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Cardiac shunting and blood flow distribution in the American alligator (ALLIGATOR MISSISSIPPIENSIS) Ostlin, Janice Christine 1997

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CARDIAC SHUNTING AND BLOOD FLOW DISTRIBUTION IN THE A M E R I C A N ALLIGATOR (Alligator mississippiensis) by JANICE CHRISTINE OSTEIN B. Sc., University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FUFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES (Department of Zoology) Wg^ccept this thesis as conforming tp]the requirejdjstand^d The University of British Columbia August, 1997 © Janice Christine Ostlin 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 written permission. Department of Zoo >Q °\Y The University of British Columbia Vancouver, Canada Date /August 2Q > 1937 DE-6 (2/88) ABSTRACT The cardiac and circulatory anatomy of the American alligator (Alligator mississippiensis) is unique in that both the cardiac and systemic circulatory systems display anatomical divisions. This situation may also be of physiological significance to the animal. The purpose of this study was to determine regional blood flow distribution in the alligator, with respect to cardiac blood flow patterns. Animals were instrumented with flow and pressure recorders, and monitored over extended time periods. Fluorescent microspheres capable of being entrapped in tissue capillary beds were introduced into both the right and left aortas under various conditions. Blood flow distribution to tissues during the presence or absence of a pulmonary to systemic (P->S shunt) was of primary interest. Tissue samples depicted a significant separation in the perfusion patterns of the right and left aortas. The right aorta primarily perfused the brain and liver, and to a lesser extent, the digestive system organs. The left aorta perfused the digestive tract organs, including the spleen. Dissection of the circulatory paths of the right and left aortas determined this anatomical division. Possible differences in the left aortic distribution of right ventricular (shunted) blood as compared to left ventricular (not shunted) bloOd were noted. T A B L E O F C O N T E N T S ABSTRACT •••• » T A B L E OF CONTENTS Hi LIST OF TABLES - iv LIST OF FIGURES v ACKNOWLEDGEMENTS vii INTRODUCTION 1 METHODS 8 General Procedures ••• 9 Surgical Technique 9 Data Acquisition 12 Data Analysis and Statistics 14 RESULTS AND DISCUSSION .. 17 Part J. Blood Flow and the Cardiac Cycle 17 Circulatory Anatomy and Regional Organ Perfusion 22 Physiological Significance? 41 Part 2. Long Term Blood Flow Observation and Variation in Flow Patterns 46 Foramen Flow 50 Conclusions 54 APPENDIX 56 B IBLIOGRAPHY 58 iii LIST OF TABLES Table I. Actual and corrected mean percent values of microspheres introduced into the right aorta ., 16 Table II. Percentage of microspheres introduced into the right and left aortas retrieved from lung tissue 16 Table III. Statistical analysis of microsphere distribution to tissues: differeces in blood flow distribution of the right and left aortas during the presence or absence of aP->Sshunt 30 Table IV. Statistical analysis of microsphere distribution to tissues: differences in blood flow distribution of the right aorta during the presence or absence of a P->S shunt 33 iv LIST OF FIGURES Figure 1. Diagrammatic anatomy of the alligator heart 2 Figure 2. Photograph of alligator aortic anatomy at the level of the abdominal anastomosis 4 Figure 3. Blood flow and blood pressure recordings in absence of a P->S shunt .. 18 Figure 4. Diagram of blood flow origin and direction during the absence of a P->S shunt 19 Figure 5. Blood flow and pressure recordings in the presence of a P->S shunt 20 Figure 6. Diagram of blood flow origin and direction during the presence of a P->S shunt ; 21 Figure 7. Photograph of the alligator abdominal circulatory anatomy 25 Figure 8. Schematic representation of the left and right aortic circulatory anatomy and major branches 26 Figure 9. Relative distribution of blood flow (% microspheres) to tissues during the absence of a P->S shunt 28 Figure 10. Relative distribution of blood flow (% microspheres) to tissues during the presence of a P->S shunt : 29 Figure IT. Relative distribution of right aortic blood flow (% microspheres) to tissues during the presence or absence of a P->S shunt 34 Figure 12. Relative distribution of left aortic blood flow (% microspheres) to tissues during the presence or absence of a P->S shunt 40 Figure 13. Profile of left aortic blood flow patterns . 4 8 Figure 14. Long term recording of left and right aortic blood flow: P->S shunt present 49 Figure 15. Profile of left aortic blood flow patterns: Flow through the foramen of Panizza 52 Figure 16. Relative distribution of blood flow (% microspheres) resulting from blood flow through the foramen of Panizza 53 vi A C K N O W L E D G E M E N T S This study would not have been possible without the guidance and assistance of numerous colleagues and friends. I would like to especially thank my supervisor, David R. Jones for his support, advice and surgical abilities, without which this study could not have been performed. I would also like to thank my labmate and friend, Amanda Southwood, who provided me with a great deal of tireless assistance and advice, and whose gator-wrestling skills are unmatched and undefeated. My particular thanks also to Thuan N.P. Nyugen for his help with the data acquisition and analysis, and for his continued understanding and moral support. In addition I would like to thank the other members of the Jones lab for their input into the project, and express my gratitude to my committee members, Bil l Milsom and David Randall, for their advice on this study. v i i Introduction The cardiac anatomy of the crocodilia (alligators and crocodiles) combines characteristics from two separate phylogenetic groups. Similarly to warm-blooded vertebrates such as birds and mammals, they possess a complete interventricular septum, effectively dividing the ventricles into two distinct pumping chambers (White, 1956: Webb, 1979; Jones, 1996). The crocodilia are the only group of reptiles which display this anatomical division of the heart, although in other groups, for example, turtles and varanids, a functional division exists to separate oxygenated from deoxygenated blood (White, 1956, 1970; Grigg, 1989). However, as seen in reptiles, both the left and right aortas are retained. This is in contrast to birds and mammals in which only one systemic aorta is retained, the right or left, respectively (Grigg, 1989). In the crocodilia, the right aorta exits the left ventricle and becomes the systemic aorta after giving rise to the common carotid artery and the subclavian artery. The left aorta exits alongside the pulmonary artery from the right ventricle, and continues at the level of the abdomen as the coeliac artery (Fig. l)(White, 1956; Webb, 1979). The aortas communicate in two locations. The first is directly outside the heart where all of the vessels exit as a common truncus. Here a small opening exists between the two aortic walls termed the foramen of Panizza (Coulson & Hernandez, 1950; White, 1956; Webb, 1979; Grigg & Johansen, 1986). This aperture has recently been observed to be capable of varying its size, and is thought to play a substantial role in the development of the complex flow patterns observed in these animals (Axelsson et al, 1995; Malvin et al, 1995). 1 F i g u r e 1. Diagrammatic Anatomy of the Alligator Heart (Jones, 1996) The right aorta (RAo) exits from the left ventricle (LV). The left aorta (LAo) and pulmonary artery (PA) exit from the right ventricle (RV). R A and L A are the right and left atria, respectively. Anastomosis The second junction is located caudal to the heart, proximal to the liver and abdominal organs (Axelsson et al, 1989). It consists of a small vessel connecting the right and left aortas, and is termed the 'abdominal anastomosis' (Fig. 1). In this study, one animal was found to lack this distinct vessel, instead the two aortas simply merged at the level of the gut before splitting into their separate vessels again (Fig. 2). It is unknown at present to what degree this anatomical pattern exists in nature or the extent to which it alters flow patterns, but it was observed that the internal anatomy of this anomaly would likely give rise to parallel flow situations similar to those observed in animals possessing a distinct anastomosis (Jones, 1996) The complex anatomy and physiology of the alligator heart sets the stage for several patterns of blood flow to occur, and as well, for these to occur in varying degrees. Most commonly, two situations exist. In the first, blood is ejected into the right aorta from the left ventricle, and into the pulmonary artery from the right ventricle, during systole. The left aortic valves do not open, and the left aorta receives no right ventricular blood. Instead, blood flows in an anterograde direction through the right aorta, feeding the carotid and subclavian arteries, then continues into the systemic aorta. A portion of this blood travels through the abdominal anastomosis into the left aorta, and travels in a retrograde fashion within it (Shelton & Jones, 1991, Jones & Shelton, 1993). Blood in the left aorta in this instance is thus oxygenated, and flow is reversed during systole. In diastole, relaxation of the heart and elastic recoil of the vessels allows for the blood in the left aorta to drain towards the body, into the coeliac artery (Shelton & Jones, 1991; Jones & Shelton, 1993). 3 Figure 2. Photograph of Alligator Aortic Anatomy at the level of the Abdominal Anastomosis (Jones, 1996). The photograph on the left of the page depicts the right aorta (RAo) and left aorta (LAo) joining via a separate structure, the abdominal anastomosis. On the right, the anatomy of one subject in this study is seen to vary in that a distinct anastomosis is lacking, instead the right and left aorta simply join. In the second situation, blood flow is anterograde in the left aorta during systole. When systemic pressure is low, blood pressure in the right ventricle can exceed that in the left aorta, allowing for ejection into the left aorta. This is termed a pulmonary-to-systemic shunt (P->S shunt) (Jones, 1996). It should be noted that in this situation, the blood flow to the lungs is uncompromised. The pulmonary valve is an active valve, and whether or not the shunt occurs following valve closure is dictated by the pressure of blood in the right ventricle and the systemic pressure (Jones and Shelton, 1993; Axelsson et al, 1995). During early systole, blood from the right ventricle is first ejected into the pulmonary artery. Aside from a passive pulmonary valve consisting of tissue cusps, the alligator also possesses an active pulmonary valve. This valve is comprised of thick fibrous nodules surrounding the lumen of the pulmonary artery (Webb, 1979). Upon active valve closure, the nodules interdigitate forming a tight seal. Because of its structure this valve is termed a 'cog wheel' valve (Fig. 1). Following closure of the cog wheelvalve, any blood remaining in the right ventricle is subject to ejection into the left aorta if right ventricular pressure exceeds that in the left aorta (Shelton & Jones, 1991; Jones & Shelton, 1993). This situation effectively allows for some deoxygenated blood to bypass the pulmonary circuit, and reach the body through the left aorta. This blood is hypercapnic, and therefore acidic in pH due to the presence of H2CO3. Therefore, in this situation, both left and right aortic blood flows are anterograde during systole, but the left aorta contains right ventricular deoxygenated blood, while the right contains oxygenated blood of left ventricular origin. 5 A third flow pattern which has recently been determined is one in which blood ejects into the right aorta and pulmonary arteries during systole, but no blood is ejected from the right ventricle into the left aorta. The anastomosis is closed, so that blood from the right aorta cannot traverse it into the left aorta, and as well, the medial cusp of the right aortic valve occludes the foramen of Panizza during systole, so that blood cannot enter the left aorta via that route (Axelsson et al, 1995; Jones etai, 1997). The anastomosis possesses extensive innervation and can vary drastically in diameter (Karila et al, 1995), and has been noted to close completely (Jones, 1996). In diastole, as the right aortic valves close, the foramen is exposed, and blood passes through it into the left aorta. In this scenario, left aortic flow patterns reveal almost no flow during systole, and a "foramen spike" indicative of blood flow from the right aorta during diastole (Jones et al, 1997). In addition, these complex patterns can occur in varying degrees as long term recordings in this study displayed, thus giving rise to multiple blood flow possibilities. P->S shunting was long thought to occur primarily during prolonged dives (White, 1969). Later studies concluded that althought the shunt can occur during voluntary diving, it was not obligately correlated (Grigg &.Johansen, 1986). Jones and Shelton (1993) found that the shunt occurred naturally in resting animals. This study elaborated on the view of Jones . and Shelton (1993), in recording blood flow patterns over extended time periods during routine activity of the animals. Previous studies have stated that the left aorta becomes the coeliac artery caudal to the anastomosis (White, 1956; Webb, 1979). However, the organs perfused by left aortic blood and the degree of perfusion have never been determined. The coeliac artery perfuses the abdominal organs in other vertebrates, primarily branching into the left 6 gastric, hepatic, and splenic arteries. The mesenteric artery arises either as a branch of the coeliac, or as a separate branch from the aorta (Romer, 1955; Folklow & Neil, 1971 ). This study attempted to determine the distribution of both left and right aortic blood, during the presence and absence of a P->S shunt in the American alligator, Alligator mississippiensis. It was hypothesized that right aortic blood would display a general systemic distribution pattern, whereas left aortic blood would be distributed to the abdominal organs, as a typical coeliac artery would supply. It was observed that a division of the systemic circulation may exist, much as the division present in the cardiac circulation, in which the distribution of the blood from the two aortas was much more limited than expected. In addition, variation in this distribution under fluctuating degrees of blood flow patterns observed in individual animals was examined. Blood flow distribution resulting from various flow conditions was investigated, and the potential physiological significance of the observed perfusion distribution discussed. 7 Methods General Procedure Systemic blood flow distribution in the American alligator (Alligator mississippiensis) was determined by means of nonradioactive 25 micron diameter fluorescent polystyrene microspheres (Interactive Medical Technologies Inc., Los Angeles, CA) injected into circulation through both the right and left aortas. The 25 micron diameter spheres were similar in diameter to the animal's erythrocytes (Nikinmaa, 1954), but, being undeformable, lodged in the tissue capillary beds. The distribution of spheres then allowed for the partitioning of blood flow to organs and tissues from the two systemic aortas to be mapped. Microspheres were injected into both the right and left aortas during both the presence and absence of a pulmonary to systemic (P->S) shunt. It was hypothesized that spheres injected into the right aorta would have a general systemic distribution, while those injected into the left aorta would be distributed primarily to the digestive organs, specifically the anterior region of the small intestine where the left aorta was thought to terminate as the coeliac artery (White, 1956; Webb, 1979). Any difference in this distribution between periods of pulmonary to systemic shunting and periods in which the shunt was absent were quantified. Animals Three subjects were utilized, two females and one male, with a mean weight of 14.6 kg (range 14.0-15.5 kg). Another animal (weight 9.5 kg) was used in blood flow measurements, and was not injected with microspheres for flow distribution determination. 8 Animals were wild caught in Louisiana, and transported to the University of British Columbia via road. They were housed at the Animal Care facility, and exposed to seasonally appropriate photoperiods, while temperatures were kept in the range from 28-32 °C. A pool with continuously flowing water was provided for submergence, and a heat lamp provided for basking. They were fed once weekly on a high protein diet consisting offish and chicken, with bone included to ensure adequate mineral requirements were met (Coulson et al., 1987). This diet was supplemented with a multivitamin powder. Solid dog chow pellets were left in the room at all times for ad libitum feeding. Surgical Technique Animals were surgically implanted with Doppler flow probes (Iowa Doppler) for blood flow measurements and heparin-treated (TD-Mac Heparin complex) vinyl catheters (Bolab size 3) to record blood pressure and inject microspheres. Anaesthesia was induced with 100 mgkg' 1 of Ketamine Hydrochloride (Ketalean, M.T.C Pharmaceuticals) injected.into the forearm musculature, and maintained with a gas mixture of 50:50 oxygen:nitrous oxide blended with 2.5-4.0% Halothane BP, initially administered by face mask and later through an endotracheal tube; Respiration was maintained during surgery at 5 breaths per minute with a tidal volume of 100 ml by a Harvard intermediate animal ventilator. Surgical anaesthesia was maintained with a 1:1 ratio of oxygen:nitrous oxide mixed with 0.8-2% Halothane BP. Body temperature was allowed to reach ambient temperature (20- 22 °C) during the procedure. A ventral midline incision was made through the skin along the length of the sternum. Lateral incisions made perpendicularly at each end of the midline incision 9 allowed for the skin to be folded back. An incision was made in the anterior portion of the sternum, and opened with a surgical retractor; This exposed the common truncus of vessels exiting the heart and the base of the heart. It should be noted that the cartilaginous nature of the sternum in these young animals facilitated this operation. Vessels were located and freed of surrounding connective tissue using blunt probes and forceps. Location of every great vessel exiting the heart was necessary to ensure correct identification of individual vessels, as the anatomy was slightly different in each subject. Doppler flow probes (Iowa Doppler) ranging in diameter from 3.2-4.0 mm were fitted to the right and left aortas. They were tied to the vessel using a length of 5-0 surgical silk running through the flow cuff wall. To implant catheters, holes were made in the vessel walls with a 21 gauge angiocath needle. The catheter was introduced into the vessel through the needle sheath and advanced to ensure placement in the common truncus just outside the heart. This distance ranged from 5-7 cm for the left aorta and 13-15 cm for the right aorta. Correct placement of catheters was required to ensure complete mixing of injected microspheres with blood. Catheters were placed directly into the left aorta, but threaded into the right aorta by way of the subclavian artery. The hole in the vessel was closed and catheters held in place by purse string sutures of 5-0 surgical silk surrounding the opening into the vessel. The catheters were also sutured at two locations along the length of the vessel. A small amount of tissue cement (Vetbond, 3M) was utilized to ensure adhesion to the vessel wall. After securing the flow probe leads and catheter tubing to surrounding tissue to prevent dislodgment, the catheters and flow leads were looped twice to allow for movement, and the ends were threaded subdermally 10 around the side of the animal to appear through an incision made in the dorsal thoracic region. This placement allowed easy access for instrumentation with minimal disturbance to the animal. The sternum was then closed utilizing 3-0 surgical silk and ethilon (Ethicon, B.C. Stevens) sutures. The skin incision was closed using the same materials by placing sutures between scales. The area was covered with Cicatrin antibiotic powder (Ayerst Laboratories) and wrapped with Vetwrap (3M) to aid in keeping the incision site dry and clean, and as well anchoring the leads to the animal's back. Following surgery, animals were kept on intubated oxygen for 60 minutes and then placed in a tank with a heat lamp. The tank contained a platform and fibergrass mat to provide, the animal with closer access to the heat source or provide it a hiding place. Topical (Flamazine) and intramuscular (Baytril or Amykacin) antibiotics were given as a preventative measure. Experimentation was not conducted for 48-72 hours following surgery, at which time water was added to the tank to encourage diving and natural behavior. The tank was initially entirely covered by black plastic to decrease visual distraction, but it soon became clear that the animals became distressed in such isolation, and therefore a window was cut in the side of this cover to allow for visual contact with the other alligators housed in the same room. The tank sides were covered with a mirrored coating, such that the subject could see out, but could not be seen. These measures were taken to simulate the conditions that the animals were accustomed to prior to surgery, such that more natural and consistent results might be produced. 11 Data Acquisition Flow and pressure leads were threaded through the wire mesh covering the tank and connected to their appropriate measurement systems located outside of the room. Contact with the subjects was kept to a minimum to avoid interference with potential results. Blood flow was measured by a Doppler flowmeter (Crystal Biotech), which fed through low-pass radio frequency filters to a computer equipped with a data collection system (Labtech Notebook, 7 t h edition). Range was initially adjusted to the highest peak flow and left unaltered for the remainder of the experiment. Catheters were attached via 3-way stopcocks to Deltran disposable pressure transducers, which were positioned at the level of the heart, or water surface for submerged animals. These were fed through signal amplifiers to the computerized acquisition system. In all subjects, left aortic blood flow was recorded along with left and right aortic pressures. In the first two subjects, right aortic and carotid artery blood flow were recorded, and blood flow measurements in the subclavian artery were attempted. However, the flow probe consistently emitted no signal. It was decided that since P->S shunting was of primary importance, only left aortic flow along with right and left aortic pressures needed to be recorded. Flow and pressure measurements were taken under both conditions of a P->S shunt and when this shunt was absent. Flow and pressure were monitored continuously for extended periods to determine the existence of any pattern related to shunting. Blood pressure was calibrated for high pressure by attaching a hand-held sphygmomanometer to the transducers and injecting air into the system to specific pressures (mrnHg). Zero pressure was taken with the transducers open to atmospheric 12 pressure when positioned at the level of the heart or at the water surface when the animal was in water. Blood flow was calibrated in situ post-mortem by cannulating the vessel on either side of the flow probe with a large diameter surgical tubing (Bolab, size 11), and placing the distal end of this tubing at some level above the heart to create a back pressure. Blood or another particulate fluid was then passed through the probe at known velocities using a syringe pump (Harvard), and the corresponding voltage signal collected onto the data acquisition system. Blood Flow Distribution Blood flow distribution was measured in three subjects. Polystyrene microspheres (Interactive Medical Technologies, Los Angeles, CA) measuring 25 microns in diameter in seven colours with different fluorescent excitabilities were injected into the right and left aortas. In all subjects, spheres were injected during periods of natural P->S shunting, and during the absence of this shunt, as determined from observation of blood flow and pressure patterns. It was necessary to wait at least 30 minutes following the injection of spheres before subsequent injections to allow for complete circulation of blood through the body circuit. Each vial of microspheres contained 5 million spheres in 2 ml of physiological saline with Tween 20 added as an antimicrobial agent. Microspheres were vortexed for 15-20 seconds to resuspend them for withdrawal into a syringe. The sphere solution was added to 3ml of heparinized saline, which was vortexed again briefly prior to injection. Microspheres were introduced directly into the catheters though 5 ml syringes with 16 gauge blunted needles attached to the 3-way stopcocks. The catheter tubing was vibrated 13 during injection by means of an aquarium air pump in order to discourage settling out of spheres. The volume was injected slowly over 60 seconds, and the original syringe rinsed three times with 5 ml of saline, which was also injected. In one subject the entire amount of sphere solution (5 million spheres) was injected during each trial, whereas in the other two subjects the spheres were diluted down to lesser concentrations. In all instances, any spheres remaining in the original bottles were returned to the manufacturer for specific quantification. At the completion of each experiment, subjects were sacrificed with an overdose of sodium pentobarbital and tissues were sampled for microsphere quantification. The digestive system organs were of principle interest as left aortic blood flow distribution was in question. However, other tissues including the brain and some skeletal muscle were also sampled. Samples were weighed and placed into polyethylene tubes, which were enclosed in plastic bags and packaged into styrofoam containers for shipment to Interactive Medical Technologies, where analysis and sphere quantification for regional blood flow perfusion was executed. Data Analysis and Statistics Tissue perfusion results were analyzed as percentages of total microspheres injected for each subject. Averages were taken of different trials in one individual, and those numbers used to calculate group averages and standard error. Right and left aortic distribution of microspheres to tissues was compared during the presence or absence of a P->S shunt. Due to low recovery of microspheres introduced into the right aorta, percentages obtained for right aortic spheres were multiplied by a factor of 7.5, to 14 approximate the greater flow rate in the right aorta as compared to the flow in the left aorta (Table. I) (Shelton & Jones, 1991). Microsphere distribution therefore depicts blood flow perfusion to the tissues sampled. In order for a tissue perfusion distribution to be considered significant, numbers of entrapped microspheres had to equal or exceed 400 spheres per tissue (Glenny et al, 1993). Tissue samples from the lung were quantified for percentages of trapped microspheres in order to determine the degree to which the microspheres escaped entrappment in capillary beds (Table II). As fluorescent microspheres had not been previously utilized for regional blood flow determination in the alligator, it was necessary to examine the lung tissue for evidence of microsphere recirculation, in order to improve upon this technique for future studies. Tissue distributions were statistically analyzed for significant difference using an unpaired t-test (p= 0.05), where possible. The distribution of the right and left aortas to tissues during the presence of a P->S shunt were compared with one another (Table III). As well, distribution differences during the presence or absence of a P->S shunt for the right aorta were analyzed (Table IV). As only one blood flow distribution was attained for the left aorta during the absence of a P->S shunt, this data is included for comparison only. 15 Table I. Actual and corrected mean percent values of microspheres introduced into the right aorta. Tissue Actual % values Corrected % Actual % values Corrected % shunt (x 7.5) shunt (x 7.5) absent present Brain 0.938 7.031 0.985 7.388 Liver 0.715 5.363 0.712 5.338 Gallbladder 0.000 0.000 6.000 0.000 Small intestine 0.095 0.713 0:078 0.587 Stomach 0.070 0.525 0.060 0.450 Spleen 0.143 1.075 0.070 0.525 Pancreas 0.000 0.000 0.050 0.375 Kidney 0.710 5.325 0.150 . 1.125 Table IT. Percentage of microspheres introduced into the right and left aortas retrieved from lung tissue. Percent values: mean and standard error Tissue RAo shunt absent N=3, n=3 RAo shunt present N=3, n=3 LAo shunt absent N=l,n=l LAo shunt present N=3, n=3 Lung 0.740 ±0.229 0.869 ±0;669 0.776 0.551 ± 0.302 16 Results and Discussion Parti Blood Flow and the Cardiac Cycle The complex blood flow patterns observed in the crocodilian circulation are a result of their unique cardiac anatomy. Two situations are common, but the extent to which either exists in isolation is highly variable. In the first situation, blood is ejected from the left ventricle into the right aorta, and from the right ventricle into the pulmonary artery, during systole (Fig. 3,4). Oxygenated blood returned to the left ventricle from the lungs enters the right aorta, which branches into the carotid and subclavian arteries, before continuing as the dorsal (systemic) aorta to the rest of the body (Fig. 4). At the level of the gut, a portion of right aortic blood traverses the abdominal anastomosis, and continues in a retrograde direction within the left aorta. Hence, flow in the left aorta during systole is retrograde and of right aortic origin (Fig. 3,4). The foramen of Panizza is effectively blocked by the right aortic valve during most of systole, thus no blood flows through it (Jones & Shelton, 1993: Franklin & Axelsson, 1994). However, recent studies suggest some variation in flow patterns through the foramen, and this will be addressed later. During diastole, elastic recoil of the left aorta causes blood to drain in an anterograde direction towards the body (Shelton & Jones, 1991; Jones & Shelton, 1993). In addition, some right aortic blood may enter the left aorta via the foramen of Panizza, to join this anterograde flow. In the second situation, blood is ejected from the right ventricle into the left aorta during systole (Figs. 5,6). In this case, following active closure of the pulmonary valves, pressure in the right ventricle exceeds that in the left aorta, and blood is effectively 17 F i g u r e 3. Blood flow and Blood Pressure Recordings in absence of a P->S shunt RAo and L A o are right and left aortic recordings, respectively. Figure 4. Diagram of blood flow origin and direction during the absence of a P->S shunt. Oxygenated blood (shown in red) is ejected from the left ventricle into the right aorta (RAo). Blood flow direction is depicted by arrows. At the level of the abdomen, some RAo blood traverses the anastomosis and travels in a retrograde direction within the left aorta (LAo). 19 5 o o £ O 2-1-RAo LAo 10s Time (s) 60 2 to S 40-RAo LAo 10 s Time (s) Figure 5. Blood Flow and Blood Pressure Recordings in the presence of a P->S Shunt. RAo and LAo are right and left aortic recordings, respectively. sc sc RAo Figure 6. Diagram of blood flow origin and direction during the presence of a P->S shunt The left hand picture depicts the situation in early systole, in which right ventricular blood (shown in blue) is ejected into the pulmonary artery (PA), and left ventricular blood (shown in red) is ejected into the right aorta (RAo). Late systole is shown on the right hand side, during which remaining right ventricular blood is ejected into the left aorta (LAo) following active closure of the pulmonary valves. CA and SC are the carotid and subclavian arteries, respectively shunted away from heart and into the left aorta, towards the body ( Shelton & Jones, 1991; Jones & Shelton, 1993). Right aortic, and hence systemic, blood pressure is somewhat reduced in this case, and left aortic pressure peaks above right aortic pressure (Fig. 5): The blood present in the left aorta in this scenario is deoxygenated and hypercapnic, having bypassed circulation through the lungs. This situation is termed a pulmonary to systemic (P->S) shunt. Circulatory Anatomy and Regional Organ Perfusion This study pioneered the technique of regional blood flow mapping through use of fluorescent microspheres in the American alligator (Alligator mississippiensis), although similar techniques have.been implemented in other species. Radioactively labeled microspheres have been utilized for decades in this type of study. Hales (1973) determined regional blood flow perfusion in the sheep using radioactive microspheres. Estimates of shunting have been determined in the lizard, Vdranus exanthematicus, along with systemic and pulmonary outputs from the heart and distribution of blood flow to tissues quantified using the radioactive method (Berger and Heisler, 1977). Validation of the use of coloured and fluorescent microspheres for measurement of regional blood flow and tissue perfusion has been provided by Kowallik et al (1991), and by Glenny et al, (1993). These accounts compare the reliability of this method to the time-honoured radioactive tracer method, and determined that it was indeed sound. Problems such as dye leaching from the spheres into tissues are insignificant even over extended periods, and as long as appropriately sized microspheres are utilized, the regional blood flow is proportional to the number of spheres entrapped within the tissue of interest. In this study 22 25 micron diameter spheres were implemented, as they most closely approximated the size of the alligator's erythrocytes (23 microns) (Nikinmaa, 1954). A. Circulatory anatomy Anatomically, the right aorta is thought to continue as the primary systemic aorta supplying blood to the body, after giving rise to the common carotid and subclavian arteries just outside the heart, whereas the left aorta, had been stated to continue as the coeliac artery caudal to the anastomosis (Fig. 1). In other vertebrates, the coeliac artery gives off major branches to the stomach (left gastric artery), liver (hepatic artery), spleen (splenic artery), and often to the intestine (superior mesenteric artery) (Romer, 1955; Warwick & Williams, 1973). The left gastric artery is the smallest branch, and supplies the cardiac region of the stomach. Some of its branches may anastomose with the splenic artery. The hepatic artery is of intermediate diameter, and primarily divides into right and left hepatic arteries which further branch into the right gastric, the gastroduodenal and the cystic arteries. The splenic artery possesses the largest diameter of the three primary coeliac arterial branches, and itself branches several times before continuing into the spleen as the terminal splenic artery. Aside from the spleen, these branches supply the pancreas (pancreatic branches), fundus of the stomach (short gastric branches), and the cranial stomach and omentum (left gastro-epiploic artery) (Warwick and Williams, 1973). In addition, the superior mesenteric artery arises either as a branch of the coeliac trunk or as a branch directly from the systemic aorta just caudal to the coeliac trunk. This vessel supplies almost the entire small intestine. 23 In this study, an extensive dissection of the left and right aortic circulatory anatomy was performed. The left aorta was found to divide into four primary branches just caudal to the abdominal anastomosis (Fig. 7,8). Two of these branches supplied the anterior region of the stomach, while the others supplied the spleen and mid-anterior region of the small intestine, respectively. The intestinal branch also gave off two smaller branches which supplied the pancreas. The anatomy of the alligator's abdominal circulation is therefore quite different than that of the typical vertebrate; A distinct coeliac trunk is absent, instead the left aorta simply terminates as four main branches. In addition, a hepatic branch in this region is lacking. Instead, the right aorta gives off a large hepatic branch soon after exiting the heart. This branch is located just caudal to the ventricles, and enters the anterior area of the liver. In addition, while supplying some minor branches to the mesentery, the right aorta gave off two other major branches, to the kidney and posterior colon, respectively (Figs. 7,8). This situation is somewhat analogous to that seen in other vertebrates, where the renal arteries branch directly from the systemic aorta caudal to the coeliac trunk, and the caudal portion of the colon is supplied by the inferior mesenteric artery, which arises from the systemic aorta just before it branches into the internal and external iliac arteries. B. Bloodflow distribution Regional blood flow distribution from the right aorta was initially expected to display a general systemic distribution, while the left aorta was expected to supply solely the abdominal organs. However, both the circulatory anatomy and the regional blood flow distribution from the two aortas suggested a division of the systemic circulation, 24 Figure 7. Photograph of the alligator abdominal circulatory anatomy. The left aorta (LAo) branches into four primary vessels caudal to the anastomosis (positioned at the arrow), supplying the stomach, spleen, small intestine and pancreas. The right aorta (RAo) is shown to divide into two vessels, supplying the kidney and intestine, respectively. SMALL INTESTINE Figure 8. Schematic representation of the left and right aortic circulatory anatomy and major branches. Right aorta (RAo) branches into hepatic, renal and mesenteric arteries; left aorta (LAo) branches into splenic, mesenteric and pancreatic, and two gastric arteries. Parallel bars represent a break in the right aorta. 26 which could arise from the division seen in the central cardiac circulation It is necessary to discuss blood flow distribution based not only on anatomical relationships, but in relation to blood flow patterns as well, as variation is possible depending on whether or not a P->S shunt is occurring. Right aortic distribution: P->S shunt absent Right aortic blood was supplied to most organs, although it's total body distribution was far more restricted than expected (Fig. 9). The common carotid artery, which branches off of the right aorta directly outside the heart, is a large diameter vessel (3.2 -3.7 mm in this study), and possesses substantial blood flow. Therefore it was expected to receive a substantial number of the microspheres introduced into the right aorta. Microspheres of right aortic origin were indeed located in the brain, ranging between 7.031- 7.388 % of those injected (Figs. 9,10). The subclavian artery, which supplies the musculature of the anterior half of the body, would also be expected to obtain microspheres mixed into the circulation just outside the heart. However, sampling of muscles from this area produced insignificant numbers of microspheres trapped in tissue, and it is thought that perfusion of these muscles may be low at rest, or alternatively that the introduction of the right aortic catheter by way. of the subclavian could have caused a blockage in this artery hence limiting the natural distribution of the spheres through this path. It should be noted, however, that the animals appeared to suffer no ill effects if such a blockage did exist, as they were still very active and displayed no signs of muscle pain or atrophy. In the future, a more complete systemic distribution of microspheres would likely be gained through direct catheterization of the right aorta. 27 Figure 9. Relative distribution of blood flow (% microspheres) to tissues during the absence of a P->S shunt. Distribution of right aortic (RAo) and left aortic (LAo) blood shown. RAo LAo 25-20-e u a. CO § 15-Legend BR Brain LI Liver GB Gallbladder SI Small intestine ST Stomach SP Spleen PA Pancreas KI Kidney 104 54 BR. LI. GB. SI. ST. Sampled Tissues SP. PA. KI. 28 Figure 10. Relative distribution of blood flow (% microspheres) to tissues during the presence of a P->S shunt. Distribution of right aortic (RAb) and left aortic (LAo) blood shown. 15-, 10-.8 Lr, BR. LI. GB. SI. ST. SP. Sampled Tissues PA. Legend BR Brain LI Liver GB Gallbladder SI Small intestine ST Stomach SP Spleen PA Pancreas KI Kidney KI. 29 Table UT Statistical analysis of microsphere distribution to tissues: differences in blood flow distribution of the right and left aortas during the presence or absence of a P->S shunt. Percent microspheres in tissues Mean and Standard error Tissue RAo shunt absent N=3, n=3 LAo shunt absent N=1,n=1 RAo shunt present N=3, n=3 LAo shunt present N=3, n=3 Significant difference (p=0.05) Brain 7.031 + 0.019 0 7.388 ± 3.488 0 not testable Liver 5.362 ±3.098 1.100 5.338 ±3.087 1.013 ± 0.527 no Gallbladder 0 0.170 0 0.512 ±0.189 not testable Small intestine 0.713 ±0.488 5.000 0.588 ±0.321 6.767 ±4.621 no Stomach 0.525 ±0.375 2:800 0.450 ±0.271 3.723 ±1.918 no Spleen 1.075 ± 0.600 23.70 1.075 ±0.303 5.993 ± 2.026 no Pancreas 0 0.670 0.375 ±0.300 0.823 ± 0.228 no Kidney 5.260 ±5.175 0.040 1.125 ±0.938 0.367 ±0.237 no 30 A low percentage of spheres reached the digestive organs, these organs were indeed supplied primarily by the left aorta (Fig. 9). This division is anatomical, as right aortic branches to the digestive system organs were observed to be few and of small diameter in the post mortem dissection. It is also possible that these vessels may remain closed due to vascular resistance or possible hormonal activity in the region. Several neuropeptides are known to have effects on the gut vasculature, for example, substance P, acetylcholine and neurotensin all cause vasodilation and hence increased blood flow through decreased vascular resistance, whereas epinephrine causes a marked reduction in gut blood flow (Axelsson et al, 1990; 1991). The only other organs consistently supplied with blood directly from the right aorta were the liver (5.362- 5.338 %) and kidney (1.125- 5.260 %)(Figs. 9,10). A major branch of the right systemic aorta was observed to perfuse the liver, which could yeild these high proportions of trapped microspheres (Fig. 8). Alternatively, the liver may possess lower vascular resistance than that of the other digestive organs, hence allowing for proportionally greater blood flow through its vessels. Caudal to the anastomosis, the right aorta gave off two primary branches, to the kidney and colon, respectively (Fig. 7). As stated, microspheres of right aortic origin were located in the kidney, however colonic samples contained insignificant numbers of trapped microspheres. In future studies, larger colon samples or tissue samples taken from close proximity to the supplying vessel may yield more complete results. 31 Right aortic distribution: P->S shunt present No significant difference existed in the right aortic blood flow distribution during the presence or absence of a P->S shunt (Table IV) (Fig. 11). In addition, in several organs, there was no significant difference between right and left aortic distributions during the presence of a shunt (Table III, Fig. 10). During the presence of a shunt, blood from the left ventricle would be ejected into the right aorta as usual, thus would be expected to travel to any organ to which the right aorta anatomically perfuses. In addition to this, any right aortic blood reaching the left aorta during the presence of a shunt would be routed along with the left aortic flow to the organs which it perfuses. At the foramen of Panizza, blood flow from right to left aorta commonly occurs during diastole ( Shelton & Jones, 1991; Jones & Shelton, 1993; Franklin & Axelsson, 1994). However, this alone would likely not be of great enough magnitude to cause a difference in blood distribution. According to Malvin (1995), some right aortic blood enters the left aorta through the foramen in very early systole. Following the principles put forth in Malvin's paper, the sudden appearance of increased left aortic blood flow parallel to the foramen could act to draw right aortic blood through it by means of the Bernoulli principle. This could add to the amount of right aortic blood present in the left aorta. Axelsson et al (1991) also suggests an increase in foramen flow during periods of increased coeliac artery flow induced by hyperemia of the gut. In addition, any flow of right aortic blood through the abdominal anastomosis during systole while the P->S shunt is in operation would be carried along with the left aortic blood to its respective organs. This is supported by Axelsson et al. (1990) who found that blood flow in the coeliac artery was always pulsatile 32 Table IV. Statistical analysis of microsphere distribution to tissues: differences in blood flow distribution of the right aorta during the presence or absence of aP->S shunt. Percent microspheres in tissues Mean and Standard Error Tissue RAo shunt absent N=3, n=3 RAo shunt present N=3, n=3 Significant difference (p=0.05) LAo shunt absent N=1, n=1 LAo shunt present N=3, n=3 Brain 7.031 + 0.019 7.388 ± 3.488 no o 0 Liver 5.362 ±3.098 5.338 ±3.087 no 1.100 1.013 ±0.527 Gallbladder 0 0 not testable 0.170 0.512 ±0.189 Small intestine 0.713 ±0.488 0.588 ±0.321 no 5.000 6.767 ±4.621 Stomach 0.525 ±0.375 0.450 ±0.271 no 2.800 3.723 ±1.918 Spleen 1.075 ±0.600 1.075 ±0.303 no 23.70 5.993 ± 2.026 Pancreas 0 0.375 ±0.300 not testable 0.670 0.823 ± 0.228 Kidney 5.260 ±5.175 1.125 ±0.938 no 0.040 0.367 ± 0.237 33 Figure 11. Relative distribution of right aortic blood flow (% microspheres) to tissues during the presence or absence of a P->S shunt. RAoNS (no shunt) and RAoS (shunt) are the right aortic blood flow distributions in the absence and presence of the P->S shunt, respectively. Legend BR Brain LI Liver GB Gallbladder SI Small intestine ST Stomach SP Spleen PA Pancreas KI Kidney BR. LI. GB. SI. ST. SP. PA. KI. Sampled Tissues 34 in an anterograde direction, even in the absence of a shunt, suggesting that some of the blood present in it is of right aortic origin. It is also possible that if systemic pressure is low during the presence of the shunt (Jones & Shelton, 1993), that vascular resistance is also low in the gut organs. The abdominal organs normally maintain a high degree of vascular tone (Folkow & Neil, 1971), and are known to undergo a great degree of reactive hyperemia postprandially and under pharmacological interventions (Axellson et al, 1990). Therefore, if vessels of right aortic origin to this area are small as previously suggested, it may be possible that a decrease in vascular resistance or some other mechanism of vasodilation could greatly affect blood flow through them. In any case it is likely that the right aortic vessels supplying the digestive tract organs, though not the primary vessels, would be required to supply oxygenated blood during hypoxic conditions or periods of prolonged shunting in order to maintain tissue vitality. Left aortic distribution: P->S shunt present The ieft aortic distribution closely paralleled its anatomical arrangement. The primary site of blood flow distribution was to the small intestine (6.767 %)(Fig. 10), which is expected as this is one site in which the left aorta terminates. The stomach and spleen also received substantial amounts of left aortic blood (3.723 and 5.993 % respectively). It was expected that the stomach should receive proportionately more of the left aortic blood than what was observed, as anatomically, it is perfused by dual branches of the left aorta. However, although the diameters of the four primary left aortic branches appeared to be approximately equal in the post mortem dissection, it is possible that the vessel diameters vary greatly in vivo. The ability of vasoconstriction and 35 dilatation of the gut vasculature has been previously mentioned for the right aortic vessels, and these properties likely hold true for the left aortic branches as well. This could account for some of the difference observed in the percent flow to these organs, The pancreas received a small proportion of left aortic shunted blood (0.823 %) (Fig. 10), which was likely due to the small diameter of the pancreatic branches present from the intestinal branch of the left aorta. The hepatic distribution of left aortic microspheres was somewhat surprising. Although the hepatic artery is a primary branch of the coeliac artery in other vertebrates, in the alligator it was observed to branch solely from the right aorta. It is possible that a small hepatic vessel from the left aorta exists, or that microspheres introduced into the left aorta reached the liver through the hepatic portal system. If any spheres were able to bypass the intestinal capillary beds, they could have access to the liver through this route by directly entering the venous portal system. In the future, thorough flushing of sampled organs would be useful in ascertaining that only . spheres entrapped within capillary beds are quantified. The liver displays an active aerobic metabolism, and must maintain strict homeostatic control in order to function properly. If a hepatic vessel of left aortic origin exists, it could supply the liver with deoxygenated shunted blood. This vessel would likely also be subject to strict control, perhaps by vasoactive metabolites, to ensure the continued functioning of the organ. In other vertebrates, the gallbladder is supplied by cystic branches of the primary hepatic artery. Some left aortic microspheres did reach the gallbladder (0.512 %) (Fig. 10), and it is suggested that a cystic branch may exist. No spheres introduced into the left aorta under 36 any circulatory condition appeared in the brain or anterior muscle, furthering the view of a divided systemic circulation. The observation has recently been made that flow may occur bidirectionally through the foramen of Panizza, and that left aortic blood may traverse it into the right aorta (Malvin et al, 1995). This is suggested to occur via the Bernoulli principle, in that blood flowing parallel to the foramen in the right aorta would set up a negative pressure difference across the aperture, and result in left aortic blood being drawn through (Malvin etai, 1995). The volume of blood which does so is likely minimal, as distribution of left aortic microspheres would be expected to parallel that of the right aorta, which was generally not the case in this study. It is possible that this occurred to sOme extent, as this would help to explain the distribution of left aortic spheres to the liver when no left aortic hepatic vessel was observed. In addition, due to the enormous variation present in the circulatory patterns in this species, it holds possible that bidirectional foramen flow may be significant under yet undefined circumstances. It is important to understand that left aortic blood is commonly of two origins. During the absence of a P->S shunt, blood present in the left aorta is of left ventricular origin, having gotten there through the abdominal anastomosis from the right aorta. This blood is therefore oxygenated. When &P->S shunt is occurring, the blood present in the left aorta is of right ventricular origin, and thus is deoxygenated and hypercapnic, having missed oxygenation at the lungs. Microspheres introduced into circulation displayed some apparent difference in distribution depending on whether or not a P->S shunt was in operation. 37 Left aortic distribution: P->S shunt absent The left aortic distribution showed some variation depending on whether or not the P->S shunt was operative. This is more surprising, as anatomically it would be expected that any microspheres injected into the left aorta would arrive at the same location, regardless of the circulatory situation occurring. During the P->S shunt, the left aorta contains deoxygenated and hypercapnic blood, thus the organs which it perfuses are subject to this. It has been noted that the digestive organs can withstand hypoxia better than other body systems, therefore could survive under these conditions (Grigg, 1989). This is also true in diving species such as the seal, in which blood flow is preferentially redistributed away from the digestive organs in favor of the brain and working muscle (Hurtord et al, 1996). Possible physiological roles for this system will be discussed in the following section. Apparently, in absence of the P->S shunt, the spleen received more blood of left aortic origin than it did when the shunt was in operation (Fig. 9). When the shunt is not operating, the left aorta contains oxygenated blood of right aortic origin, which flows towards the heart during systole and towards the body during diastole (Fig. 4). In this scenario, the spleen receives more of the left aortic microspheres (23.70 %) than it does when the shunt is in operation (5.993 %) (Fig. 12). When the shunt is present, the pulsatile force of the blood flowing through the left aorta, directing more of the spheres towards the aortic endpoints. Both the splenic and intestinal branch appear to be the main continuations of the left aorta, that is, they run in parallel with it while the two gastric branches divide off at right angles to the main aortic vessel (Figs. 7,8). Hence, in the presence of a shunt, the intestine and spleen receive the major proportions of blood. 38 In the absence of the P->S shunt, the anterograde blood flow in the left aorta comes mainly from the elastic recoil of the vessel during diastole (Jones & Shelton, 1993), and perhaps from the right aorta through the anastomosis as well (Axelsson, 1990). In this situation, the amount of left aortic spheres entrapped in the stomach and intestine are decreased from 3.723 and 6.767% in the shunting condition to 2.800 and 5.000 % (respectively) in the absence of the shunt. Although a physiological mechanism explaining the difference in left aortic blood distribution to the spleen during the presence or absence of a shunt have not been determined, it is possible that the splenic vessels undergo vasodilation when presented with non- shunted oxygenated blood, allowing for a larger blood volume to perfuse the spleen under these conditions. Folklow and Neil (1971) suggest that the spleen may possess a precapillary sphincter mechanism, which would determine the amount of blood flow to the spleen. However, the conditions under which blood flow to the spleen are regulated need to be examined. It should be noted that microspheres were introduced into the left aorta in absence of a P->S shunt in only two trials. Therefore the aformentioned data is given for comparison purposes only, as statistical analysis could not be executed. 39 Figure 12. Relative distribution of left aortic blood flow (% microspheres) to tissues during the presence or absence of a P->S shunt. LAoNS (no shunt) and LAoS (shunt) are the left aortic blood flow distributions in the absence and presence of a P->S shunt, respectively. 2 5 - , 2 0 -1 5 -1 0 -LAoS LAoNS BR. LI. GB. SI. ST. Sampled Tissues SP. Legend BR Brain LI Liver GB Gallbladder SI Small intestine ST Stomach SP Spleen PA Pancreas KI Kidney PA. KI. 40 Physiological Significance of a Divided Systemic Circulation? It is far beyond the scope of this study to determine what, if any, adaptations these blood flow distributions represent, but suggestions can be made as to the physiological "usefulness" of such a system. The physiology of individual organs will be considered along with the tissue perfusion patterns observed. J. Brain The brain received no microspheres introduced into the left aorta under any circulatory condition, during either the presence or absence of aP->S shunt. The brain is a highly aerobic organ, which cannot tolerate hypoxia for prolonged periods, therefore, periodically exposing the brain to blood low in oxygen content would be harmful. This is supported in the alligator by the redistribution of blood to the brain during prolonged dives (Anderson, 1961; Axelsson, 1991). 2. Liver Aerobic metabolism is prominent in the liver, therefore it could prove detrimental to supply this organ with periodic but substantial bouts of deoxygenated blood. In addition, key enzymes involved in glycolysis display very limited pH ranges over which they can function. Baldwin etai. (1995) depict this narrow range of functional pH in the estuarine crocodile (Crocodylusporosus). These enzymes work best at a neutral pH, and therefore perfusing the liver with large amounts of shunted hypercapnic (and therefore acidic) blood would lower the enzymatic capacity. However, a small proportion of shunted blood does reach the liver, as indicated by the left aortic microsphere distribution (Fig. 10). These animals are known to undergo an immense "alkaline tide" postprandially, 41 during which the blood alkalinity may rise up to pH 8 (Coulson & Hernandez, 1950). Shunted blood may be useful in the liver under this circumstance to supplement intracellular buffering and allow for continued enzyme function. A detailed description of the alkaline tide follows in the section on gastric physiology: 3. Gallbladder and Pancreas The gallbladder and pancreas consistently received more left aortic shunted blood than nonshunted left aortic blood. Neither organ received much right aortic blood, and it is likely that these organs can function in the presence of deoxygenated blood as suggested by Webb (1979) (Fig. 10). In the gallbladder, the presence of shunted blood, which is hypercapnic arid acidic in nature, may assist in bile acid production postprandially or stimulate the production of bicarbonate by the duct cells. 4. Small intestine The small intestine receives a large amount of left aortic blood. During the condition of a P->S shunt, this acidic blood could aid in stimulating the release of the hormone CCK-PZ, which is normally stimulated by the entrance of acidic chyme from the stomach into the duodenum during gastric emptying (Eckert et al, 1988). This hormone causes gallbladder contraction and the subsequent release of bile into the duodenum, and thus functioning of the gallbladder and small intestine could be interrelated through perfusion with acidic shunted blood. In addition, CCK-PZ decreases intestinal vascular resistance and increases intestinal blood flow (Chou et al, 1984). If shunted blood reaches the intestinal organs during digestion, it could stimulate the release of this hormone, thus acting to increase the rate of the digestive process by increasing blood flow. 42 5. Stomach As previously stated, the stomach and digestive tract organs are able to withstand lower blood oxygen levels for prolonged periods, and thus a redistribution of shunted blood to these organs would be expected in situations in which oxygen was scarce (e.g. diving) (Grigg & Johansen, 1987). However, over the extended time course of this study, P->S shunts were observed to occur frequently, and in fact, appear to represent the usual circulatory state during rest (Jones, 1996). This arrangement may be of use in maintaining a lower metabolism and in energy conservation. It seems likely that the animal derives some advantage from this situation. Alligators are gorge feeders, and consume the entire carcasses of their prey (Coulson & Hernandez, 1983). They are faced with the task of digesting substantial amounts of calcium and magnesium phosphates found in bone. In fact, bone is essential in the diet, as it provides the mineral requirements (Coulson & Hernandez, 1987). In order to digest this high mineral diet, a high concentration of gastric hydrochloric acid is required, and acidity has been recorded at pH 2 (Coulson et al, 1950). HC1 is formed by the removal of protons from the blood in exchange for bicarbonate, giving rise to the immense alkalinity observed in the blood (alkaline tide) (Coulson et al, 1950; Eckert et al, 1988). In vertebrates, this occurs in the gastric parietal cells located in the cardiac region of the stomach. In the alligator, the anterior region of the stomach was determined to be perfused by two branches of the left aorta. Blood perfusion to the stomach is significantly greater in the presence of aP->S shunt, as diagrammed in Figure 12. The perfusion of this area with acidic, hypercapnic blood during the formation of gastric HC1 would likely 43 aid in increasing the amount of available blood acid for the production of gastric acid. It is unknown at present whether the P->S shunt occurs naturally postprandially. In one study measuring gut blood flow in the alligator, coeliac artery flow was observed to increase following feeding, but no shunting was observed (Axelsson et al, 1990). However, in the aforementioned study, blood flows were assessed following force-feeding, hence it is possible that the stress of this procedure induced high enough systemic pressures to nullify the chances of a P->S shunt developing. The gut hyperemia of feeding is associated with reduced vascular resistance, and it is possible that this situation could induce P->S shunting through reduction of overall systemic pressure. The elevation in metabolic rate associated with digestion is generally supported with increased blood flow (and hence increased available oxygen to tissues). In the case of the alligator, if digestion is indeed related to the occurrence of a P->S shunt, then the digestive system blood flow distribution may be explainable. The increase in right aortic (oxygenated) blood distribution may be required to support metabolic activity, whereas the left aortic (deoxygenated and hypercapnic) may be utilized in the production of gastric acid. 6. Kidney The kidney receives very little shunted left aortic blood (0.367 %). It is unknown at present how left aortic blood reached the kidney, as the primary renal artery was observed to branch from the right aorta. It is possible that the small amount of shunted blood reaching the kidney could be utilized in increasing the urinary buffering through the production of ammonium bicarbonate (Grigg, 1989). During the presence of prolonged hypoxic conditions, this may aid in ridding the body of wastes produced during anaerobic 44 metabolism. However, until an anatomical or physiological mechanism allowing for left aortic blood to perfuse the kidney is determined, this distribution is assumed to be of minor significance. 7. Spleen The spleen appears to trap more microspheres introduced into the left aorta during the absence of a P->S shunt than during the presence. It is unknown as to why this distribution should exist. It is possible that the splenic artery undergoes vasoconstriction when faced with shunted deoxygenated blood or vasodilation in the presence of high oxygen content. It may be an organ capable of varying vascular resistance under different circumstances. Intestinal organs are noted to display metabolic regulation of circulation, and the oxygen status of blood perfusing these organs is a primary factor in how much blood perfuses capillary beds (Shepherd & Granger, 1984). The spleen is known to be a blood reservoir in several diving animals (for example, the Weddel seal), capable of contracting under the influence of catecholamines and sending stored oxygenated blood to the body during tissue hypoxia (Hurtord et al, 1996). To what extent this occurs in the alligator is yet undetermined, although the spleen was grossly observed in this study to be a muscular organ. Alternatively, storage of shunted blood could be recirculated during the presence of the alkaline tide, thus helping to neutralize the blood pH. In any case, the spleen acts to sieve out old and damaged erythrocytes with extensive capillary beds, and it is possible that the microspheres were simply entrapped there similar to damaged red blood cells, due to their inability to deform and traverse the capillary vessels. 45 Part 2. Long Term Blood Flow Observation and Variation in Flow Patterns. Each experiment in this study was conducted over a one month period, during which time blood flow patterns were montitored daily. The frequency, duration and magnitude of pulmonary to systemic shunting in the alligator has been of interest for years. The view of several authors is that the P->S shunt occurs only under specific conditions, and that the usual circulatory pattern present in these animals is that of a double-circuit circulation, in which blood from the right ventricle perfuses only the lungs, whereas left ventricular blood perfuses both the right and left aortae, the latter by means of the foramen of Panizza (Axelsson et al, 1989; Grigg, 1989). This view was supported by the observation that the left aorta contained oxygenated blood (Greenfield & Morrow, 1961). A P->S shunt, or pulmonary bypass shunt, was determined to occur only under intense situations such as forced diving, and was due to increased pulmonary vascular resistance allowing for deoxygenated blood to bypass the lung and be redirected through the left aorta towards the body (White, 1969). The presence of this shunt during periods of apnea and bradycardia associated with diving was supported by data in turtles, where a similar shunt exists during periods of apnea, however the shunt has not been necessarily correlated with voluntary diving in the alligator (Grigg & Johansen, 1987). More recent observations suggest that the P->S shunt occurs more regularly than previously believed, and that during periods in which it is not occurring, the origin of blood in the left aorta, although from the right aorta, arrives there primarily through the abdominal anastomosis and not the foramen of Panizza. Jones and Shelton (1993) determined that not only could 46 the shunt occur under natural conditions while the animal was at rest, but also for extended time periods (up to 13.2 minutes). It was later revealed that the retrograde flow present in the left aorta in the absence of a P->S shunt originated from right aortic blood which had travelled < around the loop> through the anastomosis and back towards the heart (Shelton & Jones, 1991; Jones & Shelton, 1993; Karila etal, 1995; Axelsson etal, 1996). In fact, it has recently been suggested that the shunting circulatory pattern is the predominant pattern in the resting animal (Jones, 1996). Figures 13 and 14 display the onset of a P->S shunt and its full establishment over several minutes. The waining of this pattern can be seen in figure 14. It should be mentioned here that this pattern was common, although the development of a full P->S shunt did not always occur. Often times this pattern would wax and wain, with pulses of positive flow during retrograde flow never reaching above zero. However the fact that these pulses are seen to build up to a full shunt in figure 13 determines that they are neither artifactual nor due solely to flow through the foramen of Panizza. The presence of such variable degree of shunting may be of adaptive value. In an ectothermic organism such as the alligator, the development of partial P->S shunts may aid in reducing metabolic rate. Alligators are typically "sit and wait" predators, who feed only intermittently (Coulson & Hernandez, 1983). If energetic activity is not required and food not consistently available, it would be beneficial to reduce overall metabolic rate through the reduction of the energetic expense of maintaining the digestive tract organs on highly oxygenated blood. Since it has been stated that the gut organs can withstand a higher degree of hypoxia than other bodily systems, it is possible that this situation could arise (Grigg, 1989). In addition, in the absence of the need to actively digest food, 47 A . Beginning of a P->S Shunt B. P->SShunt Occurring 00 1.0l 0.5-1 o •o . o « II 0.04 -0.54 -1.0 50 55 60 65 I 70 i,<h 0.54 o •O T -0.54 -1.0 o.o- ^%Aj\ 150 155 Recorded time (s) Figure 13. Profile of Left Aortic Blood Flow Patterns Figure 14. Long term Recording of Left and Right Aortic Blood Flow: P->S shunt present reduction of the digestive system metabolic rate through implementation of regionally hypoxic conditions would be energetically benificial. In ectothermic animals, a voluntary decrease in temperature not only reduces metabolic demand, but decreases sensitivity to hypoxia as well (Branco et al, 1993).. In fact, during periods of hypothermia in the alligator, the arterial oxygen pressure was seen to drop, which is consistent with the presence of shunting in these animals (Branco etal, 1993). As well, it is known that bicarbonate and carbon dioxide are primary regulators of hemoglobin- oxygen affinity in alligators, which do not rely on organic phosphates to modulate oxygen binding. It is possible that shunted blood, which is high in carbon dioxide, could play a role in modulating hemoglobin- oxygen affinity (Weber & White, 1986; Grigg et al, 1993) In any case, it is important to take into account the natural history of these creatures in attempting to explain possible physiological effects of their circulatory patterns. For this reason, these experiments attempted to mimic as closely as possible the conditions to which the subjects were accustomed, such that the animals might experience the entire range of circulatory possibilities. Foramen Flow Malvin et al. (1995) recently described the huge variation of flow patterns possible through the foramen, and in this study, one such example can be seen in Figure 15. In this instance, positive blood flow is occuring at the end of systole in the left aorta, due to flow from the right aorta through the foramen of Panizza as it is uncovered by the right aortic valve towards the end of active heart contraction. No retrograde flow occurs in the left aorta during systole, suggesting that the anastomosis is closed. The diameter of the anastomosis is known to vary under pharmacological intervention (Karila et al., 1995), 50 and has been observed to close under natural conditions (Jones et at., 1997). In this scenario, the blood present in the left aorta is oxygenated, having originated in the right aorta from the left ventricle. This foramen flow can be of fairly substantial magnitude, reaching values of 0.5ml s"1 in the example depicted in figure 15. Interestingly, when fluorescent microspheres were introduced into the left aorta under this condition, the tissue perfusion distribution more closely paralled that of the left aortic distribution in absence of a P->S shunt, with a large percentage of the spheres being trapped in the spleen (Fig. 16). This supports the possibility that some mechanism exists which allows for the spleen to have greater access to blood of left ventricular origin, either through simple low flow rates allowing more time for blood to perfuse the splenic artery or perhaps by some mechanism of vasoconstriction or dilatation of the splenic vasculature. How blood flow to the spleen is regulated in this animal is a topic which requires further investigation. 51 A. Beginning of foramen flow B. Enlarged View of foramen flow 0.5 o T3 -T^ O "> ° t »i 26b 26b 27b 27fe 2 8 b 2 a t ' 29b Recorded time (s) C. Foramen flow diminishing o O w PQ S 340 34$ ' 3sB ' i l l ' 36b" 3eS ' 37CI Recorded time (s) —1 • 270 Recorded time (s) Figure 15. Profile of Left Aortic Blood Flow Patterns: Flowthrough the Foramen of Panizza Figure 16. Relative distribution of blood flow (% microspheres) resulting from blood flow through the foramen of Panizza. Left aortic (LAo) blood flow distribution is depicted. 1 0 - , Legend BR Brain LI Liver Gallbladder Small intestine Stomach Spleen Pancreas Kidney Sampled Tissues 53 Conclusions " This study proved to be insightful in ascertaining the distribution of blood flow in the American alligator (Alligator mississippiensis). It was demonstrated that similar to the central cardiac circulation, the systemic circulation also displays unique partitions. The right aorta primarily supplies the brain, liver, kidney, and to a limited extent, the digestive system organs. The left aorta feeds the bulk of the digestive system organs, both during periods of pulmonary to systemic shunting and in the absence of this shunt. Some difference in the distribution of left aortic blood to the digestive organs, in particular the spleen, during the presence or absence of the shunt is apparent, and it is hypothesized that some mechanism may exist for vascular control and redistribution of oxygenated blood to the spleen. A variety of blood flow patterns were observed, including P->S shunts over extended periods, the onset and waining of such shunts, and blood flow through the foramen. If anything, this supports the idea that the variability of circulatory patterns in these animals is immense, and long term studies in as natural circumstances as possible are necessary in order to quantify the range of possibilities. The idea of a singular, distinct blood flow pattern for a P->S shunt must be expanded upon by examining blood flow over the long term. Problems encountered in this experiment were primarily related to the methods, as this was the first attempt at utilizing fluorescent microspheres to map blood flow distribution in this species. Some of the microspheres introduced into the right and left aortas apparently recirculated instead of being trapped in tissue capillary beds, as 54 evidenced by the presence of microspheres in the lungs (Table II). Although the extent to which capillary bypass vessels exist in the alligator is currently unknown, the use of larger diameter spheres would likely decrease the potential of recirculation. In addition, introduction of a larger number of spheres appears to yield more consistent results, as does introduction of the spheres under similar circulatory patterns in different subjects. Individual variation in blood flow distribution in this experiment is thought to be due to slight individual variation in circulatory anatomy or to conditions which may have resulted in temporary vasoconstriction or dilation of certain vessels. As well, it would be educational to sample the entire body of the animal in future experiments, as it is assumed that the right aorta was delivering blood to tissues which were not sampled. It should also be noted at this time that initial attempts were made to derive organ perfusion rates based on the "artificial organ method", however femoral cannulation for the purpose of blood withdrawal was unanimously unsuccessful in yeilding consistent results. It is suggested in the future that a different vessel be utilized, such as the brachial artery, as it is possible that the animals undergo vasoconstriction of their posterior vasculature, alternatively, catheterization of the aortas themselves again downstream would suffice. In general, the study was successful in determining.the anatomical difference between the perfusion sites of the left and right aortas, and in uncovering a possible physiological effect of this unique divided circulation, in the apparent difference between the left aortic distribution of right ventricular shunted blood and blood of left ventricular origin. . 55 Appendix 1. Tissue weights (g) and number of microspheres retrieved per gram of tissue in three subjects under various circulatory situations. Numbers separated by commas are separate trials. Subject 1 microsphere concentration in tissues (spheres per gram) Tissue Weight (g) RAo shunt present LAo shunt present RAo shunt absent Brain 4.5 6, 16 0,0 36 Liver 13.1 42, 143 46, 106 104 Gallbladder 8.3 41, 36 640, 923 54 Small intestine 37.4 106, 64 1857, 2272 172 Stomach 88.7 28,31 . 662, 841 54 Spleen 12.5 Ill, 113 2211,2452 344 Pancreas 12.5 7, 9 77, 136 17 Kidney 12.3 264, 1409 23, 257 1487 Subject 2 microsphere concentration in tissues (spheres per gram) Tissue Weight (g) RAo shunt present LAo shunt present RAo shunt absent Brain 4.1 2852, 3474 0,0 5530, 9538 Liver 25 1237, 1052 3445, 3884 1030, 1696 Gallbladder 7.9 5, 12 2462, 3063 15, 2 Small intestine 43.4 26, 109 21090, 32451 41, 16 Stomach 103 9, 21 3221,4163 • 8,6 Spleen 15.6 12, 30 12281, 12735 60, 9 Pancreas 36 .. 5, 179 3606, 4273 16, 3 Kidney 15.4 9,72 2299,3050 30, 9 56 Subject 3 microsphere concentration Tissue Weight (g) RAo shunt present LAo shunt present LAo foramen flow present RAo shunt absent LAo shunt absent Brain 4.1 17803 0,0 0 1840, 11519 0 Liver 22.8 6750 2977, 5548 7106 1447, 6899 5176 Gallbladder 1.4 68 6538, 5006 14555 19, 110 6103 Small 44.2 47 5581, 8606 10739 14, 102 . 17583 intestine Stomach 60.8 9 892, 1087 1906 2, 16 2325 Spleen 11.9 151 45885, 34470 37, 294 99639 23621 Pancreas 11.8 10 2333, 1532 1805 3, 19 2860 Kidney 4.2 335 1743, 1402 1602 55, 241 451 57 BIBLIOGRAPHY Andersen, H.T; 1961. 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