak (1978) who reported that the bradycardia was AVP dose-dependent but hypertension was not. Both of these studies involved maximum vasopressin infusions of ~9 mU/min. At infusion rates <9 mU/min, Irion's data (1990) appear to support those of Wiriyathian, i.e., arterial pressure but not heart rate changes in a dose-dependent manner. However, in fetal lambs, the change in heart rate with vasopressin dose occurs in a step-like pattern with approximately a ten-fold increase in AVP dose necessary to further alter heart rate (Irion et al, 1990). For 164 example, fetal heart rate was reduced -20% at doses of 2.4 and 8 mU/min, fell another 10% at AVP doses of 24 and 80 mU/min, and at 240 mU/min heart rate was 40% less than control (Irion et al, 1990). In contrast, arterial pressure increases in a curvilinear fashion: rising in a dose-dependent manner then leveling off at ~30% above control values at doses beyond 24 mU/min. Thus, when considered over a wide range of arginine vasopressin administration (~0-300 mU/min), both fetal heart rate and arterial pressure initially change in a dose-dependent manner with arterial pressure then reaching a plateau (Irion et al, 1990; Tomita et al, 1985). This is still consistent with our observations. At the doses we employed (maximum 9.0 mU/min), arterial pressure was still rising as AVP levels were not high enough to have resulted in a plateau. With increasing vasopressin levels, fetal heart rate fell rapidly and then plateaued as would be expected at our doses. Tomita's data (1985) suggest that if we had administered higher doses of arginine vasopressin (~24 mU/min), fetal heart rate would have experienced another rapid decline followed by a plateau phase, and arterial pressure would also reach a plateau. Adult sheep also exhibit hypertension and bradycardia during the exogenous infusion of arginine vasopressin. We administered AVP at doses of 25, 45, and 90 mU/min to non-pregnant ewes and noted rises in arterial pressure of 10.0 ± 5.1%, 17.9 ± 2.6%, and 35.9 ± 3.8% as well as reductions in heart rate of 28.5 ± 1.9%, 39.9% ± 1.6, and 38.0 ± 3.4%, at the respective doses. Rurak (1978) infused similar doses of AVP and achieved arterial pressures 9-16% above control and 20-45% reductions in heart rate. When vasopressin was given at 3.2 mUmin'kg', a 27% rise in arterial pressure and 48% decline in heart rate was seen in adult sheep (Harper and Rose, 1987). Thus, the cardiovascular responses seen in our ewes are similar to those observed in other studies with the exception of the degree of hypertension seen at the 90 mU/min dose (~36% above control) which is higher than previously reported. This is not due to differences in AVP doses as our levels were in the lower range obtained in the other studies. A possible difference is that our 165 infusion period lasted 90 minutes as opposed to only 30 minutes in the Harper and Rose (1987) study and 60 minutes in that of Rurak (1978). At the three vasopressin doses used in our study, adult arterial pressure appeared to increase in a dose-dependent manner (Figure 36) but the degree of bradycardia was similar at the 45 and 90 mU/min doses (Figure 37). As was seen with the fetus, when these variables were examined over the range of plasma AVP levels achieved (1-112 pg/ml), arterial pressure rose steadily (Figure 41) while heart rate initially fell rapidly and then leveled off (Figure 42). Ling (1989) noted dose-dependent changes in both arterial pressure and heart rate in adult sheep given AVP at levels similar to ours. However, in Ling's study the highest plasma AVP concentration achieved was ~70 pg/ml which is similar to the concentration we observed (71.1 ± 7.6 pg/ml) with our 45 mU/min dose. The dose-dependent nature of vasopressin-induced bradycardia was not apparent in fetal lambs until a wider range of arginine vasopressin infusion rates was used (Irion et al., 1990; Tomita et al, 1985). Similar experiments employing a wider range of infusion rates will also be required in adult sheep to determine whether the vasopressin-induced bradycardia in adults is of a dose-dependent nature. It is apparent from the present and previous studies that cardiovascular changes occur at AVP concentrations that are pathophysiologically relevant (e.g., during hypoxemia or hemorrhage) in fetal and adult sheep. The hypertension appears to be a result of marked vasopressin-induced peripheral vasoconstriction. In both the fetus and adult sheep, we noted large reductions in hindlimb blood flow during vasopressin infusion. Iwamoto (1979) also reported reductions in flow to the peripheral circulation and the gut. The mechanism by which arginine vasopressin affects the heart, however, is not certain. The increase in blood pressure observed with vasopressin infusion could stimulate the baroreceptors resulting in bradycardia However, in fetal and adult sheep, bradycardia was observed following intravenous AVP infusion even after the hypertensive response was blocked with nitroprusside or selective vasoconstrictor (V,-receptor) antagonists (Ervin el 166 al, 1992; Harper and Rose, 1987). No change in heart rate or arterial pressure is seen following infusion of a selective V2-receptor agonist (desmopressin) (Ervin et al, 1992). In addition, in fetal lambs given AVP, the fall in heart rate has been reported to precede or occur simultaneously with the pressor effect (Wiriyathian etal, 1983; Iwamoto etal, 1979). In the Iwamoto study (1979), the average time for the onset of bradycardia following AVP infusion was 4.0 + 0.4 minutes while hypertension began on average 5.3 + 1.1 minutes after the initiation of drug administration. Iwamoto (1979) also administered atropine to fetal lambs during vasopressin infusion to determine if the bradycardia was vagally mediated. Atropine failed to return heart rate to control levels. Furthermore, vasopressin administration to bilaterally vagotomized fetal lambs (Miyake etal, 1991) or adult dogs (Varma et al, 1969) results in persistent but smaller decreases in heart rate. These data suggest that the fall in heart rate observed during vasopressin infusion is not solely a reflex response to the increase in arterial pressure. Bradycardia may also be caused by vasopressin-induced constriction of coronary vessels (Varma etal, 1969) as, in adult dogs (Liard et al, 1982; Heyndrickx etal, 1976) and goats (Fernandez etal, 1998), vasopressin infusion reduces coronary blood flow. However, blood flow to the heart is unchanged during AVP administration to fetal lambs (Iwamoto et al, 1979) negating this as the cause of myocardial depression in the fetus. In adult dogs a persistent although attenuated bradycardia in seen in response to vasopressin infusion following complete denervation of the heart (Wang et al, 1987). Furthermore, vasopressin-induced bradycardia is still seen in fetal lambs following autonomic blockade with hexamethonium (Tomita etal, 1985). Thus, although the mechanism is not clear, it appears that vasopressin acts, at least in part, directly on the heart to produce bradycardia in the adult and fetus. The difficulties encountered when attempting to determine the exact role that AVP plays in cardiovascular regulation reflect the intricacies of the in vivo system. Given the numerous interactions involved in cardiovascular regulation, including those among the autonomic nervous system, renin-angiotensin system, and arginine vasopressin, it is difficult 167 to experimentally alter one variable without influencing the remaining mechanisms (Bennett and Gardiner, 1985). Elevated vasopressin levels seen under stressful conditions clearly do affect the cardiovascular system of fetal and adult sheep. The role played by AVP at basal concentrations is less apparent but likely also considerable and may involve both pressor and anti-diuretic functions (Share, 1988; Bennett and Gardiner, 1985; Cowley etal, 1983). 7.4.4 Fetal versus adult responses There are very few reports in the literature in which cardiovascular responses to vasopressin infusion have been compared in fetal and adult sheep. Rurak (1978) infused AVP into fetal lambs at 0.5 to 4.1 mUmin'kg"1 and into non-pregnant ewes at 0.4 to 2.9 mUmin'kg"1 for 60 minutes. This resulted in plasma vasopressin concentrations of ~15-505 pg/ml in the fetuses and 100-1200 pg/ml in the adults. Hypertension and bradycardia were seen in both groups, but the pressor effect of AVP was greater in the fetal lamb. Relative to the adult, the slope of the dose-response curve for arterial pressure was increased and shifted to the left. In the fetal lambs, arterial pressure rose 27 to 43% while heart rate fell 14 to 41%. Arterial pressure was elevated 9 to 16% above control values in the adult ewes while heart rate fell 20 to 45%. In contrast to the results of Rurak, Harper and Rose (1987) reported a similar rise in arterial pressure but a greater fall in heart rate in the ewe compared to the fetus. Exogenous vasopressin was infused to fetal and adult sheep at 3.2 mUmin'kg"1 for 30 minutes. Fetal plasma AVP levels averaged 113.2 ± 40.7 pg/ml but adult plasma concentrations were not reported. Arterial pressure rose similarly in the fetus and ewe (23.8% versus 26.5%) but fetal heart rate fell only half as much as the adult (26.2% compared to 47.6%). The range of arginine vasopressin doses administered in the current study were slightly lower than in the above two reports. AVP was given for 90 minutes at 0.2 to 3.2 mUmin'kg 1 (mean = 1.6 ± 0.3 mUmin'kg"1) to the fetal lambs and between 0.4 and 0.9 mUmin'kg"1 (mean = 0.6 ± 0.1 mUmin'kg"1) to the adults. Over the range of plasma 168 vasopressin concentrations studied (1-112 pg/ml), arterial pressure rose an average of 15.5 ± 1.3% in the fetuses and 14.5 ± 2.7% in the adults. Vasopressin-induced bradycardia was twice as great in the adult compared to the fetus (34.2 ± 1.7% versus 16.3 ± 1.3%). Thus our findings are similar to Harper and Rose (1987) in that exogenous AVP administration led to similar pressor effects but a greater bradycardia in the adult relative to fetal sheep. Our data were generally in the lower range of that reported by Rurak (1978) and Harper and Rose (1987) reflecting our lower AVP doses. Neither of these authors measured hindlimb blood flow, which we found to fall to a similar degree in the fetus and adult during vasopressin infusion. Although venous pressures were not available, hypertension and vasoconstriction indicate an increase in vascular resistance. A regression analysis of estimated hindlimb resistance yielded no significant difference between the fetus and ewe. There is, therefore, no evidence for a greater sensitivity of fetal vascular smooth muscle to arginine vasopressin. In contrast, the fetus does appear to be less sensitive to the negative chronotropic effect of vasopressin. This may be a protective mechanism as, compared to the adult, the fetus has a limited ability to increase stroke volume in order to maintain cardiac output when heart rate is reduced (Heymann, 1989). A possible explanation for the differing results in the above studies comparing AVP cardiovascular responses in the adult and fetus could be differences in the phase of the estrous cycle of the non-pregnant ewes. In adult rats, the pressor response to arginine vasopressin is related to both gender and stage of the estrous cycle with males and estrous females demonstrating a greater response than non-estrous females (Wang etal., 1997; Wang et al., 1996). This appears to be due to a difference in vasopressin release as metabolic clearance of AVP is not different (Crofton etal., 1988). This variation with phase of the estrous cycle suggests that the gonadal steroids may play a role. In ovariectomized rats, plasma vasopressin concentration is decreased by progesterone while estrogen has been reported to have no effect on (Crofton et al., 1985) or to increase AVP levels (Skowsky et al., 1979). In non-pregnant adult ewes, progesterone concentrations similar to 169 those observed during the luteal phase of the sheep estrous cycle (with or without appropriate levels of estradiol) cause significant reductions in arterial pressure and increase plasma volume (Pecins-Thompson and Keller-Wood, 1997; Roesch and Keller-Wood, 1997). Unlike with rats, progesterone implants in sheep are associated with slightly increased levels of AVP; however, vasopressin levels are increased to a similar degree with sham implants (Pecins-Thompson and Keller-Wood, 1997). Rurak (1978) administered vasopressin to his non-pregnant ewes at 0.4 to 2.9 mU-min'-kg"1. As the AVP doses in the current study were on the lower end and those of Harper and Rose (1987) were on the higher end of this range, we would expect his non-pregnant ewes to demonstrate increases in arterial pressure intermediate to these two studies (i.e., between 14.5 and 26.5%). However, Rurak observed only 9-16% increases in arterial pressure. This blunted pressor response may be due to his ewes being in the luteal phase of the estrous cycle (increased progesterone concentration). This explanation cannot be ruled out as in none of these studies was the estrous cycle phase of the ewes determined. Clearance of arginine vasopressin in our fetal lambs was 3.7 times as great as in the adult ewes (83.6 ± 11.2 ml-min -^kg"1 versus 22.5 ± 2.5 mlmin'kg'1). These clearance values are similar to those in previous fetal and adult sheep studies (Harper and Rose, 1987; Tomita et al, 1985; Stegner et al, 1984a; Wiriyathian et al, 1983). However, these values are higher than those reported by Rurak (1978) who calculated clearances of 23.3 ±1.6 mlmin'^ kg"1 in the fetal lamb and 8.1 ± 0.5 ml-min"1-kg"1 in the non-pregnant ewe. This may reflect that plasma AVP concentrations were determined by radioimmunoassay in the former studies while bioassay was used by Rurak. Bioassays for arginine vasopressin are much less precise as they tend to be less specific and less reproducible (Mohring etal, 1982; Johnston, 1972). An overestimate in vasopressin values would lead to an underestimate of clearance. Despite differences in the absolute values, Rurak (1978) also found fetal clearance of AVP to be ~3-fold greater than in the adult. Under basal 170 conditions, AVP levels in the adult and fetus are similar, thus the fetus must have a much higher rate of vasopressin secretion. The placenta has been suggested as the major site of vasopressin clearance as, in exteriorized fetal lambs, AVP levels in the umbilical vein are substantially lower than in the umbilical artery (Jones and Rurak, 1976). However, in lambs studied in utero, no significant plasma AVP arteriovenous concentration gradient was found across the umbilical circulation (Wiriyathian et al, 1983). Moreover, a lack of correlation between maternal and fetal paired blood samples and between twin fetal lambs indicates that arginine vasopressin does not cross the placenta (Iwamoto etal., 1979; Rurak, 1978). Elevations in fetal plasma AVP concentrations due to fetal stress (hemorrhage or hypoxemia) are associated with high levels of vasopressin in the urine (Ross et al., 1986a; Ross et al., 1986b; Daniel et al, 1984). Thus, it appears that AVP is cleared by the fetal kidney as it is in adults. This is supported by the observed increase of arginine vasopressin levels in amniotic fluid, at rates consistent with fetal urine excretion following administration of the hormone (Ross et al, 1985; Stark et al, 1984a; Wiriyathian et al, 1983). Because the amnion and chorion of the ovine fetus are extensively vascularized, substances may be transferred between the amniotic fluid and the fetal circulation via blood vessels on the surface of these membranes (Hedriana etal, 1997; Brace, 1995; Hedriana etal, 1995; Brace etal, 1992; Gilbert etal, 1991b). For example, AVP injected intra-amniotically leads to increased vasopressin levels in fetal plasma even when the esophagus is ligated (Gilbert et al, 1991b) or a glove is sewn over the fetal head (Gilbert et al, 1991a). During intravenous AVP infusion, blood flow to the fetal membranes initially increases and then declines (Hedriana et al, 1997). The increased blood flow could facilitate AVP clearance from plasma to the amniotic compartment while the subsequent decline in perfusion would reduce the rate of A VP resorption via the intramembranous pathway. However, although AVP appears able to cross from the amniotic cavity to the plasma via the intramembranous pathway, no evidence has been presented for the converse route (i.e., from plasma directly to amniotic fluid). Thus, AVP is 171 most likely cleared from the fetal circulation by the kidney while amniotic fluid may serve as a reservoir for the hormone. In the current study, arginine vasopressin infused to fetal lambs at 4.5 mU/min resulted in a reduction in hindlimb blood flow to 71.8 ± 1.9% of control. This is similar to the reduction in flow observed during mild ischemia (77.4 ± 0.3% of control) achieved by partial inflation of an occluder on the fetal external iliac artery (Section 6). There were no differences in the change in arterial Po2 (0.8 ± 0.3 versus -1.1 ± 0.6 mmHg from control), oxygen saturation (1.4 ± 0.9 versus -1.7 ± 1.1% from control), or oxygen content (0.00 ± 0.05 versus -0.21 ± 0.13 mM below control) between the vasopressin and mild ischemia experiments. In both studies, lactate was released from the hindlimb, significantly more so with mild ischemia (-3.16 ± 1.75 /imol-min"1- lOOg"1) than with AVP (-0.64 ± 0.53 /miol-min"1, lOOg"'). The greater efflux with mild ischemia likely reflects the small sample size (n=3) as lactate efflux during moderate (0.73 ±1.1 ^molmin"1- lOOg"1) and severe ischemia (-0.25 ± 1.62/miolmin"1- lOOg"1) were more consistent with the vasopressin results. Vasopressin administration was also associated with a greater hindlimb glucose uptake compared to mild ischemia (4.92 ± 0.34 versus 0.73 ± 0.57 /^ mol-min"1- lOOg"1). However, glucose flux was consistently low in all the ischemia animals, even at control. It should be noted that the lactate and glucose fluxes were not significantly different from their respective control values for either experiment. No significant bradycardia or hypertension were observed with mild ischemia. In contrast, during administration of 4.5 mU/min AVP, fetal arterial pressure was significantly elevated (to 16.5 ± 1.6% above control), and heart rate was reduced 17.2 ± 1.5% from the control value. The degree to which hindlimb oxygen delivery was reduced was similar for the arginine vasopressin infusion (72.5 ± 2.0% of control) and mild ischemia (73.1 ± 3.1% of control) experiments. In both cases, oxygen consumption was maintained due to a rise in extraction. 172 In summary, we infused arginine vasopressin to fetal lambs and adult ewes to achieve plasma hormone levels which are observed during various forms of stress including hemorrhage and hypoxemia Both the fetus and non-pregnant ewe responded similarly to the AVP administration. No changes in arterial pH or Po2 were seen. Vasopressin did reduce oxygen delivery to the hindlimb by decreasing hindlimb blood flow. Over the range of plasma AVP levels studied (1-112 pg/ml), adult and fetal sheep demonstrated similar degrees of hypertension and increased vascular resistance. However, bradycardia was more profound in the adult. Thus, we did not find evidence for a greater sensitivity of fetal vascular smooth muscle to vasopressin, but the fetal heart does appear to be less sensitive to the negative chronotropic effects of this hormone. In addition, the reductions in hindlimb blood flow and oxygen delivery observed with the 4.5 mU/min arginine vasopressin infusion were similar to those seen with mild ischemia. The fetuses responded similarly to the two perturbations: oxygen consumption was maintained by a rise in extraction and there were no differences in arterial Po2, oxygen saturation, or 0 2 content. Lactate efflux from the hindlimb was less during vasopressin administration, and glucose uptake was greater. However, neither of these variables were significantly different from their respective control values. No significant cardiovascular changes were seen with mild ischemia while exogenous arginine vasopressin resulted in fetal bradycardia and hypertension. Thus, for a similar reduction in hindlimb blood flow, the fetus responded similarly whether this reduction was caused by mechanical means (partial occlusion of the external iliac artery) or by exogenous administration of a hormonal vasoconstricting agent (arginine vasopressin). The exception to this was in the cardiovascular response. That the bradycardia and hypertension recorded during AVP infusion were not evident during mild ischemia very likely reflects the local nature of the latter perturbation compared to the whole body effects of vasopressin infusion. 173 8. SUMMARY AND CONCLUSIONS The purpose of our experiments was to determine hindlimb blood flow and metabolic responses to normal and pathophysiological conditions in the late gestation fetal lamb. The hindlimb, which is composed of skin, bone, and skeletal muscle, is considered to be representative of the fetal carcass. As the carcass, or non-visceral tissues, represents the majority of fetal mass and receives a large proportion of cardiac output, metabolic changes in these tissues can profoundly affect the fetus as a whole. We examined the fetal vascular anatomy and devised a surgical protocol which allowed us to sample blood entering and leaving the hindlimb as well as to continuously measure hindlimb blood flow using a transit-time flow transducer. Normal weight and blood flow distribution patterns could then be determined for the hindlimb. Under normal conditions, hindlimb blood flow appears to fluctuate with fetal behavioural state. As the mechanisms involved are unknown, we examined this relationship further. Despite the significant nature of the carcass, little information is available regarding its metabolic responses to abnormal situations such as reduced oxygen supply. Although, circulating lactate levels are known to rise more markedly when oxygen delivery is reduced by hypoxemia than when reduced by decreased blood flow, the role of the carcass in this response has not been examined. This prompted our study of hindlimb reactions to hypoxemia and ischemia. Arginine vasopressin (AVP) has been implicated in the cardiovascular and vasoconstrictor responses to hypoxemia in the fetus and adult. However, a direct comparison of these responses in the adult and fetus has not been previously conducted. In addition, the vasoactive properties of vasopressin presented an opportunity to compare fetal responses to vasoconstriction achieved by exogenous administration of this hormone versus that accomplished by mechanical means in the ischemia experiments. Hindlimb Blood Flow Measurement: Correlation of Radionuclide-Labeled Microsphere and Transit-Time Flow Transducer Techniques In order to study hindlimb blood flow and oxygen consumption in the late gestation fetal lamb, we needed to devise a surgical protocol for sampling blood entering and leaving the hindlimb as well as obtain continuous measurements of hindlimb blood flow. Catheters positioned in the pudendoepigastric artery and vein and advanced into the external iliac artery and vein, respectively, permitted sampling of hindlimb blood. To investigate whether a transit-time flow transducer positioned on the external iliac artery could provide accurate, continuous blood flow readings, measurements obtained by this method were compared to results achieved by injection of 15 pirn diameter radionuclide-labeled microspheres. Five fetal lambs were instrumented with tracheal and vascular catheters, a vascular occluder on the external iliac artery proximal to the circumflex iliac artery and a transit-time flow transducer distal to this vessel. Ffindlimb blood flow was altered by partial inflation of the occluder. Findings were as follows: i) . Blood flow measurements obtained by the two methods show a close correlation (r = 0.865 to 0.999). ii) . These results, in addition to evidence from vascular casting, indicate that the circumflex iliac, internal iliac, and pudendoepigastric arteries appear to be potential sources of error when measuring total hindlimb blood flow with the transit-time flow transducer. Ligation of these vessels (or catheterization of in the case of the pudendoepigastric artery) improved the correlation between the two methods. iii) . With the incorporation of ligation of these vessels into our surgical protocol, a transit-time flow transducer on the external iliac artery can be used to provide continuous accurate determinations of hindlimb blood flow in the late gestation fetal Iamb. 175 Hindlimb Blood Flow and Weight Distribution There is little information on the effects of occlusive-type catheterization on hindlimb blood flow and growth. In 6 late gestation fetal Iambs, we compared hindlimb weight and blood flow distribution under normal conditions. Longer, larger diameter (1.19 mm outer diameter) catheters were inserted into the non-study limb compared to the study limb (0.94 mm outer diameter). Blood flow was measured with a transit-time flow transducer on the external iliac artery. We observed: i) . In late gestation (~135 days), the hindlimb is approximately 7.34% of fetal body weight. ii) . The study and non-study limbs did not differ statistically in terms of hindlimb weight, but blood flow was 13.4 ± 1.8% less in the non-study limb. iii) . This difference in blood flow between the study and non-study limbs appears to be due to differences in the surgical instrumentation (longer, larger diameter catheters used in the non-study limb) rather than to an inherent difference in blood flow between right and left hindlimbs. iv) . Relative to the percentage of hindlimb weight that they comprise, fetal hindlimb skin and bone are more highly perfused than skeletal muscle i.e., receive a higher proportion of hindlimb blood flow. Although muscle comprised approximately 45% of hindlimb weight, it received only 22% of total hindlimb blood flow. 176 Behavioural State Modulation of Hindlimb Blood Flow The late gestation fetal lamb, skeletal muscle activity, in the form of breathing and body movements, are associated with specific patterns of electrocortical (ECoG) and electro-ocular (EoG) activity. In 8 experiments, ECoG and EoG activity were monitored in 7 fetal lambs between 126 and 137 days gestation. Fetal blood gas status, and metabolic and cardiovascular variables were determined during a control (pre-blockade) period and following administration of gallamine triethiodide or pancuronium bromide (post-blockade). These neuromuscular blocking agents abolish fetal breathing and body movements. Our results are as follows: i) . Neuromuscular blockade did not alter sleep cycle length or the amount of time the fetus spent in each electrocortical state. ii) . Following neuromuscular blockade, mean hindlimb blood flow was reduced 8.3 ± 3.5% (p = 0.0510) suggesting that skeletal muscle activity is associated with greater hindlimb blood flow. Blood flow tended to be higher during high voltage electrocortical activity (quiet sleep) both before and during neuromuscular blockade, but the values failed to reach statistical significance. Thus, more data is required before this relationship can be considered conclusive. The current data suggest that neuromuscular blockade did not alter the apparent behavioural state modulation of flow in the fetal lamb hindlimb. As skeletal muscle activity does not appear to be responsible for this phenomenon, this relationship may be directly mediated by the central nervous system. iii) . The difficulty in obtaining statistically significant differences in hindlimb blood flow with behavioural state or neuromuscular blockade may reflect that the changes in blood flow being considered, when expressed in either absolute or percentage terms, are very small. The average flow difference between electrocortical states was ~1.5 ml min" 177 lOOg"1 or ~5%, and neuromuscular blockade reduced flow by ~3.1 mlmin"1- lOOg"1 or ~8%. Thus, animal condition, measurement variability, and artifacts during data collection may impact the results. In addition, fetal breathing movements, which are associated with the low voltage state and are also abolished by neuromuscular blockade, may also contribute to the reduction in mean hindlimb blood flow seen following gallamine or pancuronium injections. Uterine contractures, postural changes by the ewe, and fetal arousal (awake) episodes are other potential confounding factors. iv) . Based on the polygraph recordings, hindlimb blood flow appeared to be more variable during quiet sleep and neuromuscular blockade seemed to reduce this variability. We attributed this trend to the prevalence of limb movements during high voltage activity. Variability in mean hindlimb blood flow, expressed as a coefficient of variation, fell from 7.2 ± 0.4% pre-blockade to 7.0 ± 0.5% post-blockade. During high voltage ECoG activity, blood flow variability was reduced from 7.1 ± 0.6% pre-blockade to 6.1 ± 0.5% post-blockade. However, these difference were not significant. This lack of significance may reflect that our data acquisition system did not record data frequently enough to capture the rapid variations in blood flow associated with hindlimb activity. v) . Neuromuscular blockade resulted in an increase in fetal oxygenation with fetal Po2 increasing 15.3%, oxygen saturation 17.1%, and oxygen content 17.3%. Hindlimb oxygen consumption fell 11.8 ± 4.6%. This suggests that inhibition of skeletal muscle activity, such as occurs during fetal hypoxemia, may be a compensatory mechanism which lowers fetal oxygen demands when faced with a reduction in oxygen supply. No difference in oxygen consumption was seen between the high and low voltage ECoG states. This may be the result of the low sample size (4 pairs of samples) or that the complexity and duration of hindlimb movements during the episodes of high voltage activity were insufficient to increase oxygen consumption. 178 Hypoxemia and Ischemia The objective of this study was to compare fetal metabolic and cardiovascular responses to two methods of reducing oxygen delivery to the hindlimb. Hypoxemia is, by definition, a reduction in arterial Po2 while ischemia causes a reduction in perfusion while Po2 is maintained. Because fetal Po2 is low by postnatal standards, factors which reduce Po2 even lower may affect oxygen diffusion into fetal tissues and thus lower oxygen consumption. In addition, when oxygen delivery is reduced by hypoxemia, circulating lactate levels are higher than when oxygen delivery is reduced via decreased blood flow. This led to the hypothesis that hypoxemia would be less well tolerated by the fetus than ischemia and this would be reflected in an earlier fall in oxygen consumption and/or greater release of lactate by the hindlimb which would contribute to systemic lactic acidemia. Hypoxemia studies, achieved by reducing maternal inspired oxygen levels, were conducted on 7 fetal lambs. Six fetuses were involved in the ischemia experiments, with blood flow reduced by partial inflation of a vascular occluder on the external iliac artery. The results were classified as mild, moderate, or severe based on the reduction in hindlimb oxygen delivery achieved (to ~75, 50, and 25% of the control values, respectively). Our results are as follows: i). Arterial and venous Po2 values fell significantly with increasing severity of hypoxemia. During severe hypoxemia, arterial Po2 fell from a control value of 20.2 ± 0.6 mmHg to 13.0 +1.0 mmHg and venous Po2 fell to 7.4 ±1.2 mmHg from a control value of 16.4 ± 0.5 mmHg. In contrast, arterial Po2 was unchanged with ischemia and the reduction in venous Po2 was much less (from 17.6 ± 0.9 mmHg during control to 14.3 ±1.2 mmHg during severe ischemia). With severe hypoxemia, there were also significant reductions in pH, base excess, and oxygen saturation. No changes in these variables were observed with ischemia. 179 ii) . The fall in oxygen delivery seen with ischemia was a reflection of the reduction in hindlimb blood flow. Although no change in blood flow was seen with mild hypoxemia, the moderate and severe levels were associated with reduced hindlimb perfusion which exacerbated the reduction in oxygen delivery achieved by lowering arterial Po2. iii) . A curvilinear relationship between hindlimb oxygen delivery and consumption was found with no difference in the curves for hypoxemia and ischemia For both perturbations, a rise in oxygen extraction allowed oxygen consumption to be maintained until delivery was reduced by ~50%. An earlier, but not significant, reduction in consumption seen with hypoxemia may reflect inhibition of skeletal muscle activity. Our oxygen delivery versus consumption results align well with data from other researchers despite the fact that our values are based on hindlimb rather than whole body responses. iv) . Despite greater reductions in arterial and venous (tissue) Po2 values during hypoxemia compared to ischemia, the oxygen diffusion gradient (arterial-venous Po2) was not statistically different for the two interventions. As a result, for similar reductions in oxygen delivery, hindlimb oxygen consumption was similar for both hypoxemia and ischemia v) . Glucose was taken up by the hindlimb during the control period. Glucose continued to be taken up during and after both hypoxemia and ischemia at rates not different from control. vi) . Hindlimb lactate changed from net uptake during control to net release during both hypoxemia and ischemia, with the pooled mean lactate efflux values significantly greater than the controls. Lactate was released from the hindlimb during the hypoxemia intervention, whereas with ischemia, the majority of lactate output occurred 180 during the recovery or post-ischemia period. However, there was no difference in the overall magnitude of the lactate efflux between the two perturbations. vii) . Arterial lactate values rose significantly with hypoxemia (to 5.07 ± 0.48 mM during severe hypoxemia) but were unchanged with ischemia, which reflects the local nature of hindlimb ischemia versus the whole body effects of hypoxemia. By extrapolating hindlimb lactate efflux to the entire carcass and using published values for fetal lactate clearance during normoxia and severe hypoxemia, it was estimated that carcass lactate output could account for ~29 to 100% of systemic lactate production during hypoxemia Thus, the carcass (non-visceral tissues) could be an important source of the elevated circulating lactate concentrations observed during this perturbation. viii) . Hypoxemia is associated with hypertension, bradycardia, and increased vascular resistance. In contrast, no change in arterial pressure or heart rate was observed with ischemia, although hindlimb vascular resistance rose markedly. The increase in resistance with both hypoxemia and ischemia appears to be mainly a reflection of decreased hindlimb blood flow. ix) . We conclude that, for similar reductions in oxygen delivery, hypoxemia is not associated with an earlier fall in oxygen consumption or greater hindlimb lactate efflux compared to ischemia Thus, there is no evidence that hypoxemia is less well tolerated than hindlimb ischemia in the late gestation fetal lamb. 181 Arginine Vasopressin Infusion In both fetal and adult sheep, arginine vasopressin (AVP) is released from the posterior pituitary in response to stresses such as hypoxemia and hemorrhage. In addition to its antidiuretic effects, AVP acts on vascular smooth muscle V, receptors to elicit a pressor response. In this study, we compared the effects of exogenous AVP administration in fetal and non-pregnant adult ewes. A 90 minute infusion of AVP was given to fetal lambs at doses of 0.72 (n=l), 4.5 (n=8), and 9.0 mU/min (n=l) yielding a range of 0.2 to 3.2 mU-min'-kg"1 fetal weight Two fetuses received vehicle (saline) only. AVP was also administered to adult ewes at 25 (n=2), 45 (n=3), and 90 mU/min (n=3) yielding a range of 0.3 to 1.7 mUmin'kg 1 adult weight. Plasma vasopressin levels, determined by radioimmunoassay, varied from 7-106 pg/ml in the fetuses and 1-256 pg/ml in the adults. Our observations are as follows: i) . Basal AVP levels were not different between fetal and non-pregnant adult sheep (1.8 ± 0.2 verses 1.3 ± 0.2 pg/ml). ii) . The plasma hormone levels achieved are consistent with those seen in various forms of stress including hypoxemia and hemorrhage. iii) . No change in circulating AVP levels, blood gases, or metabolic variables were seen in the 2 fetuses that received vehicle (saline) only. iv) . No change in arterial blood gases or pH were seen in the fetal or adult animals during AVP administration. Arterial glucose and lactate concentrations tended to rise during vasopressin infusion in the fetus, but the fluxes was not different from control values. Likewise, circulating glucose and lactate levels increased in the adult, but flux values could not be determined. v) . In both fetal and adult sheep, hindlimb blood flow fell significantly with arginine vasopressin infusion. This was reflected in similar reductions in oxygen delivery. 182 Hindlimb oxygen consumption was not determined in the adults, but, in the fetal lambs, consumption was maintained by a rise in oxygen extraction. vi) . Fetal and adult responses to vasopressin were compared over a range of approximately 1-112 pg/ml. The dose response curves for the 2 groups demonstrated similar degrees of hypertension and increased hindlimb vascular resistance. However, the decline in heart rate was much more profound in the adult (averaging 34.2 ± 1.7% compared to 16.3 ± 1.3% in the fetus). vii) . We conclude that there is no evidence for a greater sensitivity of fetal vascular smooth muscle to arginine vasopressin, but the fetal heart does appear to be less sensitive to the negative chronotropic effects of this hormone. viii) . As has been found by other researchers, clearance of arginine vasopressin by the fetus is approximately 3 times as great as in the adult (83.6 ± 11.2 versus 22.5 ± 2.5 mf min"1-kg"1). ix) . The reduction in fetal hindlimb blood flow and oxygen delivery seen at the 4.5 mU/min dose is similar to that achieved during mild hindlimb ischemia. The fetuses responded similarly to the two perturbations: oxygen consumption was maintained by a rise in extraction, and arterial Po2,02 saturation and content were similar. In neither case was lactate efflux or glucose uptake different from control values. However, bradycardia and hypertension were seen with AVP administration, but were not evident with ischemia. This reflects the local nature of ischemia compared to the whole body responses generated by vasopressin. Thus, with the exception of the cardiovascular effects, the fetus responds similarly to vasoconstriction achieved by a hormonal vasoconstricting agent (arginine vasopressin) or by mechanical means (ischemia). 183 General conclusions Contrary to popular belief, the fetus is very responsive to its environment. Even in normal situations, the late gestation fetus experiences cyclical changes in behavioural state, including electrocortical and electro-ocular activity, as well as fetal breathing and body movements. Blood flow, cardiovascular activity, and metabolic variables respond to these behavioural state alterations. For example, hindlimb blood flow tends to be higher and more variable during high voltage electrocortical activity, but more data are required before these relationships can be considered conclusive. However, our data suggest that neuromuscular blockade, which abolishes skeletal muscle activity, does not alter the apparent behavioural state modulation of hindlimb blood flow. Thus, this relationship may not be due to a linkage between skeletal muscle activity and ECoG state but rather may result from direct action of the central nervous system. The fetus is also relatively adaptable to pathophysiological situations. When faced with a reduction in oxygen delivery, the fetus attempts to maintain oxygen consumption by increasing the extraction of oxygen from the blood. Blood flow may also be preferentially redistributed to the vital organs, such as the heart and brain, and away from peripheral tissues like the carcass. However, these compensatory mechanisms can be limited by the development of systemic and tissue lactic acidemia In addition, when oxygen supply is diminished, total body oxygen demands are lowered by reducing or abolishing fetal breathing and body movements. For similar reductions in oxygen delivery, hypoxemia and ischemia appear to be equally well tolerated: the oxygen delivery versus consumption curves are similar and the magnitude of hindlimb lactate efflux is not different for the two perturbations. However, circulating lactate concentrations are significantly greater with hypoxemia The carcass contribution to this situation could be substantial, accounting for ~29—100% of the total systemic lactate production during this perturbation. Hypoxemia has whole body effects while ischemia is primarily of a local nature. This is evident in the cardiovascular responses 184 to these interventions: no significant cardiovascular changes are evident with ischemia, while hypoxemia is associated with fetal bradycardia and hypertension. Arginine vasopressin has been implicated in the cardiovascular responses to hypoxemia observed in both the fetus and adult. In our study, over a similar range of arginine vasopressin levels, fetal and non-pregnant adult sheep exhibited similar degrees of hypertension and peripheral vasoconstriction. However, the fetus appears to be less sensitive to the negative chronotropic effects of this hormone. In addition, when similar reductions in hindlimb blood flow were compared, the fetus responded similarly whether this was achieved hormonally (arginine vasopressin) or by mechanical means (ischemia). Vasopressin infusion elicited systemic cardiovascular effects (hypertension and bradycardia) which were not evident with ischemia Thus, both hypoxemia and arginine vasopressin generate whole body responses in the fetus while hindlimb ischemia is a local phenomenon. In conclusion, we were able to catheterize and instrument the hindlimb of the late gestation in utero fetal lamb in order to monitor its responses to normal and abnormal conditions. As the hindlimb is composed of skin, skeletal muscle, and bone, it is considered to be representative of the fetal carcass (non-visceral tissues). Our studies illustrate that the hindlimb, and thus the carcass, plays a major role in the dynamic nature of the fetus under normal conditions as well as in its ability to adapt to pathophysiological situations. 185 9. REFERENCES Akagi, K. and Challis, J. R. G. (1990a). Hormonal and biophysical responses to acute hypoxemia in fetal sheep at 0.7-0.8 gestation. Canadian Journal of Physiology and Pharmacology 68(12): 1527-1532. Akagi, K. and Challis, J. R. G. (1990b). Threshold of hormonal and biophysical responses to acute hypoxemia in fetal sheep at different gestational ages. Canadian Journal of Physiology and Pharmacology 68(5): 549-555. Alexander, D. P., Bashore, R. A., Britton, H. G., and Forsling, M. L. (1974). Maternal and fetal arginine vasopressin in the chronically catheterised sheep. Biology of the Neonate 25: 242-248. Apatu, R. S. and Barnes, R. J. (1991). Blood flow to and the metabolism of glucose and lactate by the liver in vivo in fetal, newborn and adult sheep. Journal of Physiology 436: 431-447. Archie, J. P., Jr., Fixler, D. E., Ullyot, D. J., Hoffman, J. I. E., Utley, J. R, and Carlson, E. L. (1973). Measurement of cardiac output with and organ trapping of radioactive microspheres. Journal of Applied Physiology 35(1): 148-154. Assali, N. S., Brinkman, C. R., Jr., and Nuwayhid, B. (1974). Comparison of maternal and fetal cardiovascular functions in acute and chronic experiments in the sheep. American Journal of Obstetrics and Gynecology 120(3): 411-425. Baer, R. W., Payne, B. D., Verrier, E. D., Vlahakes, G. J., Molodowitch, D., Uhlig, P. N., and Hoffman, J. I. E. (1984). Increased number of myocardial blood flow measurements with radionuclide-labeled microspheres. American Journal of Physiology 246: H418-H434. Bard, H., Battaglia, F. C , Makowski, E. L., and Meschia, G. (1972). The synthesis of fetal and adult hemoglobin in sheep during the perinatal period. Proceedings of the Society for Experimental Biology and Medicine 139: 1148-1150. Bendeck, M. P. and Langille, B. L. (1992). Changes in blood flow distribution during the perinatal period in fetal sheep and lambs. Canadian Journal of Physiology and Pharmacology 70(12): 1576-1582. Bennett, T. and Gardiner, S. M. (1985). Involvement of vasopressin in cardiovascular regulation. Cardiovascular Research 19: 57-68. Bocking, A. D., Harding, R, and Wickham, P. J. D. (1985). Relationship between accelerations and decelerations in heart rate and skeletal muscle activity in fetal sheep. Journal of Developmental Physiology 7(1): 47-54. Boddy, K. and Dawes, G. S. (1975). Fetal breathing. British Medical Bulletin 31(1): 3-7. Boyle, D. W., Hirst, K., Zerbe, G. O., Meschia, G., and Wilkening, R. B. (1990). Fetal hind limb oxygen consumption and blood How during acute graded hypoxia Pediatric Research 28(2): 94-100. 186 Boyle, D. W., Meschia, G., and Wilkening, R. B. (1992). Metabolic adaptation of fetal hindlimb to severe, nonlethal hypoxia. American Journal of Physiology 263: R1130-R1135. Brace, R. A. (1983). Blood volume and its measurement in the chronically catheterized sheep fetus. American Journal of Physiology 244(4): H487-H494. Brace, R. A. (1995). Current topic: Progress toward understanding the regulation of amniotic fluid volume: water and solute fluxes in and through the fetal membranes. Placenta 16: 1-18. Brace, R. A. and Brittingham, D. S. (1986). Fetal vascular pressure and heart rate responses to nonlabor uterine contractions. American Journal of Physiology 251: R409-R416. Brace, R. A., Gilbert, W. M., and Thornburg, K. L. (1992). Vascularization of the ovine amnion and chorion; A morphometric characterization of the surface area of the intramembranous pathway. American Journal of Obstetrics and Gynecology 167(6): 1747-1755. Bristow, J., Rudolph, A. M., Itskovitz, J., and Barnes, R. (1983). Hepatic oxygen and glucose metabolism in the fetal lamb: Response to hypoxia Journal of Clinical Investigation 71(5): 1047-1061. Buckberg, G. D., Luck, J. C , Payne, D. B., Hoffman, J. I. E., Archie, J. P., and Fixler, D. E. (1971). Some sources of error in measuring regional blood flow with radioactive microspheres. Journal of Applied Physiology 31(4): 598-604. Burd, L. I., Jones, M. D., Jr., Simmons, M. A., Makowski, E. L., Meschia, G., and Battaglia, F. C. (1975). Placental production and foetal utilisation of lactate and pyruvate. Nature 254: 710-711. Burton, R. G. and Gorewit, R. C. (1984). Ultrasonic flowmeter. Uses wide-beam transit-time technique. Medical Electronics 15(2): 68-73. Cameron, V., Espiner, E. A., Nicholls, M. G., Donald, R. A., and MacFarlane, M. R. (1985). Stress hormones in blood and cerebrospinal fluid of conscious sheep: effect of hemorrhage. Endocrinology 116(4): 1460-1465. Campbell, S. S. and Tobler, I. (1984). Animal sleep: A review of sleep duration across phylogeny. Neuroscience and Biobehavioral Reviews 8: 269-300. Carter, A. M. (1989). Factors affecting gas transfer across the placenta and the oxygen supply to the fetus. Journal of Developmental Physiology 12: 305-322. Chestnut, D. H., Weiner, C. P., Thompson, C. S., and McLaughn, G. L. (1989). Intravenous administration of d-tubocurarine and pancuronium in fetal lambs. American Journal of Obstetrics and Gynecology 160(2): 510-513. Cissik, J. H., Ehler, W. J., Hankins, G. D., and Snyder, R. R. (1991). Cardiopulmonary reference standards in the pregnant sheep (Ovis aries): A comparative study of ovine and human physiology in obstetrics. Comparative Biochemistry and Physiology 100A(4): 877-880. 187 Clapp, J. F., Ill, Szeto, H. FL, Abrams, R., Larrow, R., and Mann, L. I. (1980). Physiological variability and fetal electrocortical activity. American Journal of Obstetrics and Gynecology 136(8): 1045-1050. Clewlow, F., Dawes, G. S., Johnston, B. M., and Walker, D. W. (1983). Changes in breathing, electrocortical and muscle activity in unanaesthetized fetal lambs with age. Journal of Physiology 341:463-476. Cohen, W. R., Piasecki, G. J., and Jackson, B. T. (1982). Plasma catecholamines during hypoxemia in fetal lambs. American Journal of Physiology 243: R520-R526. Cohn, H. E., Sacks, E. J., Heymann, M. A., and Rudolph, A. M. (1974). Cardiovascular responses to hypoxemia and acidemia in fetal lambs. American Journal of Obstetrics and Gynecology 120(6): 817-824. Court, D. J., Parer, J. T., Block, B. S. B., and Llanos, A. J. (1984). Effects of beta-adrenergic blockade on blood flow distribution during hypoxemia in fetal sheep. Journal of Developmental Physiology 6(4): 349-358. Cowley, A. W., Jr., Quillen, E. W., Jr., and Skelton, M. M. (1983). Role of vasopressin in cardiovascular regulation. Federation Proceedings 42(15): 3170-3176. Crofton, J. T., Baer, P. G., Share, L., and Brooks, D. P. (1985). Vasopressin release in male and female rats: effects of gonadeetomy and treatment with gonadal steroid hormones. Endocrinology 117(3): 1195-1200. Crofton, J. T., Share, L., and Brooks, D. P. (1988). Pressor responsiveness to and secretion of vasopressin during the estrous cycle. American Journal of Physiology 255(6 part 2): R1041-R4048. Curran-Everett, D. C , Claybangh, J. R., Miki, K., Hong, S. K., and Krasney, J. A. (1988). Hormonal and electrolyte responses of conscious sheep to 96 h of hypoxia. American Journal of Physiology 255(2 part 2): R274-R283. Daniel, S. S., Stark, R. I., Husain, M. K., Sanocka, U. M., and James, L. S. (1984). Excretion of vasopressin in the hypoxic lamb: comparison between fetus and newborn. Pediatric Research 18(3): 227-231. Daniel, S. S., Stark, R. I., Zubrow, A. B., Fox, H. E., Husain, M. K., and James, L. S. (1983). Factors in the release of vasopressin by the hypoxic fetus. Endocrinology 113(5): 1623-1628. Davis, L. E. and Hohimer, A. R. (1991). Hemodynamics and organ blood flow in fetal sheep subjected to chronic anemia American Journal of Physiology 261(6 part 2): R1542-R1548. Dawes, G. S. (1985). The control of fetal heart rate and its variability in lambs. In Fetal Heart Rate Monitoring: Clinical Practice and Pathophysiology, edited by W. Kunzel. Berlin: Springer-Verlag. Dawes, G. S. (1992). Central nervous control. In Fetal Behaviour: Developmental and Perinatal Aspects, edited by J. G. Nijhuis. Oxford: Oxford University Press. 188 Dawes, G. S., Fox, H. E., Leduc, B. M., Liggins, G. C., and Richards, R. T. (1972). Respiratory movements and rapid eye movement sleep in the foetal lamb. Journal of Physiology 220: 119-143. Dawes, G. S., Gardner, W. G., Johnston, B. M , and Walker, D. W. (1983). Breathing in fetal lambs: the effect of brain stem section. Journal of Physiology 335: 535-553. Dawes, G. S., Lewis, B. V., Milhgan, J. E., Roach, M. R., andTalner, N. S. (1968). Vasomotor responses in the hind limbs of foetal and new-born lambs to asphyxia and aortic chemoreceptor stimulation. Journal of Physiology 195: 55-81. Drummond, W. FL, Rudolph, A. M., Keil, L. C., Gluckman, P. D., MacDonald, A. A., and Heymann, M. A. (1980). Arginine vasopressin and prolactin after hemorrhage in the fetal lamb. American Journal of Physiology 238: E214-E219. Edelstone, D. I. (1984). Fetal compensatory responses to reduced oxygen delivery. Seminars in Perinatology 8(3): 184-191. Edelstone, D. I., Darby, M. J., Bass, K., and Miller, K. (1989). Effects of reductions in hemoglobin-oxygen affinity and hematocrit level on oxygen consumption and acid-base state in fetal lambs. American Journal of Obstetrics and Gynecology 160(4): 820-828. Edelstone, D. I. and Rudolph, A. M. (1979). Preferential streaming of ductus venosus blood to the brain and heart in fetal lambs. American Journal of Physiology 237(6): H724-H729. Ervin, M. G. (1988). Perinatal fluid and electrolyte regulation: Role of arginine vasopressin. Seminars in Perinatology 12(2): 134-142. Ervin, M. G., Ross, M. G., Leake, R. D., and Fisher, D. A. (1992). Vl-and V2-receptor contributions to ovine fetal renal and cardiovascular responses to vasopressin. American Journal of Physiology 262(4 part 2): R636-R643. Fernandez, N., Garcia, J. L., Garcia-Villalon, A. L., Monge, L., Gomez, B., and Dieguez, G. (1998). Coronary vasoconstriction produced by vasopressin in anesthetized goats. Role of vasopressin V1 and V2 receptors and nitric oxide. European Journal of Pharmacology 342(2-3): 225-233. Fisher, D. J., Heymann, M. A., and Rudolph, A. M. (1982). Fetal myocardial oxygen and carbohydrate consumption during acutely induced hypoxemia. American Journal of Physiology 242(4): H657-H661. Fletcher, D. J., Hanson, M. A., Moore, P. J., Nijhuis, J. G., and Parkes, M. J. (1988). Stimulation of breathing movements by L-5-hydroxytryptophan in fetal sheep during normoxia and hypoxia. Journal of Physiology 404: 575-589. Gilbert, W. M., Cheung, C. Y., and Brace, R. A. (1991a). Oral-nasal membranes are not the major route for fetal absorption of amniotic fluid arginine vasopressin. American Journal of Obstetrics and Gynecology 165(6 parti): 1614-1620. 189 Gilbert, W. M , Cheung, C. Y., and Brace, R. A. (1991b). Rapid intramembranous absorption into the fetal circulation of arginine vasopressin injected intraamniotically. American Journal of Obstetrics and Gynecology 164(4): 1013-1020. Giles, R. C , Jr., Berman, A., Hildebrandt, P. K., and McCaffrey, R. P. (1977). Use of 59Fe for sheep erythrocyte kinetic studies. American Journal of Veterinary Research 38(4): 535-537. Gilman, A. G., Rail, T. W., Nies, A. S., and Taylor, P., eds. (1990). Goodman and Gilman's The Pharmacological Basis of Therapeutics, eighth ed. New York: Pergamon Press. Gleason, C. A., Roman, C , and Rudolph, A. M. (1985a). Hepatic oxygen consumption, lactate uptake, and glucose production in neonatal lambs. Pediatric Research 19(12): 1235-1239. Gleason, C. A. and Rudolph, A. M. (1985). Gluconeogenesis by the fetal sheep liver in vivo. Journal of Developmental Physiology 7(3): 185-194. Gleason, C. A., Rudolph, C. D., Bristow, J., Itskovitz, J., and Rudolph, A. M. (1985b). Lactate uptake by the fetal sheep liver. Journal of Developmental Physiology 7: 177-183. Gleed, R D., Poore, E. R, Figueroa, J. P., and Nathanielsz, P. W. (1986). Modification of maternal and fetal oxygenation with the use of tracheal gas infusion. American Journal of Obstetrics and Gynecology 155(2): 429-435. Groome, L. J. (1991). A theoretical analysis of the effect of placental metabolism on fetal oxygenation under conditions of limited oxygen availability. BioSystems 26: 45-56. Hacker, N. F. and Moore, J. G. (1986). Essentials of Obstetrics and Gynecology. Philadelphia: W B Saunders Company. Hanson, M. A. (1988). The importance of baro- and chemoreflexes in the control of the fetal cardiovascular system. Journal of Developmental Physiology 10(6): 491-511. Harding, R., Hooper, S. B., and Van, V. K. (1993). Abolition of fetal breathing movements by spinal cord transection leads to reductions in fetal lung liquid volume, lung growth, and IGF-II gene expression. Pediatric Research 34(2): 148-153. Harding, R, Poore, E. R, Bailey, A., Thorburn, G. D., Jansen, C. A. M., and Nathanielsz, P. W. (1982). Electromyographic activity of the nonpregnant and pregnant sheep uterus. American Journal of Obstetrics and Gynecology 142(4): 448-457. Harding, R., Poore, E. R., and Cohen, G. L. (1981). The effect of brief episodes of diminished uterine blood flow on breathing movements, sleep states and heart rate in fetal sheep. Journal of Developmental Physiology 3: 231-243. Harding, R, Sigger, J. N., Poore, E. R, and Johnson, P. (1984). Ingestion in fetal sheep and its relation to sleep states and breathing movements. Quarterly Journal of Experimental Physiology 69(3): 477-486. 190 Harding, R., Sigger, j. N., and Wickham, P. J. D. (1983). Fetal and maternal influences on arterial oxygen levels in the sheep fetus. Journal of Developmental Physiology 5: 267-276. Harper, M. A. and Rose, J. C. (1987). Vasopressin-induced bradycardia in fetal and adult sheep is not dependent on an increase in blood pressure. American Journal of Obstetrics and Gynecology 157(2): 448-453. Hartman, J., Koemer, J., Lancaster, L., and Gorczynski, R. (1985). In vivo calibration of a transit-time ultrasound system for measuring ascending aorta volume flow. The Pharmacologist 27(3): 217. Hassaart, T. H. M. and de Haan, J. (1985). Effects of fetal breathing movements on umbilical venous blood flow in fetal lambs. European Journal of Obstetrics & Gynecology and Reproductive Biology 20: 121-129. Hay, W. W., Myers, S. A., Sparks, J. W., Wilkening, R. B., Meschia, G., and Battaglia, F. C. (1983). Glucose and lactate oxidation rates in the fetal lamb. Proceedings of the Society for Experimental Biology and Medicine 173: 553-563. Hedriana, H. L., Brace, R. A., and Gilbert, W. M. (1995). Changes in blood flow to the ovine chorion and amnion across gestation. Journal of the Society for Gynecological Investigation 2(6): 727-734. Hedriana, H. L., Gilbert, W. M., and Brace, R. A. (1997). Arginine vasopressin-induced changes in blood flow to the ovine chorion, amnion, and placenta across gestation. Journal of the Society for Gynecological Investigation 4(4): 203-208. Heymann, M. A. (1989). Fetal cardiovascular physiology. In Maternal-Fetal Medicine: Principles and Practice, edited by R. K. Creasy and R. Resnick. Philadelphia: W. B. Saunders Co. Heymann, M. A., Payne, B. D., Hoffman, J. I. E., and Rudolph, A. M. (1977). Blood flow measurements with radionuclide-labeled particles. Progress in Cardiovascular Diseases 20(1): 55-79. Heyndrickx, G. R., Boettcher, D. H , and Vather, S. F. (1976). Effects of angiotensin, vasopressin, and methoxamine on cardiac function and blood flow distribution in conscious dogs. American Journal of Physiology 231(5): 1579-1587. Hjelmqvist, H , Ullman, J., Gunnarsson, U. , Lundberg, J. M., and Rundgren, M. (1991). Haemodynamic and humoral responses to repeated hypotensive haemorrhage in conscious sheep. Acta Physiologica Scandinavica 143(1): 55-64. Hohimer, A. R. and Bissonnette, J. M. (1991). Vascular lactic acid infusions do not alter the incidence of fetal breathing movements or their inhibition by acute hypoxemia. Pediatric Research 29(5): 483-486. Irion, G. L., Mack, C. E., and Clark, K. E. (1990). Fetal hemodynamic and fetoplacental vascular response to exogenous arginine vasopressin. American Journal of Obstetrics and Gynecology 162(4): 1115-1120. 191 Itskovitz, J., Goetzman, B. W., Roman, C , and Rudolph, A. M. (1984). Effects of fetal-maternal exchange transfusion on fetal oxygenation and blood flow distribution. American Journal of Physiology 247(4 Part 2): H655-H660. Itskovitz, J., LaGamma, E. F., and Rudolph, A. M. (1983). The effect of reducing umbilical blood flow on fetal oxygenation. American Journal of Obstetrics and Gynecology 145(7): 813-818. Itskovitz, J., LaGamma, E. F., and Rudolph, A. M. (1987). Effects of cord compression on fetal blood flow distribution and C»2 delivery. American Journal of Physiology 252(1 Part 2): H100-H109. Iverson, P. O., Nicolaysen, G., and Benestad, H. B. (1992). Blood flow to bone marrow during development of anemia or polycythemia in the rat Blood 79: 594-601. Iwamoto, H. S. and Rudolph, A. M. (1985). Metabolic responses of the kidney in fetal sheep: Effect of acute and spontaneous hypoxemia. American Journal of Physiology 249: F836-F841. Iwamoto, H. S., Rudolph, A. M., Keil, L. C., and Heymann, M. A. (1979). Hemodynamic responses of the sheep fetus to vasopressin infusion. Circulation Research 44(3): 430-436. Iwamoto, H. S., Rudolph, A. M., Mirkin, B. L., and Keil, L. C. (1983). Circulatory and humoral responses of sympathectomized fetal sheep to hypoxemia. American Journal of Physiology 245: H767-H772. Jansen, C. A. M., Krane, E. J., Thomas, A. L., Beck, N. F. G., Lowe, K. C , Joyce, P., Parr, M., and Nathanielsz, P. W. (1979). Continuous variability of fetal PC»2 in the chronically catheterized fetal sheep. American Journal of Obstetrics and Gynecology 134(7): 776-783. Jansson, T. (1991). The microsphere technique in anaesthetized and awake pregnant guinea-pigs: Reproducibility of repeated blood flow measurements. Acta Physiologica Scandinavica 142(2): 237-245. Jensen, A., Bamford, O. S., Dawes, G. S., Hofmeyr, G., and Parkes, M. J. (1986). Changes in organ blood flow between high and low voltage electrocortical activity in fetal sheep. Journal of Developmental Physiology 8(3): 187-194. Jensen, A. and Berger, R. (1991). Fetal circulatory responses to oxygen lack. Journal of Developmental Physiology 16(4): 181-207. Jensen, A., Hohmann, M., and Ktinzel, W. (1987). Dynamic changes in organ blood flow and oxygen consumption during acute asphyxia in fetal sheep. Journal of Developmental Physiology 9: 543-559. Jensen, A., Roman, C , and Rudolph, A. M. (1991). Effects of reducing uterine blood flow on fetal blood flow distribution and oxygen delivery. Journal of Developmental Physiology 15(6): 309-323. Johnston, C. I. (1972). Radioimmunoassay for plasma antidiuretic hormone. Journal of Endocrinology 52: 69-78. 192 Jones, C. T. (1977). The development of some metabolic responses to hypoxia in the foetal sheep. Journal of Physiology 265(3): 743-762. Jones, C. T. and Ritchie, J. W. K. (1978). The cardiovascular effects of circulating catecholamines in fetal sheep. Journal of Physiology 285: 381-393. Jones, C. T. and Ritchie, J. W. K. (1983). The effects of adrenergic blockade on fetal response to hypoxia. Journal of Developmental Physiology 5(4): 211-222. Jones, G. T. and Robinson, R. O. (1975). Plasma catecholamines in foetal and adult sheep. Journal of Physiology 248: 15-33. Jones, C. T. and Rurak, D. (1976). The distribution and clearances of hormones and metabolites in the circulation of the foetal sheep. Quarterly Journal of Experimental Physiology 61: 287-295. Jones, D. P. and Kennedy, F. G. (1982). Intracellular oxygen supply during hypoxia. American Journal of Physiology 243(5): C247-C253. Kendrick, K. M., Keverne, E. B., Hinton, M. R, and Goode, J. A. (1991). Cerebrospinal fluid and plasma concentrations of oxytocin and vasopressin during parturition and vaginocervical stimulation in the sheep. Brain Research Bulletin 26(5): 803-807. Kennedy, F. G. and Jones, D. P. (1986). Oxygen dependence of mitochondrial function in isolated rat cardiac myocytes. American Journal of Physiology 250: C374-C383. Kiaer, T., Dahl, B., and Lausten, G. S. (1993). The relationship between inert gas wash-out and radioactive tracer microspheres in measurement of bone blood flow: Effect of decreased arterial supply and venous congestion on bone blood flow in an animal model. Journal of Orthopaedic Research 11(1): 28-35. Kitterman, J. A. (1988). Physiological factors in fetal lung growth. Canadian Journal of Physiology and Pharmacology 66(8): 1122-1128. Knight, K., Hall, C., and Rurak, D. W. (1997). Hindlimb (HL) blood flow and metabolic responses to hypoxemia and ischemia in the fetal lamb. Paper presented at The 5th Annual Western Perinatal Research Meeting, February 14-16, at Banff. Knight, K. E., Hall, C., and Rurak, D. W. (1996). Tissue blood flow distribution and the effect of chronic vascular catheterization in the hindlimb of the fetal lamb. Canadian Journal of Physiology and Pharmacology 74(11): 1270-1276. Koong, L. J. and Rattray, P. V. (1975). A description of the dynamics of fetal growth in sheep. Journal of Animal Science 41(4): 1065-1068. Koos, B. J., Sameshima, H., and Power, G. G. (1987a). Fetal breathing, sleep state, and cardiovascular responses to graded anemia in sheep. Journal of Applied Physiology 63(4): 1463-1468. Koos, B. J., Sameshima, H., and Power, G. G. (1987b). Fetal breathing, sleep state, and cardiovascular responses to graded hypoxia in sheep. Journal of Applied Physiology 62(3): 1033-1039. 193 Kwan, E., Rurak, D. W., and Taylor, S. M. (1995a). Blood volume restitution and growth in fetal lambs after acute hemorrhage. American Journal of Physiology 269: R749-R757. Kwan, E., Rurak, D. W., and Taylor, S. M. (1995b). Oxygen consumption, acid-base status, and behavior during and after acute, severe hemorrhage in fetal lambs. American Journal of Physiology 269: R758-R766. Leake, R. D., Weitzman, R. E., Weinberg, J. A., and Fisher, D. A. (1979). Control of vasopressin secretion in the newborn lamb. Pediatric Research 13(4 part 1): 257-260. Liard, J. F., Schelling, D. R, and Thibonnier, M. (1982). Cardiac output distribution during vasopressin infusion or dehydration in conscious dogs. American Journal of Physiology 243: H663-H669. Ling, W. D., Brooks, D. R, Crofton, J. T., Share, L., and Bohr, D. F. (1989). Pressor responses to vasopressin in pigs and sheep with DOCA hypertension. American Journal of Physiology 256(1 part 1): H101-H104. Llanos, A. J., Block, B. S. B., Court, D. J., Germain, A. M., and Parer, J. T. (1988). Fetal oxygen uptake during uterine contractures. Journal of Developmental Physiology 10: 525-529. Llanos, A. J., Court, D. J., Block, B. S., Germain, A. M., and Parer, J. T. (1986). Fetal cardiorespiratory changes during spontaneous prelabor uterine contractions in sheep. American Journal of Obstetrics and Gynecology 155(4): 893-897. Luscher, T. F. and Wenzel, R. R. (1995). Endothelin and endothelin antagonists: Pharmacology and clinical implications. In Mediators in the Cardiovascular System: Regional Ischemia, edited by K. Schror and C. R. Pace-Asciak. Basel: Birkhauser Verlag. Makowski, E. L., Meschia, G., Droegemueller, W., and Battaglia, F. C. (1968). Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero. Circulation Research 23: 623-631. Marsal, K., Lindblad, A., Lingman, G., and Eik-Nes, S. H. (1984). Blood flow in the fetal descending aorta; intrinsic factors affecting fetal blood flow, i.e. fetal breathing movements and cardiac arrhythmia Ultrasound in Medicine and Biology 10(3): 339-348. Meschia, G., Battaglia, F. C , Hay, W. W., and Sparks, J. W. (1980). Utilization of substrates by the ovine placenta in vivo. Federation Proceedings 39(2): 245-249. Meschia, G., Cotter, J. R., Breathnach, C. S., and Barron, D. H. (1965). The hemoglobin, oxygen, carbon dioxide and hydrogen ion concentrations in the umbilical bloods of sheep and goats as sampled via indwelling plastic catheters. Quarterly Journal of Experimental Physiology 50: 185-195. Meschia, G., Cotter, J. R, Makowski, E. L., and Barron, D. H. (1966). Simultaneous measurement of uterine and umbilical blood flows and oxygen uptakes. Quarterly Journal of Experimental Physiology 52: 1-18. 194 Milley, J. R. (1988). Uptake of exogenous substrates during hypoxia in fetal lambs. American Journal of Physiology 254: E572-E578. Miyake, Y., Murata, Y., Hesser, J., and Tyner, J. (1991). Cardiovascular responses to norepinephrine and arginine vasopressin infusion in chronically catheterized fetal lambs. Journal of Reproductive Medicine 36(10): 735-740. Moessinger, A. C. (1983). Fetal akinesia deformation sequence: an animal model. Pediatrics 72(6): 857-863. Mohring, J., Bohlen, R, Schoun, J., Mellet, M., Suss, U., Schmidt, M., and Pliska, V. (1982). Comparison of radioimmunoassay, chemical assay (HPLC) and bioassay for arginine vasopressin in synthetic standards and posterior pituitary tissue. Acta Endocrinologica 99: 371-378. Moore, P. J. and Hanson, M. A. (1992). Animal investigations. In Fetal Behaviour: Developmental and Perinatal Aspects, edited by J. G. Nijhuis. Oxford: Oxford University Press. Moore, P. J., Hanson, M. A., and Parkes, M. J. (1989). Almitrine inhibits breathing movements in fetal sheep in utero. Journal of Developmental Physiology 11: 277-281. Moreno, L. F., Stratton, H. H., Newell, J. C , and Feustel, P. J. (1986). Mathematical coupling of data: correction of a common error for linear calculations. Journal of Applied Physiology 60(1): 335-343. Natale, R, Clewlow, F., and Dawes, G. S. (1981). Measurement of fetal forelimb movements in the lamb in utero. American Journal of Obstetrics and Gynecology 140(5): 545-551. Nathanielsz, P. W., Bailey, A., Poore, E. R, Thorburn, G. D., and Harding, R. (1980). The relationship between myometrial activity and sleep state and breathing in fetal sheep throughout the last third of gestation. American Journal of Obstetrics and Gynecology 138(6): 653-659. Nathanielsz, P. W., Yu, H. K., and Cabalum, T. C. (1982). Effect of abolition of fetal movement on fetal intravascular P O 2 and incidence of tonic myometrial contractures in the pregnant ewe at 114 to 134 days' gestation. American Journal of Obstetrics and Gynecology 144(5): 614-618. Nijhuis, J. G., Prechtl, H. F. R., Martin, C. B., Jr., and Bots, R. S. G. M. (1982). Are there behavioural states in the human infant? Early Human Development 6: 177-195. Nuyt, A. M., Segar, J. L., Holley, A. T., O'Mara, M. S., Chapleau, M. W., and Robillard, J. E. (1996). Arginine vasopressin modulation of arterial baroreflex responses in fetal and newborn sheep. American Journal of Physiology 271: R1643-1653. Oddy, V. H., Brown, B. W., and Jones, A. W. (1981). Measurement of organ blood How using tritiated water. I. Hind limb muscle blood How in conscious ewes. Australian Journal of Biological Sciences 34(4): 419-425. 195 Parer, J. T. (1980). The effect of acute maternal hypoxia on fetal oxygenation and the umbilical circulation in the sheep. European Journal of Obstetrics & Gynecology and Reproductive Biology 10(2): 125-136. Parer, J. T., Court, D. J., Block, B. S. B., and Llanos, A. J. (1984). Variability of basal oxygenation of the fetus—causes and associations. European Journal of Obstetrics & Gynecology and Reproductive Biology 18(1-2): 1-9. Pecins-Thompson, M. and Keller-Wood, M. (1997). Effects of progesterone on blood pressure, plasma volume, and responses to hypotension. American Journal of Physiology 272(1 part 2): R377-R385. Peeters, L. L. H., Sheldon, R. E , Jones, M. D., Jr., Makowski, E. L., and Meschia, G. (1979). Blood flow to fetal organs as a function of arterial oxygen content. American Journal of Physiology 135(5): 637-646. Reuss, M. L., Parer, J. T., Harris, J. L., and Krueger, T. R. (1982). Hemodynamic effects of alpha-adrenergic blockade during hypoxia in fetal sheep. American Journal of Obstetrics and Gynecology 142(4): 410-415. Richardson, B. S. (1989). Fetal adaptive responses to asphyxia. Clinics in Perinatology 16(3): 595-611. Richardson, B. S., Carmichael, L., Homan, J., Tanswell, K , and Webster, A. C. (1989). Regional blood flow changes in the lamb during the perinatal period. American Journal of Obstetrics and Gynecology 160(4): 919-925. Richardson, B. S., Patrick, J. E., and Abduljabbar, H. (1985). Cerebral oxidative metabolism in the fetal lamb: Relationship to electrocortical state. American Journal of Obstetrics and Gynecology 153(4): 426-431. Robillard, J. E. and Weitzman, R. E. (1980). Developmental aspects of the fetal renal response to exogenous arginine vasopressin. American Journal of Physiology 238(5): F404-F414. Robillard, J. E., Weitzman, R. E., Fisher, D. A., and Smith, F. G., Jr.,(1979). The dynamics of vasopressin release and blood volume regulation during fetal hemorrhage in the lamb fetus. Pediatric Research 13:606-610. Roesch, D. M. and Keller-Wood, M. (1997). Progesterone rapidly reduces arterial pressure in ewes. American Journal of Physiology 272( 1 part 2): H386-H391. Rosenfeld, C. R. (1977). Distribution of cardiac output in ovine pregnancy. American Journal of Physiology 232(2): H231-H235. Ross, M. G., Ervin, M. G., Leake, R. D., and Fisher, D. A. (1985). Amniotic fluid ionic concentration in response to chronic fetal vasopressin infusion. American Journal of Physiology 249(3 part 1): E287-E291. Ross, M. G., Ervin, M. G., Leake, R. D., Habeeb, O., and Fisher, D. A. (1986a). Isovolemic hypotension in ovine fetus: plasma arginine vasopressin response and urinary effects. American Journal of Physiology 250: E564-E569. 196 Ross, M. G., Ervin, M. G., Leake, R. D., Humme, J. A., and Fisher, D. A. (1986b). Continuous ovine fetal hemorrhage: sensitivity of plasma and urine arginine vasopressin response. American Journal of Physiology 251: E464-E469. Ruckebusch, Y. (1972a). Development of sleep and wakefulness in the foetal lamb. Electroencephalography and Clinical Neurophysiology 32: 119-128. Ruckebusch, Y. (1972b). The relevance of drowsiness in the circadian cycle of farm animals. Animal Behaviour 20: 637-643. Ruckebusch, Y., Gaujoux, M., and Eghbali, B. (1977). Sleep cycles and kinesis in the foetal lamb. Electroencephalography and Clinical Neurophysiology 42: 226-237. Rudolph, A. M. (1984). Oxygenation in the fetus and neonate~A perspective. Seminars in Perinatology 8(3): 158-167. Rudolph, A. M. (1985). Distribution and regulation of blood flow in the fetal and neonatal lamb. Circulation Research 57(6): 811-821. Rudolph, A. M. and Heymann, M. A. (1967). The circulation of the fetus in utero: Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circulation Research 21: 163-184. Rudolph, A. M. and Heymann, M. A. (1970). Circulatory changes during growth in the fetal lamb. Circulation Research 26: 289-299. Rudolph, A. M. and Heymann, M. A. (1984). Methods for studying the circulation of the fetus in utero. In Animal Models in Fetal Medicine, edited by P. W. Nathanielsz. Ithaca: Perinatology Press. Rudolph, C. D., Roman, C , and Rudolph, A. M. (1989). Effect of acute umbilical cord compression on hepatic carbohydrate metabolism in the fetal lamb. Pediatric Research 25(3): 228-233. Rurak, D., Stobbs, K., Hepburn, K., and Kwan, E. (1992). Behavioural state modulation of hindlimb blood flow in the fetal lamb. Paper presented at Society for the Study of Fetal Physiology 19th Annual Meeting, August 25-28, at Niagara-on-the-Lake. Rurak, D. W. (1978). Plasma vasopressin levels during hypoxaemia and the cardiovascular effects of exogenous vasopressin in foetal and adult sheep. Journal of Physiology 277: 341-357. Rurak, D. W. (1994). Fetal oxygenation, carbon dioxide homeostasis, and acid-base balance. In Textbook of Fetal Physiology, edited by G. D. Thorburn and R. Harding. Oxford: Oxford Medical Publications. Rurak, D. W. and Gruber, N. C. (1983a). The effect of neuromuscular blockade on oxygen consumption and blood gases in the fetal lamb. American Journal of Obstetrics and Gynecology 145(2): 258-262. Rurak, D. W. and Gruber, N. C. (1983b). Increased oxygen consumption associated with breathing activity in fetal lambs. American Journal of Applied Physiology 54(3): 701-707. 197 Rurak, D. W. and Gruber, N. C. (1984). The effect of vasopressin on fetal oxygenation in sheep. Canadian Journal of Physiology and Pharmacology 62: 27-30. Rurak, D. W., Kwan, E., Patrick, J. E., and Richardson, B. S. (1989). Fetal tolerance to reduced oxygen delivery: Effects of hemorrhage and hypoxemia. Paper presented at Society for the Study of Fetal Physiology Annual Meeting, at Reading UK. Rurak, D. W., Richardson, B. S., Patrick, J. E., Carmichael, L., and Homan, J. (1990a). Blood flow and oxygen delivery to fetal organs and tissues during sustained hypoxemia. American Journal of Physiology 258: R1116-R1122. Rurak, D. W., Richardson, B. S., Patrick, J. E., Carmichael, L., and Homan, J. (1990b). Oxygen consumption in the fetal lamb during sustained hypoxemia with progressive acidemia. American Journal of Physiology 258: RI 108-R1115. Rurak, D. W., Selke, P., Fisher, M., Taylor, S., and Wittman, B. (1987). Fetal oxygen extraction: Comparison of the human and sheep. American Journal of Obstetrics and Gynecology 156(2): 360-366. Rurak, D. W. and Taylor, S. M. (1986). Oxygen consumption in fetal lambs after maternal administration of sodium pentobarbital. American Journal of Obstetrics and Gynecology 154(3): 674-678. Schumacker, P. T. and Samsel, R. W. (1989). Oxygen delivery and uptake by peripheral tissues: Physiology and pathophysiology. Critical Care Clinics 5(2): 255-269. Share, L. (1988). Role of vasopressin in cardiovascular regulation. Physiological Reviews 68(4): 1248-1284. Sheldon, R. E., Peeters, L. L. H., Jones, M. D., Jr., Makowski, E. L., and Meschia, G. (1979). Redistribution of cardiac output and oxygen delivery in the hypoxemic fetal lamb. American Journal of Obstetrics and Gynecology 135(8): 1071-1078. Shelley, H. J. (1961). Glycogen reserves and their changes at birth. British Medical Bulletin 17(2): 137-143. Simmons, R. A. and Charlton, V. E. (1988). Substrate utilization by the fetal sheep lung during the last trimester. Pediatric Research 23(6): 606-611. Singh, S., Sparks, J. W., Meschia, G., Battaglia, F. C , and Makowski, E. L. (1984). Comparison of fetal and maternal hind limb metabolic quotients in sheep. American Journal of Obstetrics and Gynecology 149(4): 441-449. Skowsky, W. R., Swan, L., and Smith, P. (1979). Effects of sex steroid hormones on arginine vasopressin in intact and castrated male and female rats. Endocrinology ' 104(1): 105-108. Slotten, P., Phernetton, T. M., and Rankin, J. H. G. (1989). Relationship between fetal electrocorticographic changes and umbilical blood flow in the near-term sheep fetus. Journal of Developmental Physiology 11(1): 19-23. 198 Smith, G. N., Brien, J. F., Carmichael, L., Homan, J., Clarke, D. W., and Patrick, J. (1989). Development of tolerance to ethanol-induced suppression of breathing movements and brain activity in the near-term fetal sheep during short-term maternal administration of ethanol. Journal of Developmental Physiology 11: 189-197. Sparks, J. W., Hay, W. W., Jr., Bonds, D., Meschia, G., and Battaglia, F. C. (1982). Simultaneous measurements of lactate turnover rate and umbilical lactate uptake in the fetal lamb. Journal of Clinical Investigation 70(1): 179-192. Stark, R, Daniel, S., Husain, M. K., Milliez, J., Morishima, H. O., James, L. S., and Van de Wiele, R. (1981). Release of vasopressin by the fetal lamb during premature parturition induced with corticotropin. Pediatric Research 15(9): 1261-1265. Stark, R, Daniel, S. S., Husain, M. K., Sanocka, U. M., Zubrow, A. B., and James, L. S. (1984a). Vasopressin concentration in amniotic fluid as an index of fetal hypoxia: mechanism of release in sheep. Pediatric Research 18(6): 552-558. Stark, R. I., Daniel, S. S., Husain, K. M., James, L. S., and Van de Wiele, R. L. (1979). Arginine vasopressin during gestation and parturition in sheep fetus. Biology of the Neonate 35(5-6): 235-241. Stark, R. I., Daniel, S. S., Husain, M. K., Zubrow, A. B., and James, L. S. (1984b). Effects of hypoxia on vasopressin concentrations in cerebrospinal fluid and plasma of sheep. Neuroendocrinology 38(6): 453-460. Stark, R I., Wardlaw, S. L., Daniel, S. S., Husain, M. K., Sanocka, U. M., James, L. S., and Vande Wiele, R. L. (1982). Vasopressin secretion induced by hypoxia in sheep: developmental changes and relationship to beta-endorphin release. American Journal of Obstetrics and Gynecology 143(2): 204-215. Stegner, H., Leake, R. D., Palmer, S. M., Morris, A. M., and Fisher, D. A. (1984a). Arginine vasopressin metabolic clearance and production rates in fetal sheep, pregnant ewes, and lambs. Developmental Pharmacology and Therapeutics 7(2): 87-93. Stegner, H., Leake, R. D., Palmer, S. M., Oakes, G., and Fisher, D. A. (1984b). The effect of hypoxia on neurohypophyseal hormone release in fetal and maternal sheep. Pediatric Research 18(2): 188-191. Stratford, L. L. and Hooper, S. B. (1997). Effect of hypoxemia on tissue glycogen content and glycolytic enzyme activities in fetal sheep. American Journal of Physiology 272(1 part 2): R103-R110. Stratton, H. H., Feustel, P. J., and Newell, J. C. (1987). Regression of calculated variables in the presence of shared measurement error. Journal of Applied Physiology 62(5): 2083-2093. Szczepanska-Sadowska, E. (1973). Hemodynamic effects of a moderate increase of the plasma vasopressin level in conscious dogs. Pfliigers Archiv European Journal of Physiology 338: 313-322. Szeto, H. H. (1992). Behavioral states and their ontogeny: Animal studies. Seminars in Perinatology 16(4): 211-216. 199 Szeto, J. J. and Hinman, D. J. (1985). Prenatal development of sleep-wake patterns in sheep. Sleep 8(4): 347-355. Tan, W. P. (1997). Circulatory and metabolic studies of normally grown and growth restricted fetal sheep before and during spontaneous labor and at delivery. Ph.D., University of British Columbia. Tobler, I. (1984). Evolution of sleep processes: A phylogenetic approach. In Sleep Mechanisms (Experimental Brain Research Supplementum 8), edited by A. A. Borbley and J. L. Valatx. New York: Springer-Verlag. Tomita, H., Brace, R. A., Cheung, C. Y., and Longo, L. D. (1985). Vasopressin dose-response effects on fetal vascular pressures, heart rate, and blood volume. American Journal of Physiology 249: H974-H980. Townsend, S. F., Rudolph, C. D., Wood, C. E , and Rudolph, A. M. (1989). Perinatal onset of hepatic gluconeogenesis in the lamb. Journal of Developmental Physiology 12(6): 329-335. Van Bel, E , Roman, C , Iwamoto, H. S., and Rudolph, A. M. (1993). Sympathoadrenal, metabolic, and regional blood flow responses to cold in fetal sheep. Pediatric Research 34(1): 47-50. Varma, S., Jaju, B. P., and Bhargava, K. P. (1969). Mechanism of vasopressin-induced bradycardia in dogs. Circulation Research 24: 787-792. Vizsolyi, E. and Perks, A. M. (1976). Neurohypophyseal hormones in fetal life and pregnancy. I. Pharmacologic studies in the sheep (Ovis aries). General and Comparative Endocrinology 29: 28-40. Walker, A. M., Fleming, J., Smolich, J., Stunden, R., Home, R., and Maloney, J. (1984). Fetal oxygen consumption, umbilical circulation and electrocortical activity transitions in fetal lambs. Journal of Developmental Physiology 6(3): 267-27'4. Walker, D. W. (1984). Brain mechanisms, hypoxia and fetal breathing. Journal of Developmental Physiology 6: 225-236. Wallace, L. R. (1948). The growth of lambs before and after birth in relation to the level of nutrition. Journal of Agricultural Science 38(3): 17; 244-302. Wang, B. C , Ginter, G. F., and Goetz, K. L. (1987). Augmented pressor response to vasopressin in awake dogs after cardiac denervation. American Journal of Physiology 252(1 part 2): R145-R152. Wang, Y.-X., Crofton, J. T., Bealer, S. L., and Share, L. (1997). Sexual dimorphism in regional blood flow responses to vasopressin in conscious rats. American Journal of Physiology 273(3 part 2): R1126-R1131. Wang, Y.-X., Crofton, J. T., Liu, H , Sato, K., and Share, L. (1996). V2-receptor blockade enhances pressor response to vasopressin: gender difference. Life Sciences 59(8): 695-703. 200 Wilkening, R. B., Boyle, D. W., and Meschia, G. (1988). Measurement of blood flow and oxygen consumption in the pelvic limb of fetal sheep. Proceedings of the Society for Experimental Biology and Medicine 187: 498-505. Wilkening, R. B., Boyle, D. W., and Meschia, G. (1989). Fetal neuromuscular blockade: Effect on oxygen demand and placental transport. American Journal of Physiology 257: H734-H738. Wilkening, R. B. and Meschia, G. (1983). Fetal oxygen uptake, oxygenation, and acid-base balance as a function of uterine blood flow. American Journal of Physiology 244(6): H749-H755. Wintour, E. M., Moritz, K. M., and Potocnik, S. J. (1995). Cardiovascular, hormonal, and metabolic responses to severe prolonged hemorrhage in adult sheep. American Journal of Veterinary Research 56(9): 1232-1240. Wiriyathian, S., Porter, J. C , Naden, R. P., and Rosenfeld, C. R. (1983). Cardiovascular effects and clearance of arginine vasopressin in the fetal lamb. American Journal of Physiology 245(1): E24-E31. Wlodek, M., Thorburn, G. D., and Harding, R. (1989). Bladder contractions and micturition in fetal sheep: their relation to behavioral states. American Journal of Physiology 257(6 Part 2): R1526-R1532. Wood, C. E , Keil, L. C , and Rudolph, A. M. (1982). Hormonal and hemodynamic responses to vena caval obstruction in fetal sheep. American Journal of Physiology 243: E278-E286. Woods, L. L., Cheung, C. C , Power, G. G., and Brace, R. A. (1986). Role of arginine vasopressin in fetal renal responses to hypertonicity. American Journal of Physiology 251: F156-F163. Yeleswaram, K , Rurak, D. W., Kwan, E., Hall, D., Doroudian, A., Wright, M. R, Abbott, F. S., and Axelson, J. E. (1993). Transplacental and nonplacental clearances, metabolism and pharmacodynamics of labetalol in the fetal lamb after direct intravenous administration. Journal of Pharmacology and Experimental Therapeutics 267(1): 425-431. 10. APPENDIX. Unsuccessful fetal surgical preparations 202 s fa U a G © co eg CD OS 2 3 e i T3 3 co •c co e CO 8 a "8 CO co 2 3 co 00 l H 3 CO 00 3 •§ T 3 .1 "2 CO 3 .CO co 3 8 co CO co 3 O "8 8, o I 3 3 i "5 2 a 3 3 i co 1 8 co 2 3 3 i =3 CO 3 8 CO CO co 3 O 1 & J O , 3 3 •a CO 2 a 3 3 "6 CO 3 8 8 CO 2 I 3 2 B 3 3 "8 "8 "6 T3 .fi .3° CO 3 ^ s a O CN c o 00 in vo CN T - H c n wo CN CN CN CN VO CN " t CO £> 00 CN CN o o CO q WO "fr CN CO CO T - H T - H O N T - H CO l > CO CO "fr CO C M 2 « U Ci) CD CO 3 fa Z CO CN CN CN CN CN CN CD MO fi CO S *•> eg w CO 3 CO Q co oo 3 00 O A CN CO CN "fr 00 CN T - I o CN VO CO CN CN CO CN o CN CN VO CN 00 O N CN T - H WO O N CN — i O N CN WO CN ON CN wo CN CN O N CD CD £ } * s W 3 CO $ 2 g — ^ 2 CN X i — i CO X CN 3 CN CN o CN CN a 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0089240/manifest

Comment

Related Items