AN ABSTRACT OF THE THESIS OF JOSEPH EDWARD MCCARTHY for the M. S. (Name) (Degree) in Radiation Biology presented on / l í (Major) Title: VASCULAR AND EXTRAVASCULAR FLUID VOLUMES IN THE PACIFIC HAGFISH, EPTATRETUS 5TOUTII(LOCKINGTON) Redacted for Privacy Abstract approved: _Dr: Frank P. Conte The partitioning of body fluids in the Pacific hagfish was in- vestigated using several experimental techniques. Direct measure- ment of the blood volume was obtained through the simultaneous use of tagged red blood cells and plasma proteins labeled with Evan's blue dye. Blood cells obtained from donor animals were incubated with L- methionine -methyl in a physiological saline for two hours. The labeled cells were washed three times and injected into either the frontal or caudal area of the subcutaneous sinus of experimental animals. Blood samples were collected over a period of 104 hours through an indwelling catheter in the posterior portion of the sub- cutaneous sinus. Dilution curves were obtained. Fluid volume calcu- lations were based upon values extrapolated to "zero" time. The packed blood cell volume for five animals was 4. 9 ± 2. 0 percent of the body weight. The plasma volume was 13. 8 ± 3. 1 percent of the body weight. A second group of experimental animals was used to estimate the extracellular fluid volume, as measured by the dilution of inulin- carboxyl -C14 The average of 25. 9 ± 5. 1 percent of the body weight was obtained. The interstitial fluid ( lymph) being the difference between the extracellular fluid volume and the total blood volume was 7. 2 percent of the body weight. Total body water was determined following the dehydration of individual animals to a constant weight of 104 ± 1°C. The value for five animals was 74. 6 ± 3. 4 percent of the body weight. Vascular and Extravascular Fluid Volumes in the Pacific Hagfish, Eptatretus stoutiii(Lockington) by Joseph Edward McCarthy A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science June 1967 APPROVED: Redacted for Privacy Associate Professor of General Science in charge of major Redacted for Privacy C airman of the '5epartment4f General Science Redacted for Privacy Dean of Graduate School Date thesis is presented \\,\0 °\ tC10 Typed by Marion Palmateer for Joseph Edward McCarthy ACKNOWLEDGEMENT I wish to extend my thanks to my major professor, Dr. Frank P. Conte for his patience and assistance in the preparation of this thesis. This research was supported by funds from the General Science Department, Oregon State University. TABLE OF CONTENTS Page INTRODUCTION 1 Anatomical Aspects of the Hagfish 2 Evolutionary Aspects of the Cardiovascular System 6 MATERIALS AND METHODS 10 Collection of Hagfish (Eptatretus stoutii) 10 Maintenance of Experimental Hagfish (Eptatretus stoutii) 10 Catherization of the Blood Sinuses 12 Total Body Water 14 Excellular Water 16 Plasma Volume 19 Packed Red Blood Cell Technique 21 RESULTS 24 Total Body Water 24 Extracellular Water 25 Plasma Volume 26 Packed Red Blood Cells 27 DISCUSSION 28 The Dilution Technique 28 Labeled Blood Cell Technique 29 The Fluid Compartments of Vertebrates 30 CONCLUSIONS 40 BIBLIOGRAPHY 42 APPENDICES 47 APPENDIX I 47 APPENDIX II 48 LIST OF FIGURES Figure Page 1 A horizontal section of the caudal heart of the Pacific hagfish, Eptatretus stoutii, adapted from Greene, 1900. 5 2 The Pacific hagfish, Eptatretus stoutii. 13 3 The indwelling catheter. 15 4 Determination of the extracellular water volume using inulin - carboxyl -C14. 18 5 Determination of the plasma volume using Evan's blue dye (T-1824). 18 6 Determination of the red blood cell mass using radio- isotope- labeled blood cells. 20 7 The in vitro uptake of L- methionine- methyl -C14 by red blood cells of the fish Salmo gairdneri (Appendix I). 20 LIST OF TABLES Table 1 Total body water of the hagfish, Eptatretus stoutii. 24 2 The extracellular water of the hagfish, Eptatretus stoutii. 25 3 The plasma volume on the hagfish, Eptatretus stoutii. 26 4 The blood cell mass of the hagfish, Eptatretus stoutii. 27 5 A comparison of blood volumes in the classes of vertebrates. 32 6 Body fluid measurements of the fishes expressed as percent of total water. 39 VASCULAR AND EXTRAVASCULAR FLUID VOLUMES IN THE PACIFIC HAGFISH, EPTATRETUSSTOUTII(LOCKINGTON) INTRODUCTION The hagfish, Eptatretus stoutii, is considered to be one of the most primitive living vertebrates. Despite this unique position in the phylogenetic classification of the vertebrates, the hagfish has been largely ignored by biologists even from a standpoint of evolu- tionary interest. There has been little work carried out with the hagfish in the last hundred years. Most of our present knowledge concerning the hagfish have been reviewed by Brodal and Fänge (1963). Once one has become familiar with the habits of the hagfish, the reasons for the lack of general laboratory experimentation on this animal becomes apparent. The vast majority of these animals are found on the bottom of the open ocean at depths ranging from 50 to 1000 meters. This makes the hagfish relatively inaccessable for experimentation compared to the many forms of life which may be readily cultured in the laboratory. Many unique problems concern- ing the animals have appeared. These have ranged from determina- tion of its proper phylogenetic classification to the early attempts to maintain the animal under laboratory culture. Early attempts at holding the hagfish in underwater traps and in the laboratory did not 2 prove very successful (Palmgren, 1927). From recent publications, McFarland and Munz (1958), Munz and Morris, (1965) and in the present author's experience, there is ample evidence that the main- tenance of the hagfish under laboratory conditions can be relatively simple, so long as an ample supply of clean, refrigerated (5-10° C) standard seawater is available. Anatomical Aspects of the Hagfish The hagfish has been an anatomical and physiological puzzle for many years (Cole, 1926; Palmgren, 1927). Current textbooks classify the hagfish in the order Myxinoidea and the class Cyclo- stomata. The lampreys, order Petromyzontoidea, are also placed in this class with the hagfish. The basis for the classification is the similar external appearance of the animals. Both are eel -like with rasping, jawless mouths, but beyond this superficial similarity there exists little comparison between the two groups. The ecology, physiology, and internal anatomy of the two animals differ exten- sively. These differences have been recognized and the hagfishes have been separated from the lampreys as the class Myxine (Berg, 1958). The hagfish, Eptatretus stoutii, possesses many anatomical features which are unique among the vertebrates. One of the most radical departures from the higher vertebrate development is the 3 cardiovascular system. The hagfish possesses, as do the other vertebrates, a central systemic (branchial) heart. This heart is a completely aneural structure (Greene, 1902; Fänge and Ostlund, 1954; Augustinsson et al., 1956; Jensen, 1958; Jensen, 1961) and is composed of a loose arrangement of a sinus venosus, atrium, and a ventricle. The ventricle has no coronary arteries (Jensen, 1961) and contains a high concentration of adrenalin and noradrenalin (Ostlund, 1954) in special secretory cells of the heart (Bloom et al. , 1961). The hagfish has been observed to survive even when the ventricular portion of the heart does not contract (Jensen, 1961). Immediately posterior to this branchial heart is a small pulsating vessel, the portal heart (Retzius, 1822; as cited by Chapman, Jensen and Wildenthal, 1963). This valved heart, also rich in noradrenaline secretory cells, (Ostlund, et al. , 1960; Euler and Fänge, 1961) col- lects venous blood from the anterior cardinal vein, the supraintesti- nal vein, and the common portal vein. The blood from these vessels is pumped into the branchial heart. Another important feature of the cardiovascular system is the caudal heart (Retzius, 1890; as cited by Chapman, Jensen, and Wildenthal, 1963) which collects blood from the posterior subcu- taneous sinus and moves it forward to the portal heart. The caudal heart is under neural control (Greene, 1900) and the animal can survive its surgical removal (Greene, 1900) or the complete 4 removal of the tail segment containing the heart (Jensen, 1960). This heart is actually a paired structure (Figure 1). It is made up of two thin -walled vessels separated by a cartilaginous tee. Muscles attached to each side of the cross bar of the tee alternately contract. Each contraction causes blood to flow out of one side of the heart and concomitantly, a filling of the opposite side. From this author's observation, the caudal heart does not have a regular rythmn. The last heart to be observed was the cardinal heart recorded by Cole (1926). This heart, like the caudal heart, is a paired struc- ture found in the hyoid region of the animal. It is formed by swell- ings of the left and right anterior cardinal veins. In addition to this strange array of pumping mechanisms, the hagfish has developed another means of aiding the proper circulation of blood. It has been found that muscular contractions of the gill sacs help maintain a positive pressure in the dorsal aorta (Johansen, 1960). In spite of the gill contractions and the four hearts, the hag- gish has a relatively low pressure (3 - 8 mm Hg) cardiovascular system (Johansen, 1960). This fact is readily observable when an ani- mal is tipped end to end causing the blood to course back and forth beneath the skin. The hagfish cannot maintain a high blood pressure and a rapid circulation due to another unique anatomical feature and that is the presence of a large number of blood sinuses or lacunae. 1 . .ry 2 _ 2 11 ` ^ - ` ^ 2 ï\ ; 3 :' ., } ;. : :,,, ... 1 A B C, Figure 1. A horizontal section of the caudal heart of the Pacific hagfish, Eptatretus stoutii, adapted from Greene, 1900.