OCTOPUS MICROCIRCULATION: AN ULTRASTRUCTURAL STUDY A thesÍs submitted for the degree of Doctor of Philosophy Ín the University of Adelaide by Jay Browning, BSc (0tago) asc Hons (Adetaide) September, 1981 CONTENTS SUMMARY DECLARATTON A CKNO11/LEDGEMENTS BIBLIOGRAPHY OF PUBLTCATIONS FROM THIS THESIS SECTION I: INTRODUCTION pag e l.l The cephalopods - a viable alternative to the vertebrate plan 1 I.2 The refinement of circu-Latory systems 2 I.3 The advantages of a closed system 6 L.4 The vertebrate microcirculation I 1. 5 The cepha lopod ci rcu J.atory sys tem - a résumé of organization and function I4 I.6 An outline of the specific investigations 1B SECTION II: SOURCE OF ANIMALS AND EXPERIMENTAL PROCEDURES 2"I Capture and classification of species inves tigated 20 2.2 Experimental- procedures 2I 2.2rI 0perative procedures 2I ( Contents continued ) 2"212 Perfusion 22 2,2ri El-ectron microscopy 27 SECTION III: SCANNING ELECTRON MÏCROSCOPY OF VASCULAR CASTS 3. I Introduction 26 7.2 Material-s and methods 27 7.3 Resu lts 28 t.3 , I , The peri phera 1 venous vascu l-ature 29 7.7r2 The central arterial supply 30 3.7r3 The interposing microvascular network 7I 3.3r4 The vasculature of the suckers and other regions 32 3.4 Discussion 71 3.4,1 How rei-iable is the casting technique? 41 3.4 12 General comments on the 0ctopus casts 42 SECTION IV: OCTOPUS MICROVASCULATURE; MORPHOLOGY AND PERMEABILITY 4.I IntroductÍon 45 4.2 Materials and methods 48 4.3 Results 49 4.7, I General observations 49 4.3r2 Ferritin and carbon permeabilitÍes 51 (Contents continued ) 4 4 Discussion 60 4.4,I Tissue integrity and the location of. tracer molecules 60 4. 4,2 Permeabi lity of vertebrate capi llaries 60 4.413 Permeability of 0ctopus microvessels 63 4.4,4 Comparisons with the physiological data on 0ctopus 67 4. 4 r 5 Specu lations on the evo J-ution of microvascular structure 70 SECTION V: THE DIMENSIONS AND DENSITY OF EXCHANGE VESSELS IN BRACHIAL TTSSUES OF OCTOPUS 5. L Introduction 74 5.2 Materials and methods 75 5.2,I The Krogh-Erlang equation 77 5 .7 Resu 1ts 79 5.3, I App lication of the Krogh-Erlang equation 82 5 4 Discussion 84 5.4, I What constitutes an 0ctopus exchange vessel B4 5.4r2 The comparative vas cu larity of 0ctopus tissues 87 5.4, I The distributive Po tential of the 0ctopus vascu J-ature 90 ( Contents continued ) SECTI0N VI: TISSUE CHANNEL MORPH0L0cY IN 0CT0PUS 6. I Introduction 97 6.2 Materials and methods 95 6.2,I Quantitative methods 96 6.3 Resu lts 100 6.7,I Quantitative resu.Lts 104 6,4 Discussi.on 104 6.4rI Visualization of tissue channels 104 6.412 rmeation of the test ions in 0ctopus tissues I13 6.4 r7 Reg ional differences in 0ctopus tissue channels 114 6.4r4 Comparisons between tissue channel org anization in 0ctopus and marnma Ls 117 6.4r5 What roles are tissue channels likely to play in 0ctopus 1t9 SECTION VII: CONCLUDING REMARKS 126 REFERENCES 133 APPENDICES: Attached eopies of papers published from thj.s thes is . I SUMMARY Cepha lopods have evo lved a sophisticated circu latory system. However litt le rvas known about the microcircu lation where the singu lar ly most important function of the circuì.atory system, that of metabol-ic exchange, is carried out. This thesis report.s a study of the microvascul-ature of 0ctopus r uñdertaken primarily at the ultrastructural leve1, with the aid o1' el-ectron microscopy. Most emphasis was directed toward the muscle and neura'l tissues of the arms. Forthcomi-ng comments refer to this somatic vascu lature. Other tissues vlere Ínvestigated, but more superficÍa1ly. Cephalopods show a general ana J-ogy with the vertebrates , âod it is with t.he latter r râther than with other invertebrates, that comparisons couLd be most usefulJ-y drawn. An overview of the angioarchitechture was reveal-ed following scanning electron microscopy of vascul-ar corrosion casts. As a result, vesse ls encountered in u ltrathin section cou l-d be readi Iy associated with the three-dimensional- alrangement of which they are a part. Microvas cu l-ar fine structure and aspects of vascu lar permeabi J-.ity were investigated concomitant J.y . The endot.helium is never complete and is of little direct. conseqUence to vascul-ar permeability. The basement membrane is substantial and continUoUs, and constitutes the primary blood barrier. The pericyte layeI ¡ras always found to be continuous. Pericyte junctÌons constitute the major avenue for movement of hydrolytic materials acloss the vessel- wa11. Junctional permeability to ferritÍn, and to ferrocyanide ions ttraS shown; the fotmer, a compalatively large protein (hydrodynamic radius about 5.5 nm), could travelse the vesse I wa l-l within l" to 2 minutes . The junctions (about 12 nm wide) are too naIIow to aIlow the passage of colloidal carbon. A vascular impermeabíJ.ity to the haemocyanin compl-exes is inferred. Major differences between the croSS-sectional morpÌrologies of microvesse.l-s in octopuses and vertebrates are discussed and related t primarily, to developnent of erythrocytes which only occul i-n the latter. The density and dimensj.ons of the microvesse ls r^rere measured. It was decided that on ly vesse ls with a minimum perÍcyte wiclth equal tor or less than, O.25 um and a lumenal diameter of less than 1,5 um wele to be classified aS exchange vessels. About 45 exchange vessels tvere found pe, .r2 of tissue, the average dÍameter of wh-ich waS about B. O um. These VeSse ls , in terms of wall width and lumenal diametet, aIe simÍlar to those found Ín Lower vertebrates. Microvessel density, however, is an order of magnitude or two lower in 0ctopus. The Krogh-Er lang analysis indicated that the 0ctopus tissues studied, un like comparative vertebrate tissYut ' have little vascular capacity in IeServe to meet tissue demands once metabolic rates move above the resting level. The system of tissue channels in 0ctopus was also ínvestigated quantitatively, and ít proved broadly comparable with the mammalian system ( informat,ion about other vertebrates is l-acking). Little remains of tl-re mole open systems characteristic of the other mo.l-luscs and most other invertebrates including the primitive chordates. Various aspects of, and specu lations about , the development and function of both microvesse ls and tissue channels are discussed. Cephalopods evolved a closed and high ly pressurized vascu lar system independent ly o f the on ly other comparab Ie system, that found in the vertebrates. The structure and function of the microcirculatory system in the cephalopod 0ctopus is now better described. I' declare that this thesis contains no material which has been accepted for the award of any other degree in any university and that, to the best of my knowledge and belief, it contains no material previously published or written by another person, except where due reference is made ín the text. Jay Browning ACKNOIt/LEDGEM ENTS Mr. A. Franks, Mr. C. SmÍth and Mr. T. Winokurow' professional fisher.man, obtained the nrajority of animal-s used in this study. It would have been difficull- to have proceeded without their help. Ms. H. Smi.th, of the South Austr'a lian Department of Fisheries , a lso obtained some o f the animals. Mrs. A. Vincent helped w-ltlr the first series of operations. Mr. L. Jo11 heJ-ped with the taxonomy. Dr. L. Jarvis of Flinders University, South Australia, supervised use of the Quantimet. Mrs. R. Gaffney constructed a proglamme to handle some of the quanLitative data. ( I unwittingly omitted acknowledgement of her valuab le assistance in a relevent paper . ) The material-s for the vascul.ar casting vlere supplÍed by Dr. B. Gannon' of Flinders University; Mr. P. Rogers supplied the experimental. expertise . Mr. K. Crocker, of the EÌectron Microscope Unit , Uîiversity o f Ade laide , was a lways wiJ.ling to offer technical- help and advice. Mr. T. MacKenzie of the Zoology Department workshop also gave time and expertise wil-.tinglY, and Mr. P. Kempster helped with photography. My supervisor, Dr. J. R. Cas ley-Smith, helped of design course with general approaches, experimental ' crit.icism of manuscripts etc. However, and perhaps more important Iy , Dr . Cas ley-Smith has the perceptive abi fity to realÍze when encoulagement is particularly needed, whíle Iemaining one of the l-east interfering persons I know. Mrs. T. Piller helped with experiments, typÍng and in many other ways. similarly Mr. P. Christy helped in many ways, often proof reading and contributing useful comments. FinallY I thank mY wife Anke Oekinga, largelY for her patience , Pârticu J-ar lY during the finishing stages of this thesis when we al-so ha.d our todd J.ing son Yanto to contend wÍth. This thesis was completed desPite Yanto's it to best effotts, and if for no other reason ' I dedicate him. BIBLIOGRAPHY OF PUBLICATIONS FROM THIS THESTS [ (a)abstracts and (b)papersl (a.1) Browning, J. and Casley-SmÍth, J. R. (I979) Fine structural studies of the permeability of 0ctopus microvasculature to macromolecules. Bibl. Anat. i 18, 38. ( b. 1) Browning, J . (I979) 0ctopus microvasculature: Permeability to ferritin and carbon. Tissue Ce11, lI; 37I-383. (b .2) Browning, J. (fggo) oemarcation of tissue channels by ferrocyanide deposits: Use of an alternative preci.pitant. Microvasc. Res., 19i 38O-184. ( b. l) Browning, J . ( 1980) The vascu.l-ature of Octopus arms: A scanning electron microscope study of corrosion casts .