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 . Zoomorph. , 96; 243-251. (0. ¿r) Bror.rning, J. and Casley-Smith, J. R. ( 1981) Tissue channel morphology in _QcLcE_e. Cefl

Tiss. Res " , 2I5 i )53- 17O. (¡.¡) growning, J. (1981) The density and dimensions

of exchange vessels in 0ctopus pa llidus ( Hoy le ) .

(accepted for publication; J. Zoo logy , Lond. ) 1

SECTION I: INTRODUCTION

1.1 THE CEPHALOPODS _ A VIABLF: ALTERNATIVE TO THH

VERTEBRATE PLAN ( Cephalopoo, t) o..upy a special posítion amongst the Ínvertebrates. While possessing some distinctive molÌuscan characteristics, they aIe in most respecLs very unlike other non-backboned animals. Rather it is with the vertebrates that the cephalopods show similarities in the compJ-exity of neuromuscular function and precJatory mode of existence.' For instance, comparisons between the cephalopod and vertebrate eye make for one of the best known examples of analogoLls evoluti.on ín biological texts' and much of our initial understanding of neural impulse propagation followed f::om the work of Hodg kin and tluxJ.ey and collegues (Gordon, 1972) on the giant axons of squid. Many details of the complex and densely innervated nelvous system of squid and octopuses lrave been elucidat'ed, largely as a result of studies by Young (see, e.9., Young I97I, Ig76). This neural complexity is associated w-lth a high inte lligence . The extensive literature on recognition ancJ learning abilities and general behavi.our of pglgpus has been recently revievred (we1ls, I978).

(1) Throughout this tþesis the term cephalopod is loosely used to inclucie the comrnomonty studied octopuses' squid and cuttlefish. Specific reference is made of the nautiloids, the onJ-y other'extant cepttalopocl group. 2

It is generally less well appreciated that the extant cephalopods, while a group far reduced from the palaeozoic times when they ruled the oceans (Morton, 1967), nevertheless remain a viab le a lternative to the marine rrertebrates. Catch records in 1952 show (in terms of biomass ) that more squid of a single species was taken than were herring from the North Atiantic and adjacent, seas (Packard, 1972).

Cephalopods, then, have developed to a degree where t,h'ey success f u J-ly compete with the marine vertebrates in an ecoJ.ogical sense, and they uti lize a similarly v¡ell developed neuro-muscul-ar capability to do so. An important conponent of this successful body-pJ-an is the possession of a sophÍsticated blood-circuLatory system. This thesis reports an inquiry into the cephalopod circulatory system, ât the l-evel at which exchange occurs between blood and tissues.

I.2 THE REFINEMENT OF CIRCULATORY SYSTEMS l{ith the development of multicellular organizat,ion in animals there arose a fundamental problem: nuLrÍents and metabolites neecied to be transported between the tissues and the excretory, respiratory and nutrient surfaces (or organs ) . The increasing inadequacy of dj.ffusive processes in this regard, and the necessity for 3 some type of circulatory system is normalJ-y discussed in introductory statements on vascul-ar development and respiratory physiology (see, Ê.9.r Kampmeier, 1969; Jones, I972; Prosser, I977; Cliff, I976). Apart from the obvious inability of cells to function if starved of nutrients (or alternatively if poisoned by metabolic by-products ) , specialization and refinment of UiocÁemical interacLiorrs coutd only occul concomitently with deveJ.opment of better homeostatic mechanisms. The concept of the need for a relative constancy Ín the interna.l- environment was first realized, with any clarity, by Claude Bernard a century ago. The degree of homeostatic control is, in large part, a function of the vascuLar system.

It/hile cause and effect cannot be factually defended, as noted by Cliff (I976), there is an obvious relationship between the specÍalization and elficiency of vascular systems and the evolution of what ale commonly understood to be the higtrer forms of anima I li fe . Impairment of vascular function in these hi.gher forms usually leads to rapid and general malfunction: the need of a continuous b lood supply for the function o f the mammalian brain is well known.

The least specialized multicel-lular animals, the spongeS and coel-enterates, direct ambient water acloSS much of their surface, âñd in this sense actively 4 circulate fluid (see, Ê.9.r Barnes, 1968; Kamprneier, 1969). There is no suggestion of an irrternal vascuLar organization. The pri.mitive triploblastic platyhelminths do not have any real vascular organization either. Free living fllatworms possess a system of canals which lead from the body core to the periphlty. These canals, holever, are excretory structures. Cliff (Igle) makes the safient point that this emphasises the general importance of circulatory systems to the removal of waste products. 0lher major invertel¡rate groups, apart from the asche tmintns ( which inc l-ude the roti fers and nematodes ) have easiJ-y recognizab-le vascu.l-ar systems. The most simple are composed of short muscuLar tubes which propel a haemoJ-ymph, although not always in a unÍdirectional fashion (see, ê.g., the ascidians). At the other extreme there are we ll deve l-oped systems with one or more hearts and arterial and venous vesse ls linked by an interposing network of cJ-osed exchange vessels. In such systems blood and tÍssue f lu j.d are physicalJ.y separated. Many of the annelids, oñe class of the molluscs (the cephaLopcrds ), and the holothurian echinoderms possess closed vascul-ar circulations (Prosser, I973) , as do all but perhaps the most primitive of the vertebrates. In other members of the rnajor invertebrate groups the vascular systems are often only sJ-ightly less ref ined. Many gastropods ancl crustaceans, for example, have vessels nearing the dimensions o I vertebrate capi llaries , but 'b l-ood ' is 5 eventually released into the extracellular tissue space from where it drains agaÍn j.nto recognizable vessels. In these systems, then, blood and tissrle f luÍd are largely indistinguishab le .

A simi lar progression Ín complexity of vascu Ìar morphology is shown in the chordate phy togenetic series . Primitive chordates, such as the ascidians, have a very simple vascular organization as mentioned previously. The cephalochordate amphioxus possess a more refined vascul-ar ) system but it may be described to be truly open (Cas ley-Smith, I97 I) . In hagfish, which are perhaps the most primitive vertebrates in existence, the microvasculature is better defined although quÍ.te sinusoidal (¡ofransen and Martin, lg65); open junctions are found between the vascu.l-ar endothel-ia1 cells (Ca.sley-Smith and Casley-Smith, I975) . Capillaries in the other vertebrates, f rom the e.l-asmobranchs upwards, share many similarities. The generalized vertebrate patteln will be dÍscussed l-ater (Section 1.4) .

There wou ld appear to be a general relationship between the possession of increasingly refined vascuLar systems, the degree of rnoti lity and the structural and functional complexity of the animal- - aLthougl'r the insects have made exceptionaJ. advances in these regards whÌle retaining a very open vascul-ar system. Insects, however, 6 are not limited by the necessari ly s low b lood circu lation rates inherent with an open system; instead they possess a ramifying. system of air-filled tubules which allow f,or efficient respiratory exchange (see Cha.pman, lgsgl . ït is pertinent to note that trachea can only functiorr effeciently over short distances. This factor coupled with the problem of the disproportionate weight-to-strengtlr ratio of increasíngIy large exoskeletal supports, prevents the attainment of Ìarge body size in insects. It cannot, be coincidence that the largest invertebrates possess , closed blood systems. Annelid oligochaetes, for example, may be greater than two metres in length (see Dales, 1961) and the size of the huge benthic squid ís legendary; one specimen r¡/as measured to be seventeen metres long and four metres in body girth (see Barnes, 1968).

I,3 THE ADVANTAGES OF A CLOSED SYSTEM To begin, it should be noted that, no system is tru 1y c losed . It can be presumed that a lL microvesse l beds wi.ll leak (or receive) f luid to some extent, and investigations of anima ls with closed systems invariab ly reveal reqions where the vasculature Ís more or Less s-inusoidal, and therefore quite 'open'. Closed systems are, however, functionally distinct flronl open ones, if the extrace Ìlu lar body fluids are effectively separated into the vascular and interstitial compartments. The separation 7 of tissue fluid and b lood need not be maintained by a cellular layer a basement membrane (sometimes called a basal lamina) may serve this purpose ( Abbott , I97O;

Cas ley-Smith, l9B0) .

The basic requirement of a circulatory system in which fluid is circulated is the presence of a hydrostatic pressure source (this may have a negative component as found in elasmobranch hearts; Satche1l, 1971). Perhaps the two crucial advantages of a closed system are that (a) pressure is required to move a much reduced quantity of fluid, and (b) this fluid, because of its physical separation from the main extrace llu lar compartment , can deveJ.op specialized transport functions. Circulation of blood (vol/unit time) can occur more rapidly if the volume -is small and the flow is confined within a unidirectional system of tubes. Further, control over the supply vesse ls aÌlows aLmost the immediate diversion of b lood to tissues with the increased requirements - because bloocj remains within the tubes, there is not the delay of collecting it from the j-nterstitial space.

The blood of cephalopods contains a high concentration of haemocyanin (as will be discussed more ful1y l-ater ) . ThÍs respiratory protein exists in plasma solution. Taking the circul.ation of this protein as an example, it is difficult to envisage how an open system I could operate as efficiently as a closed one. To maintain similar concentrat,ions of haemocyanin in the blood (now haemolymph), four to five times as much would need to be produced. Indeed if the concentration in the vessel-bound portions of the haemolymph were to be as high, then even more wou ld be required for much wou ld stagnate in the random array of Ínterstitial channe ts and in Íncreasing amounts with time as the result of molecular sievir-rg (this effect has been discussed in broad terms by Casley-Smith, 1980) . There rvould no ef fective method ol' preferentially, and rapidly, returning deoxygenated protein to the circulation. 0nce 'bloodr had left the vessels, control- over its flow lvould be minimal. Similar problems arise if the transport of other nutrients and removaL of metabolic wastes is considered.

To reiterate, it may be fairly concluded that the presence of a cJ-crsed blood system confers a great advantage allowing animals to function as complex aggreqates of highly differentiated cel-ls whiLe developing a large body size, and pursuing an active, motile existence.

I.4 THE VERTEBRATE MICROCIRCULATÏON The microcircu latory sy stem of 0ctopus and the cephalopod circulatory system in general, can be use fu 1ly compared with the vertebrat,e system. Reasons for this are 9 given in Sectíon 1.5 and elsewhere. The investigations of the Octopus system reported in this thesis are largely based on established experimental approaches and the results are compared with the wide body of information and ideas available from the extensive literature concerned with the vertebrate sys-tem. As a resu lt ' a brie f description of our understanding of the vertebrate microvascular system is included here.

A recent and lucid review of the history of the investigations into the vertebrate microcirculation is presented by Courtice ( 1981). Harvey (1628) produced evidence that blood circulated in mammals, but the pathways postulated to exist between arteries and veins could not be visualized. Malpighi (166I) first observed blood flow in capiLlaries of the frog lung under one of the early 11ght microscopes. This capillary system is now described and understood in very great detai-1.

Krogh who began his studies in the ear J-y 1900's ' made a huge contribution t,o our understanding of capiJ.lary function. He measured, amongst other things, câpiJ.J.ary density in various tissues, related this to tissue demands for oxygen and determined that vessels wete recruited (i.e., more were perfused) when tissue demands for oxygen increased (seer Ê.g., Krogh, 19I9a, b, c). Krogh also investigated capillary permeabJ. j.ity to macromolecules. 10

His worl< remains pertinent and is extensively referred to in Section V. Starling (1S97) propos'ed his now cLassical ideas about f luid llow acloss capill-ary wa1ls. Essentially it was hypothesised that at the arterial side of the capil-laries the high btood ' hydrostatic plessure forces fLuid out, across' the capillary wall, but the nature of the wall is such that the colloÍds (i.e. pJ-asma proteins) are retained and slightly concentrated. The fal"l in lrydrostatic pressure which will occul along the capillary,, coupled with the colloid osmotic plessure ensures uptake of fluid orÍginally lost to the interstitial space. There followed ext.ensive physiological study ' and measurentents were obtained of capí llary pIessuIeS and permeabiìi.ty characteristics, and the inter-relationships between t.hese investigated. The names of Landis and Pappenlreimer are paramount here (see, e.g., Landis and Pappenhei.mer , 1963). Pappenheimer (1957) proposed a pole theory o1'the capillary wa11 which woulcl account for the good given physioloç¡ical findings. Recent reviews aIe ' for examp-le by \{olff (L977), Tweifach (1977), Renkin and Curry ( 1978) and Ren[

In 1627, As e 11i pub lished an account of 'mi 1ky veins' , found about the viscera of animals recentlY fed t l1 and the anatomy of the ma jor J.ymph vessels was known. Tlre function of the tymph system remained a mystery nevertheless. Hewson (1774 - cited in Courtice, 19B1), determined that lymph originated from blood. It is norv known that while the Starling hypothesis remains essentially correct, there is a net J-eakage o1" f luid and protein from the blood to the interstitial space. The lymphatics are largely responsible for returning these t,o the main circulation. Drinker (L942) showed that in the dog the equivalent of the entire volume of blood plasma, and about a half of the blood proteJ-n, is circul-ated daily via the lymphatic system. The important point to note is that the lymphatics do not merely constitute a fine adjustment to the general circulatory system, but are an integral part of it. While the issue is not fully resolved, -lymphatic vessels appear to have f irst evolved in some of the elasmobranchs and are found in all of the higher vertebrates ( reviewed Cas ley-Smith , 198 la ) .

Prior to electron microscopy, PhYsiologists necessarily treated capillary beds a.s black boxes. Palade (1953) first presented information about the capillary wali. at the ultrastructural level. The lumens of vessels are defined by a J"ayer of endothelial cells. These contain numerous vesicles, and in many regions (particularly visceral) are fenestrated. Furthermore, 12 endothelial cell junctions are complex, and vary from tissue to tissue. In most somatic tissues the cells are held toge.ttrer over about 95% of their junctional length by zonulae occludentes (CasIey-Smith et â-1., I975) where contact is intimate, ancl the opposing membranes are to some extent fused. For the rest of th.e juncl-ion the cells are usua 1J-y separated by a small gap ( close junctions ) .

In some organs (e . g. spleen ) and in injured tissues , endothelial cells are often wideJ.y separaLed (open junctions),. WhiIe areas of contention remain, it is now generalJ.y agreed that the hypothetical small and lalge pores of Pappenheimer are largely represented by the junctions and vesicles respectively (reviewed Renkin, I979). Thj.s is discussed in more detail in Section IV. Fenestrae are known to occur where the capí llaries are more permeab le. Evidence has been presented to support the hypothesj-s that the fenestrae are also responsible for inward movement of some o1' the spi J.led f luid and protein i . e. they act in concert with the lymphatics to help correct the problems which arose with the development o1'a highly pressurized blood f Iow (Casley-Smitlr, 198la).

Two fundamenta I and opposinq requÍrements are encountered in the development of the closed vascular system. In order that blood and tissue fluid can remain distínguishabl-e the v¡al-Ls of the exchange vessels need to be relatively tight, and able to withstand the hydrostatj.c I3 pressures necessalily suffered if the blood is to circulate quickly. 0n the other hand, the vessels need to be permeable if the exchange potential ís to be maximized. Ì./e now have a good appreciation of how this basic compromise is reached in vertebrates.

It is not possible to usefull-y summarize in these few pages a 1l that is norrl estab lished about the microcircu latory system in vertebrates . Rather , âî attempt has been made to describe some of the important aspects oi capillary strtlcture and function on which ouI increasingly more complehensive understanding is based, and from which the present studies on the 0ctopus system ffiây, to some extent, be rationalized and perhaps put in perspective. The cephalopod microcirculation was known to be c-losed, and some qualitative obselVations had been recorded o f the microvas cu lar ultrastructure . Very Litt le or nothing had been Iecorded, for example, about (a ) exchange vessel size and density, (b ) the permeability charactelistics of the microvascu Lar lining ' either determined physio,togicalJ.y or anatomÍca1-ty, (c) 1-he hydrostatic and osmotic pressures under which the microcircu l-ation operates , (d ) flow rates in the exchange vesse-Ls and how these alter during exereise, and (e ) the organization of the extravascular flu-id compartment. Some of these questions are investigated in this thesis. Before out linÍng the particu lar areas of research entered T4

into, a brief and general account of Lhe cephalopod circulatory system is presented.

1.. 5 THÊ CEPHALOPOD CTRCULATORY SYSTEM A RESUME OF

ORGANTZATION AND FUNCTION The arrangement of the hearts and major vessels of the cephalopod system has been described in detail for some time ( Isgrove, 1908; Grj.mpe , 19 It) and has been il-lustrated since (WeLls, I978). The major components of the circuîatory sys Lern of cephaJ.opods are schematica 1ly illustrated in fig. 1-L. Similar diagrams have oft.en been shown (see, e .9. r Johansen and Martin, 1962; Potts, 1965) . A centraL ventricle propels blood through arteria-l vessels and into the exchange pathways, from where it is collected by the venous system. The major veins drain into the brachial hearts. These boost blood pressure, forcing blood through the gi11s. Efferent branchial vessels channel- aerated blood back Ínto the ventricle. This system avoids a shortcoming of the fish circulation, where the high resistance bed of the gi J.ls interposes between the active pump and the exchange network of the general body tissues. Previous evidence strongly suggested that the somatic vascu.lature j-n cephalopods is closed, i . e. b lood is effectively separated from tÍssue fluid by elements recognizably vascuLar. Arterial- pressures of as high as 120cm HzO have been recordecJ from severely agitated 15

Fig.l-I. A schematic representation of the major components of the 0ctopus circulatory system; ventral view.

l{ater flows are indicated by dotted arrows. Water drawn into the mant le cavity via the two lateral, valved openings circulates about the gi11s and is then expelled via the centrally placed funnel. Blood flows are indicated by solid arrows. Blood from the general body circu lation eventually drains Ìnto the vena cava (VC) which bifurcates, sending branches to each of , the brachial hearts (BH). The branchial hearts propel bLood to the gills via the afferant brachial vessels (ABV). Blood arrÍves to the central ventricle (VN) from the gills via the efferant branchial vessels (EFB). The a ventricle propels blood to the body via two vessels, the larger of which is the anterior aorta (AA). The posterior of the animal (POST), that is the regÍon furthest away from the head and arms, is indicated. 'F

I , \ \t.-

t I VC + EFB

\ t BV ,

BH

POST

FIG 1.1 I6

0ctopus - they had been held out of water naÍled to a board | (Fuchs, 1895; cited in Johansen and Martin , 1962) . Pressures of 45-70cm ,rO were Iecorded from indwe Iling catheters in q. dofleini which vlere untestrained and showed no adverse react ion to the measuring apparatus (¡onansen and Martin, 1962). Little is knovrn about microvascular blood pressures, but venous pressure is known to increase along the major vessels as a result ofl strong peristal-tic activity (Smith, 1962b). Although not understood in great detail, it is obvious that the entire system is under complex neuro-hormonal" control ( ¡otlansen and Huston, 1962; Johansen and Martin, 1962; We1ls, l97B).

The respiratory protein haemocyanin, while encountered in a variety of invertebrate groups ' .1s found in híghest coneentration in cephalopods (Ghiretti, 1966). The oxygen-carrying function of the haemocyanin has been shown critica.l to cephalopods (neOf ield and Goodkind, 1929), in contrast to the often partiaJ. respirat,ory reliance oo r or indeed ambiguous function of, similar proteins in various other invertebrates (cnirett-i, 1962;

Prosser, 1973) .

Members of various invertebrate phyla are said to contain closed systems (see Section I.3). However in the annelids ¡ pêrhaps the nlost advanced amongst these and about which most is known , the separation o f b.lood f rom 17

tissue fluid may be maintained only by a basement membrane (reviewed Casley-Smith, 1980) without the continuous pericyte layer f ound in cephalopods. The more substantia-l vascular wall in cephalopods is undoubtly associated with the generation of higher blood pressures. None of the other systems are required to serve animals the size ol the larger cepha.ì-opods, and the lower oxygen consumption rates are indicative of a generally lower vascular e ffici.ency. In regard to oxygen consumption rates and arterial blood pressures, c€phalopods are similar to the lower urrî.brates. Indeed these are o ften higher in cephalopods than they are, for example, in the pelagic teleosts (coinpare tables I93 and I75, Altman and Dittmer,

le7 r) .

This entire introductory section has been kept broad, and a detailed review of the l-iterature pertaining to the cephalopod microvascu lar system purposefu J-ly omitted. The major research projects (outlined in Section 1.6) are presented with specific introductions and concluding remarks. It was considered more appropriate to leave detailed comment and refeÌences to the literature with each of the major projects, where these were most rel-evant. l_8

I.6 AN OUTLTNE OF THE SPECIFIC INVESTIGATIONS Thj.s thesís reports a study of microvascular sLructure and organization in Octopus. As noted by Cas ley-Smith ( 1980) , the arbitrary distinction between structure and function is particular ly irrelevant in the study of the microvasculature. General observations were made on the microvessel-s in a number of tissues. However it was decided to limit the bulk of the investigation to the exchange vesse ls and tissue channe l-s in the arms , and to therefore obtain a more compÌete understanding of the micloci.rculation in one of the most ímportant somatic tissues.

The study is divided Ínto four major sections. (a ) A study of the three-dimensional organization, or angioarchitechture, of the brachial circulation. This involvecl scanning electron microscopy of vascular corrosÍon casts. (b ) An investigation of microvasculature permeability to electron-dense tracers, and concomitant observations of vascular ult,rastructure. (c) A quantitative study of the density and dimensions of mj-crovessels, and the extrapolat-ion of these results to gaÍn some j.dea as to the distributive potential of the vas cu lature . (d) A quantitative study of the density and dimensions of tissue channels, with caÌculations on the like.ty hydraulic 19

and diffusive conductívities of these. (Tissue channeLs are a generally neglected, yet necessary component of a closed vascular system. )

Each section has a speci fic introductíon , description of materials and methods used, description of results obtained and a discussion r âñd. each has in essence been published separately. Copies of all papers written from this thesis are included as appendices. 20

SECTION II: SOURCE OF ANIMALS AND EXPERIMENTAL

PRO CEDUR ES

2.I CAPTURE AND CLASSIFICATION OF SPECIES I N VEST I GA TED

Octopus flinderi austral-is and pa J-lidus v/elie mostly obtained from the bottom trawling nets of comrnercial pravrn fishelmen working in the St. Vincents GuLf , Sout.h AustraLia. A 1"ew animals were also obtained from similar trawls undertaken by the South Australian Department of Fisheries. The nomenclature of these species is apparently , in question (.loLly, pers. coffifl. r 1979) . Nevertheless corrections or aLternati-ves have not been pubtished to my knowledge r ârìd the species listed are identifiable wilh those described by Cotton and Godfrey ( 1940). All three species were utilized while techniques were deveJ.oped and a familiarity with the ultrastructure of the tissues (and partÍcu lar ly b lood vesse ls ) was attained. Further cornments on the lack of notable qua lital-ive var j-ations in the microvascu lar arrangements amongst octopuses are therefore made with some justification. However 9.. flinderi proved too large and too di fficu tt to obtain for regu lar study , and will not be mentioned further. 0. austra -1is althcugh most common r âre sma1l and proved difficult to experirnent with. 0. pallidus are of intermediate size and could be obtained with just suffj-cient regularity to al_loyr systematic study. For the sake of consistency, tlris species was sole-ly used in the quantitaLive stuclies. 2I

SampIe numbers were unavoidably small, but adequate. The animals involved weighed between 150 and 500 gm.

0ctopuses taken from the nets rvere p laced into bins of seawater which were then irrtermittently aeratecl by hand. Anima ls were transported within the bins to the laboratory, and then placed into fresh and consistently aerated and filtered seawater. Animals have lived in this system for up to two months, although the experiments described normally undertaken days of ,were within I - 3 capture.

2.2 EXPERIMENTAL PROCEDURES Specific aspects of experimental procedure are described with the separate Sections ( Ill, IV, V and VI). Comments are made here on frequently used procedures and on the development of these.

2.2, I Operalive Procedures: All animals were anaesthetized before being operated oo, apart from the quick removal of sma l-l portions of arm tissue from two animals for control stucJies. A¡raesthesia \\,as achieved by graded additions of ethyi- alcohol over I - 2 hours, to a final concentration of approx, 3%. Ethyl alcohol is one of the most commonly used forms of anaesthetic for cephalopods (see, e.g., Johansen and Martin, 1962; Smith,

I962a) . 22

In the first experiments the branchj.al hearts were exposed and injected with the tracer-containing soLutions. This Ínvo-l-ved Little interference with the vascul-ar system or body tissues and, if care was taken, âñimals once revived, woul-d continue to live in the tanks. Hoy.Jever, only smal-l amounts of injectant could. be introduced. In another procedure , the e fferent branchj-a I vesse I was cannulated, aJ-lovring the perfusant to be sent straight to the central ventricl-e, âfld in greater vo.Lumes. Both procedures, all.owed access to the gene::a1 circulation. Later , and mosl- commorr J.y , the anterior aorta was cannulated (see fi.g. 4-l). Thj.s allowed direct access to

the brachial circu lation. The aorta was exposed by a medj.an, Iongitudinal cut through the dorsal- mantle, just. behind the eyes. The aorta behind the site o1" canrìu lation was left un ligated. This, coupled with the operative cuts of the mantle, allowed for suffici.enL outflow. The lasL two procedures did irrevocab le damage as very rnajor vesse ls were cut. Anima ls were periodically washed with seawal-er, ot- immersed in seawater if the time intervals between tissue sampling were extended.

2.2 2 Perfusion: Some tissues were processed rvithout príor perfusion. Most often, colLoidal carbon (approx. 0.02mI/nI rÍnger; Pelikan CII/ I43l-a) and ferritin (tO"¿ in ringer; Ca l-biochem, A grade , 2x cryst. , cadmj.um free ) were perfused through the animals. The ferritin had been 23

pr eviously centrifuged, thus allowing for removal of

ap oflerritin in the discarded supernatant (Davis et .11., 19 6B). The ringer was made up according to Smith (1962a), an d contained per 100 flf , 2.O59 NaCl, O.4g MOS0O, O.779 Mg O.249 CaCl and O.759 KC1. osmot,ic C1_,2'2 The total pr essure of cephalopod blood is close to that of sealater (P otts and Todd , 1965). Between 2 and 4 ml of perfusant co uld be readily introduced via the efferent branchial AI tery, the amount being effectively limited by the fr equency perf ,of contractions of the venl-ricle. A usant VO lume of l0 ml cou ld be eas ly perfused through an 0. oa I li dus of 3009 via an aortic cannr-ll_a over a period of 4 - 6 minutes. All perfusions were uncJertaken by hand, \'' and normally only a gentt/ Oressure was required. I I

2.2 3 Electron Microsco For routine e lectron microscopy, tÍssues r,lere cut under primary fixative into pieces with a mini-mum thickness of l-ess than one mm. These tissue samples were then placed in prÍmar.y fixative for I.5 - 2.Q hours, washed in buffet (2x, l0 min), and post-fixed with osmÍum. The tissues were then washed again in buf'fet (2x, l0 min) ancl dehydrated. Initially, dehydration was achieved through a series of alcohols (loa, Bo%, 9o%, loopí), wÍth one l0 - ú min. wash in each di lution and two washes in the roo%. The tissues were immersed in epoxypropane for t0 min and then int,o l: I epoxypropane and Spurs embedding media. In Later 24

experiments tÍssues vvere embedded in Taab embedding resin. This resin proved easier t.o use and is thought to have less toxic components. Here, tissues were usually dehydrated in a graded series of acetones and then into 1:1 acetone and resin, before being infiltrated in pure resin. Tissues were slowly and continuously agitated thoughout these procedures.

The primary fixative routinely used contained 2.5?6 glutaraldehyde and 2.oo/o paraforma.ldehyde in Sorerrsen's phosphate buffer (see GIauert, I975 ) at a pH of approx. 7 .O and with I8% g lucose. In pilot studies Ít r,las decided, on qua litatÍve grounds , that the use o f g lucose to increase the osmotic pressure gave better resul_Ls than the use of Nacl or Ringer solutions of símilar osmotic activity. The quantity of gJ.ucose coupled wÍth the other constituents guaranteed that the fixative was hyperosmotic to 0ctopus bod y fluids. The use of hyperosmotic fixatives gave better fixation. The waslring buffler contained the same phosphate salts and I8% glucose. The secorìdary fixative contained r% osmium tel-roxÍde in the washing buffer. DifferenL perfusates and fixatives were employed in the study of tissue channels and these are described e lsewhere ( Section VI ) .

An ETEC Siemens Au to s can scannÍng e l-ectron mic r os co pe was used for the study of the vascular casts 25

(Section III). The transmission electron microscope involved throughout the study was a Siemens Elmiskop I. Ultrathin sections were cut wÌth a Reichert OmU7 u ltramicrotome and placed on copper grids . Large ( 100 mesh ) grids were speci fica lly used in the quantitative study of vessels (section IV), and these were necessarily coated with carbon-strengthened formvar (see Hayat, I97O).

More usuaJ-1y smaller, unsupported, grids of 400 or 2OO mesh vlere used. All sections observed in Section VI vlere unstained. Sufficient contrast was obtained provided that ) (a) care was taken to cut thin sectÍons (about 60 nm ), (b) a smaLl objective aperture (ls um) lvas used, and (c) the sections lay on unsupported grids. Other sections were stained with Ìead citrate (modified from Reynolds, 1963) and, in some instances, also with uranyl acetate (sX aqueous; see Hayat, I97O) . 26

SECTI0N III: SCANNING ELECTR0N MICR0SC0PY 0F

VASCULAR CASTS

3.I INTRODUCT ION The advanced circulatory systems of commonJ_y studied octopuses, squid and cuttlefish, have drawn much interest. The gross. morphology of the vasculature is quite well understoocl r âñd was first described for a number of species some time ago (see for example, Wi lliams , I9O2; Isgrove, 1908; Grimpe , 19 I3) . However, information about the arrangement of microvasculature is generally Iimited, the blood system of central- neural tíssues excepted ( Young, L97 I) . It was decided that an invesligation of the vascular arrangement (or angioarchitechture) of the region of prÍncipal interest, i.e. the brachial tissues, woul-d be a useful- prelude to the ultrastructuraL studies. A corrosion cast technique rvas subsequently utilized.

Although the study reported here was directed towards the brachial- circulation, good casts of various other regions were obtained in the process. The arrangement of the major vessels in 0ctopus arms lvas well described by Smith (1967) , but many finer detai ls remained beyond the scope of h-Ìs study. - Apart lrom the investigation into the haemal and coe lomic circu latory systems of crinoÍds reeently reported (GrÍmmer and 27

Holland, I979), scanning e lectron microscopy ( SEtl ) o f vascular corrosion casts of other invert.ebrate anima ls has, to my knowledge, not been unde::taken . The resu lts o f the present investÍgation show the vascu l-ature org anization of 0ctopus arms with a c larÍty not obtained previously, and the technique cou ld certain 1y be use f u IJ.y employed in investigation of the vascu lature o f other cephalopod tissues.

3.2 MATER IALS AND METI.IODS Animals were caught and anaesthetised as described previousJ.y (Sectj.on II). Both 0. pallidus and 9. australis were perfused with plastic, but casts from the l-atter were less comp lete and are not i llustrated. Seawater was washed through the animal via the anterior aorta onJ.y until the outf l-ow f irst appeared colourl-ess. Liquid plastic was then immediateJ.y introduced, and rnore than 20 ml could be perfused through a 2OO gm animal before the plastic began to harden. The perfusion pressure was exerted by hand. The whole preparatÍon was next placed in warm water for about 12 hours to ensure complete polymerization of the plastic, and then into 25% KOH untj.l the tissues had been dissol"ved. A period of 5 - 10 days was ample.

The casts were washed gentty with water before 28 blocks were cut and mounted. The mounted samples were subjected to osmium vapour for approx. 36 hours (Murakami et a1., I97t), coated with gold and carbon, arrd viewed in an ETEC Siemens Autoscan scanning electron microscope at an accel-erating voltage of 10 kV. Following the suggestion of Murakami (L975 ) parts of the casts were immersed in water, which was then frozen. The fine pJ-astic networks could then be cut while supported by ice.

The perfusate consisted of 70% Mercox ( Japan Vilene Co. ) and tO% inhibitor-free methyl methacrylate monomer, mixed a few minutes be fore use . To this tvas added 3% of the catalyst, 2,4 dichlorobenzoyl peroxide 50% w/v in dibutyl phthalate, immediateLy prior to per:f us-ion.

The addition of red pigment (110494, Polysciences Inc. ) allowed the progress of the perfusate to be easily seen. The materia ls and preparatory procedures employed in this study are those most recently developed by Gannon and eollegues (see the acknowledgements ), and the ent-i-re process is based on the ear l-ier work of" Murakami (I97I) .

3.t RESULTS Perfusion via the anterior aorta resulted in initÍal filling of' the brachial circulation, but the success of the technique was such that casts ofl the entire anj.mal were obtained (fig. 3-I). 29

The brachÍa 1 vas cu lature can be separated into three major components (fig. 3-2)z the periphera.l- circu-lation containÍng the notably large venous return (figs. 3-i to 3-6), the central arterial supply (figs. 3-7 to 3-9), and the vasculature of the suckers at the base of the arms (figs. 3-IO to 3-I2). A network of mostly finer vesse-ls oecurs between the central and peripheral regions (fis. 3-e). t.3. I The oerioheral venous vasculature: The peripheral vascufature is the most obvious aspect of the brachial circulation, and the Ìarge longitudinal venous vessels are observable without microscopical aid (fig. 7-7). However, the various, detailed intercommunications of vessels in this region are revealed by SEM with a ctarity not previous ly attaíned. A general observation of the vasculature here is that vessel communications often bear littIe relationship to a classical hierarchy based on vessel díameter. For example, both small and large vessels Lead into the major longitudinaL vessels ( fig , 3-t) , there are c Losed loops of large and small vessels, and anastomoses of various díameters and lengths connect between the major vessels (fig. 3-5). The venous vessels are larger nealr the skin; the largest vessels are the most exposed. This peripheral vasculature, ât least laterally and outwardly, is morphologically distinct, and easily separabl.e from the rest of the braclrial 30

circulation ( fig . 3-6) . It incLudes the most peripheral of the microvascular networks, a contorted mass of relatively large vessels (diameters of 30 - 50 um are representative).

3.3,2 The central arterial supplV: The primary arterial supply is centrally placed in each arm and closely aligned with the core of neuropil tissue. The major artelial vessel ( figs . 3-7 , 3-B) runs aJ.ong the top of a compact tube of microvessels that is encLosed and deflined by arterial branches. The main arterial vessels and enclosed network can also be separated physicaJ.ly as a distinct unit. The centraJ- microvessels are rnore regularly arranged than much of the peripheral network, and generally of smaller diameter (commonly 5 - 20 um). It is un likely that the sma l-ler diameter is a resu It o f inadequate perfusion because the vesse ls are so close to the arterial supply. Concomitantly, it is unlikely that the peripheral vesseLs mentioned earli.er were markedly dis tended because per f us ion pressures must have been l-ou¡er here.

Casts of the major arterial vessels show a striated surface with a number of what must be presumed to be protruding endothelial nuclei (fig. 3-B). The ÍndentatÍons are generally Ìess abundant elsewhere and thus un likely to be o f pericytic origin because the 3I pericytes are permanent structures of all the vessels. Casts of larger venous vessels showed fewer indentations, and the striations, if present, were not as marked. 0n the basis of these differences, whi,ch could either reflect different morphologies or be artifactual, the major arteries and veins were separable. It is of interest to note that venous and arterial vessels Ín amphibian and mammalian tissues are aJ.so morphologically distinct

( Lametschwandtner et â1. , I978; Gannon, pers. comm. , f981). Arterial vessel-s could be traced to the lateral and lower sucker bea.ring aspect of the arm. No deflínite examples were found of arteries branching anterior ly to the upper surface.

3.3r3 The interposing microvascular network: The microvascular network (figs. 3-2, 3-9) interposed betvreen the surface and central regions (and Lherefore associated with the predominant body of muscular tissue of the arms) was not uniform-ly dense in al-l arms studied. It occasionally became notably less dense toward the tip of an arm. However, it rvas apparent that perfusion had been inadequate in these instances. Generally, both the surface and central regions vvere well outlined, and thus the few large interconnecting vesse ls that lvere found probably function as channels of preferential f1ovi. Although the data was not quanti fied, the interposing microvasculature was largely composed of vessels similar 32

in diameter to those of the central region, and appeared to be more dense laterally and toward the lower surface. It is obvious that the exchange vesse ls in 0ctopus are arranged , essentÍa 1ly , in random fash ion un like the more parallel arrays of capillaries found in vertebrat,e muscle.

3.3,4 The vasculature of t,he suckers and other reqions: The vascuLature of the suckers is comparatively extensive (figs. 3-10 to 7-12), indicating the importance of the muscular activity of these structures. Arterial vessels could be traced from the central supply to the dense microvascu lar network about the base of suckers. The venous return, beginning at the sucker rim, is easily identified (fig. 3-LZ) and drains into the large lateral venous vessels seen in figs. 7-2, t-3.

Although the brachÍal circulation rvas of immediate interest, vascular casts of other regions, particularly anterior to the site of cannulation (eyes, brain, mouth etc. ) appeared complete. In fig. 7-I3, for example, a part of the vasculature of the eye regÍon reveal-s a specialized parallel array of vessels (corresponding to the ciliary ring vasculature described by Young; I97I), adjacent to a two dimensional neLwork of inter- communicating vessels. Whereas tissues posterior to the site of cannulation were, ât least superficialJ.y, well perfused, even more complete casts would prclbably result vt

Íf specific arterial supplies had been cannulated.

The informationaL gain obtairrable from mÍcrographs of corrosion casts can be considerably increasecl if stereo paÍrs are prepared (Gannon, 1978). Use of this procedure helped to establish connections between vesseLs whích tvere otherwise unseen or ambiguous. Further, the 3-dimensional- visualization sometimes revealed that smaller vessels were broken or had been inadequately filted when otherwise thi.s was not obvious. The example shown in fÍ9. 7-14 iltustrates this point. The vessels are part of t,he interposing microvascular netvrork. 0ther microvascu lar casts were more complete.

3.4 D ISCUSS ION The corrosion cast techni.que was successfuJ-ly employed in this study of the Clctopus arms , â f lowing who J.e segments of the entire vasculature (or angioarchitechture) to be readily observed. As noted by Gannon et a1. (I977) the alternative method of reconstructing three-dimensional images from two-dimensional images is difficult, particularly in regard to establishing microvascular connections. l,lhereas the perfusates are becoming mole satis factory according to the criteria listed by Gannon ( 1978) , nevertheless there are problems with interpretation of the results obtained. t4

Fig. 7-I Vascular corrosion cast, showing the entire 0ctopus bod v. A cannula (arrow) is inserted into the anterior aorta. Perfusate was introduced about L cm. anterior to the o cular region (0) . Large, longitudinal veins and '' transverse venous anastomoses (V) are vÍsible at the surface. (Bar=1cm)

t5

Fig. 7-2 Median longitudinal section of a brachial cast. PerÍpherally are the large venous vessels (V) seen in fig. 3-I. The principal arterial vessel (A) runs centrally. Major arterial bran"i.. (B) can be traced to the region of the suckers. The interposing microvascu lature beds are shown incomp lete ly here , â llowing re lationships between the peripheraJ-, central and basal regions to be more easily seen. (Bar = 2OO um)

Fig. 7-7 Brachial vasculature. Lateral view of the subepidermal vasculature which is composed largely of venous vessels. The large vessel- (V) is one of the longitudÍna1 vessels observed in fig. 7-I. Both large and smaLl vesse ls (arrow) lead into a major vessel. (Bar = 2OO um)

36

Fí9. 7-4

Higher magnification of a major venous vessel, showing the characteristically smooth cast. (Bar = 100 um)

Fig.3-5 Transverse connections (or anastomoses) between large venous vessels. The arrow indicates an indentation left by a nucleus, most probably of an endothelial celI. (Bar = 100 um)

Fig. 7-6 Cross-section of the subepidermal vasculature. The re gÍ on inwards ( In) is largely avascular. Venous vessels become larger more peripherally. (Bar = 100 um) \il ln \. ì \ f

;\ ) qr\. t 1 ^ ì 1 ¡

â-t I l I / - 37

Fig.3-7 Central aspect of the brachial vasculature. The principal arterial vessel (A)' runs longitudinally. There Ís a microvascular network associated with the arterial supply. (Bar = 100 um)

Fig. 3-8 Higher magnif ication of the central arteriaL suppJ-y. RadiaI arterial vessels lead from the major longitudinal vessel (A). Indentations of endothelial nuclei (arrows) occur in small and large vessels, and striations in the latter, are shown. (Bar = L00 um)

38

Fig. 7-9 Transverse section through the brachial cast, showing the deflnitive centrally placed vascular bed with the arterial vessels (A), and the interposing network of Írregularly arranged microvessels. A large arterial branch (B) leads towards the region of the suckers. (Bar = 2OO um)

t9

Fig.r-10 Cross-section of the sucker vasculature, indÍcating how the form of the sucker is indicated by the remaining blood vessels. (Bar = 100 Lm)

Fig. 7-II Top view of the sucker vasculature. (Bar 2OO um)

'F19. t-I2 Lateral view of the sucker vasculature. The beginnings of the venous drainage (V) can be traced from the rim of the sucker. (Bar = 100 um)

40

Fí9. 7-I3 Vasculature of the eye. The specia lized nature of the tissue is asso ciated wi th a hÍghly di fferentiated vasculature. (Bar = 2oo um)

Fig. 3-14 Stereo pair of microvasculature within the brachial 'tissues. Vascular pathways can be readily traced. Some smaller vessels appear to be bLínd-ended (B), others appear not to have been adequately perfused (U). (Bar = 20 um) B

¿r l- \ 4I

3.4,I How reliable Ís the castincl technique? 0ne of the major prob lems with the casting procedure is independent of the nature of the perfusate: it is that J.iquids tend to pass through channels of least resistance and inadequate f i.tling of other regions may occur. Presumably these regions can be fÍ 1led if high perfusion pressures are used, although the thin-vralled vessels may rupture. Leaked p lastic, norma 1ly quite distinctive under the microscope, resul-ts from vessel rupture or indicates an

"open" vascular lining. Cas ley-Smith and VÍncent (I978) , for examp-le, used a low víscosity perfusate which passed out of fenestrated mammalian capillaries and into channels in the immediate extravascu lar matrix. Accurat.e descrÍption of vessel dimensions is limited owing to possible dístension, or otherwÍse, of the vessels in response to the perfusater âñd because there are inherent Limitations in obtaining quantitatÍve data from scanning electron micrographs. llowever, these difficulties are partially overcome when inlormat,ion is obtained from other sources. The cross-sectional dimensíons of vessels, such as lumenaJ- d:'.ameter, are better estimated w-1th stereology of transmj.ssion electron micrographs (as reported in Section V), and similarly, the likelihood of non- artifactual leakage of the perfusate can be assessed from ultrastruct.ural- observation of the vessel wall. While the mj.crovasculature of 0ctopus has not been investigated as extensively as Ín the vertebrates, the pericyte layer of 42 the vasculature of somatic tissues appears to be corrtinuous and is not fenestrated (see Section IV; Barber and Graziadei, 1965), suggesting that any extravasation is very probab 1y a resu 1t o f disruption of the vesse I wa 11 and is not indicative of a relatively open system. Incomplete fi l1ing is probab ly inevitab le, and the existence of blind-ended microvesse ls in Octopus is not determined with certairrty. Nevertheless, a good indication of the reLative conìpleteness of the casts can be made on the basis of factors illustrated (fig. i-14) and dis cusseO in the resu l-ts .

3.4,2 GeneraL comments on the 0cto pus casts: While Smith (De=) gave a good descripl-ion of Lhe brachial circul-ation in 0ct,o_pus, the investÍgat j-on of the organization of ilre smaller vessel-s was limited by the use of the light m:'.croscope. This is also true of young's (197I) investigat.Íons into the vasculature of neural tissues of 0etopus. A more complete picture of the detaiLed vascul_ar intercomrnunications within these, and other ti.ssues, would resul-t from the use of the techniques described Ín the present study.

The findings of Snrith (196j) are substantiated and extended, with the various regional differences in vessel morphology shown, and mole information gained about the microvasculature. 0n the basis of vessel organization arrd 43

frequency, the microvascular networks of the perípheral and more central regions obvious ly constitute the major exchange potential of the system, although the latter is largely composed of comparatively wide vessels. The presumed reduction in exchange efficiency of these wider vessels Ís related, perhaps, to a reduced metabolic need of the subcutaneous tissues, and foll"ows the general trends toward J.arge vessel size, and probable capacitance function, of the peripheral circulation. The numerous connections between the large and exposed vesse l-s are probably important in channeling blood should injury, or some other factor, necessitate it. The vessels of the microvascular networks appeared to be randomly arranged, although parts of the central network contained some vessels that were more radially orientated. The casts add strong evidence for the closed nature of the somatic vasculature of 0ctopus. Furthermore the casts are very simi lar in general appearence to those obtained from vertebrate tissues, emphasising the convergent, development of vascu.Lar organization in the two groups. Studies of the ontogeny of the 0ctopus vascular sys tem wou Ld be Íl1uminating, for it is not known Íf this similarity in gross structure re flects a simi larity in deve lopment processes.

Keller et a1. (L972) wrote in the introduction to a study of vascular corrosion casts of the mammalian 44 carotid body that knowJ.edge of the vascular organization was indÍspensible to interpretation of observations on the distribution of oxygen partiat pressures, blood flows and distribution of flows. This preliminary study with 0ctopus vlas the first to involve scanning electron microscopy ofl vascuLar casts. Although very much more coulcl be gained from further investigations with the technique, the primary aim of the study was achÍeved. That is, an overview of the vascular arrangement was obtained, giving some perspective to the results of the ) rest of this thesis where vessels and tissues are visualized at the ultrastructural leve1. A number of findings made r or substantiated, here were of specific interest. The random arrangement of the interposing microvessels contrasts with the vertebrate muscle capi llary beds as previous ly described, which is of relevance to the study on vessel density (Section V). The large and thin-wa1led vessels often found subcutaneously were obviously venous, while the thicker walled vessels encountered r,lithin, or near, the neuropil were arterial. These regional differences allowed the major vessel types to be initia 1J.y recognised, and then described, with confidence. In this regard the region of the suckers is Iess useful; both venous and arterial vessels occur here. 45

SECTION IV: 0CTOPUS MICROVASCULATURE: MORPHOLOGY AND PERMEABILITY

4.I INTRODUCT ION Cephalopods have a blood system which resemb.l-es the bLood system of vertebrates in gross morphololgy. The vascular casts of the preceding sluCy (Section III ) further emphasize this convergence. Both systems are closed, and similar differences exist between the cast morphologies of venous and arterial vessels. However, little j.s'known about capillary function in cephalopods, and in this respect the degree of similarity between the two divergent groups has not been established. There has been, to my knowledge, only one physiological investigation of microvascular function in cephalopods, undertaken by Smith (1962a) in an attempt to determine the exÍstence in Octopus ca pilJ-aries of a condition comparable to that described by the StarlÍng equilibrium in mammals. ExperimentaL difficulties and a paucity o1" results make the determinations questionable. Further, as noted by Smith (1962a), tlre interpretation of results was limit,ed by a lack of anatomical- evidence.

The ultrastructure of smaller vessels in Sepia and 0cto pus has been described by Barber and Graziadei (1965, 1967 a, b) and illustrations of cephalopod vesse.l_s Ín electron micrographs of various glands and tissues are 46 quite numerous (see for example Yamamoto et âf., 1965; Gray, 1969; Kawaguti, 1970). I wiIl' use the descriptive terminology of Barber and Graziadei ( 1965): ceLls ly1.ng Lumenally to the basement membrane of the vessel are endoLhelial, whiLe those positioned ablumenally are pericytes.

The basement membrane often serves as the primary blood barrier since the endothelial J-ayer is rarely, Íf ever, complete. The vascuÌar vrall proper is conposed ofl pericytes, never more than a single cell layer thick. Neural tissue within the braÍn (Froesch, I974) , in communication with the vena cava (Martin, 1968; Berry and CottrelL, I97O) and in certain other regions (f'roesch and Mangold, I976a) , occasionally repl-aces sections of the pericyte wall and is then direct ty /pposea to the basement membrane. Barber and Graziadei (1967a, b) and Gray (1969) did not observe suclr neurohaema -1" contacts in their studies of cephalopod neural tissue, althouglr both report the infrequent occurrence of blood-fi1led tissue spaces. The pericyte Iayer of smaller vessels in cephalopod musculature has always been found to be complete.

Pericyte junctions are clearly recognizable Iegro ns with an invarying gap of approximately I2 nm. Froesch and Mangold (I976b) refer to rfenestrated regions' in the pericyte wall of Octopus capi llaries. The paper of 47

Froesch (tglt+) was referred to for evidence of such fenestrae (Froesch, pers. comm. ) . However, the term was used imprecise ly here to describe gaps in the discontinuous endothelial layer. True fenestrae, âs commonly seen in mammalian visceral capillaries, have not been found in pericytes. It can be inferred that the permeability characteristics ofl cephalopod exchange vessels must then differ markedJ.y from vertebrate capi lJ-aries, which show a variety of jurrctiona.l contacts between endotheli.al cells of the vascular lining and, in some regions, the occurrence of fenestrae and numelous vesicl-es. Permeability studies with cephalopod vessels have not been speci fica 1ly undertaken with the aid o f electron microscopy, although Froesch and Mangold (I976b) did report brie fly that vesse Ls in the optic g land of 0cto ous are permeable to ferritin.

The aim of this e -Lectron microscope study lvas to gain more in formation about the genera 1 norpho logy and permeabi lity characteristics of the microvascu lar lining in 0ctopus ; the latter aspect involved tracing the fate of carbon and ferritin introduced into the circulat,ory system. Most emphasis vlas placed on the microvasculature of the body muscu lature and brachia I neuropi 1 tissue . Preliminary investigation of other glands and tissues allowed for some comparisons to be made. The results presented begin to lay the basís from whj.ch results of any 48

further physio logica I studies on vesse I permeabi lity in eephalopods may be interpreted.

4.2 MATERIALS AND METHODS Animals were anaesthetized as described previously (Section II). Normally, carbon and ferritin were introduced into the genera I circu lation via the the efferent branchial vessel so that a range of tissues could be looked at for comparative purposes, but cannulations of tlre anterior aorta were also undertaken (fig. 4-1). Contractions of the ventricle were fo llowed by an immediate blackening of the entire animal. During the operaLion, the exposed mantle cavity was periodically washed with seawater. The UrJcniafI hearts and ventricle continued to beat over the experimenta 1 period and extremely effective vasoconstriction prevented the mantle muscuLature from blackening for 0.5 - 1.0 cm about the edges of the operative cut. One animal, cannulated and re-immersed in seawater, was allowed to a lmost complete ly revive be fore the so lution containing Ferritin lvas introduced. These observations indicate that the functioning of the vascul-ar system was not adversely affected to a critical degree by the operative procedure. Various tissues were processed for electron microscopy as described previous ly (Section I I ) . Sections were stained w.ith lead citrate but urany 1 acetate was not used as an 49

additional stain when sections were viewed at the hÍgher magnifications, for although the increased contrast is

favourable for viewing tissue under lower magni flication ,

the position of sma lL tracer mo lecu l-es can be more accurately determined when contrast is lower and there are fewer staining artifacts.

4.3 RESULTS 4.3 I General- observations: The basic morphology of the microvessels containing carbon and ferritin is comparable to that described by Barber and Graziade i (1965, 1967a) for 0ctopus vulqaris and Sepia o ffician lis r êîd did not dif f er from the microvasculature of control animaJ.s, i. e. those not subjected to operative stress. The endothelial layer is never complete. The pericyte wa11 is continuous. The pericytes, s€parated by junctions with a consÍstent gap of II-J2 flfrr normally interdigitate to a large extent, particularJ-y in the thicker walled vessels (figs. 4-2, 4-3). The appearance of the pericyte wall is remarkably similar in vesseÌs of different tissues (figs. 4-2 to 4-7). The observation of Kawaguti (I97O), that pericytes in all vessels of Sepia esculenta and Watasenia scínti llans contaÍned myofibrils, was not found to be true of 0ctopus species here, nor is it true of other cephaJ-opods (Barber and Grazi-adei, 1965). Myofibrils were notably absent flrom many of the smalJ.er, thin-walled vessels. Kawaguti's 50

( lgzO) use of the term runicellular muscle layerl asa description of pericytes cannot be supported.

Few vessels in muscu.Lar tissue were closely encircled by muscle. Almost invariabJ-y all, or a substantial portion, of the vessel wall was surrounded by a co llagenous matrix as seen in fig. 4-2. Immediate extravascular regions in the non-muscular tissues of the eye and brachial neuropil were similarly collagenous ( fig . 4-7) . The aldehyde fixative caused strong contraction of body muscu.Lature and most probably of myofibriLs. This would account for the usually collapsed, or parti.ally collapsed, nature of vessels seen to contain myof ibri.ls. ContractÍon of muscle tissue presumably had an effect on vessel diameter since collapsed vessels, without obvious myofibrils were normally found near muscle blocks (fig. 4-6)i those surrounded by large areas of collagenous tissue vlere often open (fig. 4-7). QualÍtative differences were noted between ferritin ancl carbon concentrations in co llapsed vesse ls from the same block. These vesse ls had been exposed to the tracers for more than 5 minutes, thus discounting the possibility that the tracers had insufficient time to permeate the vascu lar' system. Such observations give an indication of which vessels in vivo lvere operative and which were, to sonìe extent, inoperative. 5l

4.3,2 Ferritin and carbon permeabi lities : All vesse.l-s

Ínvestigated were found to be permeab le to ferritin , including the thicker walled type of vessel shovrn in figs. 4-2 and 4-3. Ferritín was found in the extravascular matrix within I - 2 minutes of being Íntroduced ( fig. 4-B) , and in greater concentral-ions within 5 - 6 minutes ( fig . 4-9) . carbon was never found to penetrate past the basement mernbrane or enter the pericyte junctions, nor was it transported across the pericyte in vesicl-es (figs. 4-10 to 4-12). Endothelial cells did iake up carbon into large vesicles (or vacuoles) over the longer experimental periods (figs. 4-ro to 4-rz). The basement membrane , although obvious 1y permeab-ì-e to ferritin, does constitute some barrier to protein movement. since the highest ferriLÍn concentrations occur in the lumenal aspect of it (figs . 4-8, 4-11). The discontÍnuous endothe lia 1 layer p lays litt le part in reducing vascu lar permeability to ferritirr. The concentrations of ferritin in the regions where the basement mernbrane is separated from the vesse L rumen by an endothe ria I ce lt are as high as elsewhere (figs. 4-10, 4-11). Moreover, carbon can be found between the endotherial layer and the basement membrane, being perhaps associated with the basement membrane, although never deep wÍthin Ít (fig. 4-10).

The pericyte junction appeared to be the main avenue for movement of ferritin from the vessel lumen to 52 extravascular tissues. Ferritin often penetrated the basement membrane to a greater extent in the vicinity of an opening to a pericyte junction ( f igs , 4-8, 4- !2, 4-14) , perhaps indicating a differentialJ-y hj-gher permeability of the basement membrane in these regions but, more probably, indicating regions of greater fluid flow (which swept the ferritin molecules along). The extent of the movement of ferritin through pericytes is dif f icu-l-t to ascertain. Easily recognizab le vesic les were occas iona J.ly seen i.n endothelial cells (figs. 4-10, 4-11), but far less f requent ly in the pericytes ( f ig . 4- I7) . Perhaps tlre vesicular structure is not well- preserved with fixation. Alternatively it is possible that ferritin exisLs freeJ.y in solutÍon in the pericyte cytoplasrn. In fig. 4-It+, ferritin is either within a vesi .rc þ a pericy te junct j.on which has been obliquely sectioned . many instances, il^rn ferritin found in the cytoplasm could be associated with obliquely sectj.oned junctions (fig. 4-5). It is probable that movement of ferritin resulting from knife drag ( cas ley-Smith , 1962) accounts for some o f the ferritin found in the extravascular matrix and apparently freely in the cytoplasm of the pericytes. However, the importance of this drag in large-scale movements is largely discounted in fig. 4-9 where ferritin , in comparative ly high concentration in the extravascular matrix, is seen tc¡ be totalJ.y contained by the membranes of the opposing neuropil tissues. 57

Fig. 4-I Octopus pallidus preparation, ventral aspect; showing the cannulation of the anterior aorta ( large arrow) and vessels out lined by the carbon containing perfusant (sma11

'arrows). E = eye. Each large division on the scale rule is a cm.

54

Fig. 4-2 A collapsed thicker-wa1led vesse I from brachÍa I musculature. (Bar = 0.5 um)

Fig. 4-t A simÍlar type of vessel from the brachial neuropi 1. (Bar = 0.5 um)

In both vessels the lumen is defined by the discontinuous -endothelial layer (E), the basement membrane (B) and the pericytes 1ayer, here showing evidence of myofibrillation (l'ty). There are occasional mitochondrÍa (Mi) in the pericytes, and numerous sections through pericyte junctions (arrows). In both figures about one quarter of the vessel is shown. Both lie in a colJ-agenous matrix (Co). tt-;

I ¡:lh' ..{r 55

Fig.4-4 Thin walled vesse 1 Ín the subcutaneous tissues of the arm. Portions of the pericyte wall are less than 0.04 um thick (arrow), although the vessel diameter vlas estimated to be about L5 um. The endothelial cell (E) contains what appears to be glycogen granules. Reflective units of the subcutaneous chromatophores are seen (R). (gar = 0.5 um)

Fig.4-5 Thin walled vessel situated in salivary gland sinus. There is carbon (C) and ferritin (dark dots) both within the vessel lumen (L) and a sinus (S). The ferritÍn concentration within the vessels appears hiqher. There Ís ferritin in an oblique section through a pericyte junction (arrow). The basement membrane (B) between the endothelium (E) and pericytes is obvious. There is also a less dense basement membrane lying ablumenally along the pericyte wa11 and along the opposite boundary of the sinus. Ferritin (upper right hand corner) is also seen in the region between the outer muscle layer (Mu ) and secretory tissue of a g land , whi le carbon is not . (Bar = O.z5 um) ¿"

: ixdii.:!-i: ' "i::'*i' 56

Figs. 4-6 and 4-7 Exchange vessels in ,brachial muscLe. The lumens contain carbon ( C) . The pericyte J.ayer ( P) is again seen to be continuous, while the endothelium (E) is not. The vessel in the top figure is collapsed, probably as a result of contraction of the adjoining muscle (Mu). (Bar = 0.5 um) The vesse 1 in the bottom figure is open ' probab ly due in part to the partial apposition of neural tissue (N) which shares the same connective tissue corridor, and he lps to iso late the vesse I from the adj oining musc le . (Bar = 0.5 um) l.r\ 57

Fig.4-8 Vessel in brachial musculature, I - 2 minutes after the Íntroduction of ferritin and carbon. The lumen (L) of the vessel contains much ferritin. The ferritin is in lower concentration in the basement membrane except at the lumenal opening of the perÍcyte junction (arrow). In the time period, ferritin has moved across the pericyte wall and some distance (encircled) into the collagenous matrix (Co). (Bar in figs 4-8 to 4-10 = 0.1 um) Fig. 4-9 Vessel ín the brachial neuropil, 5 - 6 minutes after the introduction of the tracers. There is high coneentration of ferritin in the collagenous matrix (Co) outside the pericyte wa11 (P). The ferrÍtin is contained withÍn the extravascular matrix by the adjacent membranes of the neuropil (N). Fig. 4-10 VesseI in brachial musculature, about 20 minutes after the introduction of tracers. A vesicle (V) in the endotheLium contains ferritin. The carbon and ferritin aggregation

within the same ce 1-L is probab ly a 1so in a vesic le . Carbon (C) is seen to lie between the endothelÍum and basement membrane (B). Single ferritin particles (encircled) are seen at the entrance to a pericyte junction and on the outer perieyte wall adjacent to the extravascular matrix. P = pericyte; ExV = extravascular matrix. ¡ '.'.' 4-8

I

oR .¡¡ 4-9 a o N O Ç ,t' -å ¡ aa .L .ìF G.9 g f

1

I \ 58

Fig.4-11 Vessel in the funnel musculature, about 25 mÍnutes after the ÍntroductÍon of the tracers. A vesicle ( V) in the endothelium contains ferritin. Ferritin is al-so seen in junctions (arrows) in the pericyte wall (P) and extravascularly. B : basement membrane. (Bar in figs. 4-11 to 4-I4 = 0.1 um)

Fig. 4-12 Vessel in the brachial neuropil, abouL 2S minutes after the Íntroduction of the tracers. Ferritin has penetrated the basement membrane to the greatest extent about the entrance of a pericyte junction (pj ) and is found within the junctÍons (encirc led ) . The arrolvs point to ferritin which Ís possibly within vesic les in the pericyte. ExV = extravascular matrix; E = endothelium. 59

Fig. 4-13 Vessel in the retina, about 25 minutes after the introduction of the tracers. Ferritin has accumulated in the basement membrane (B), and is seen within the perÍcyte layer both in a vesicle (V), and apparently freely Ín the cytoplasm. There appears to be less ferritin in the endothelial cytoplasm (E).

Fig.4-I4 Vessel in the brachial musculature, I - 2 minutes after the introduction of the tracers. Ferritin in the pericyte is either within a vesicle or an obliquely sectioned junction (arrow). There appears to be marked penetration of ferritin into the basement membrane about the opening to a pericyte junction (pj l. L = lumen of the vessel; ExV = extravascular matrix. fiI

I

a '\ ''ill' .l o

t- b. I 60

4.4 D TSCUSS ION 4.4,1 Tissue inteqri.ty and the location ofl tracer molecules: Changes in tissue permeabilities most probabJ.y occuÌ as a result of operative stress and during the fixation process. There have been few studies concerning the time taken be fore tissue subjected to fixatÍon is immol:ilized; it perhaps takes as Lorrg as 30 seconds (Simionescu et al., I973). However, there are indications that such objections may not be of major importance here. There was the continued funclion of the circulatory system in anaesth'et ized animals, the revival of operatecl a.nÍ.mals, and morphological correlation with the microvasculal-u::e of contro.L anima l-s . Tissues appeared to be adequale ly f ixed and not osmotically stressed, according to the normal- criteria of general tissue integrity and swe.l-ling, shrinking or disruption of mitochondria and nuclear' membranes. Protein tracers are cross-lirrkecl l-ry the a ldehyde fixatj,ve, to all other proteins in the Ímmediate vicinity, and knife drag of f errit. j.n can be largely discounted. Provided there is not much fluid movement irr the tissues due to osmotic stress prior to the arrival of the fixative, there is litt le reason to presume that the positÍon of protein tracers wi 1l great ly change in the fixation process.

4.4,2- PermeabiLity of vertebrate capi ll,ar j es: Emphas is in the present study centred on the somatic microvascu lature 6I of 0ctopus , âhd although the results are rudimentary and stand in Ísolation, useful comparisons can be made, in particular, with non-fenestrated capillaries found in the higher vertebrates. These non-fenestrated or continuous capillaries are possibily the most prevalent type, permeating musc-l-e and other non-visceral tissues (reviewed Lu ft , 1973; Renkin , I978) . Ferritin , with a no J.ecu lar dÍameter of approx. lL ñffi, is one of the largest tracers employed in permeability studies of vascular walls. Bruns and Palade (1968) found that ferritin, two minutes after introduction, could only be found in the more l-umenaIly positioned vesicles of capil-laries in the rat diaphragm. Ferritin was never found within endothe Lial junctions or within the cytoplasmic matrix. Simionescu et al. (1973) could not find evidence for a junctÍona1 permeatritity to myoglobin (hydrodynamic radius approx. 4 - 5 nm) in capillaries of rat diaphragm. Transport across the vessel- wall- lvas entirely vesicular and the first ablumenal release occurred some 45 - 50 seconds after the initial injection. Further studies of Simionescu et a l. (IllS, I978), where a variety of tracers were employed, largely confirm this vesicular transit time. Unlike ferritin, microperoxidase was also reported by these authors to pass through endothelial junctions of venules, accounting for the earLiest appearance of extravascular tracer (O 70 seconds ) . Junctional permeability to microperoxidase had been previously reported in capil laries of rat diaphragm 62

(Wissig and Williams , I974). Casley-Smith ( 19Blb) observed that ferrocyanide ions, while taken up in vesicles, aLso moved across the endothelial barrier most quickly via junctions (within 15 seconds ) . It is now generally agreed that water-soluble molecules of up to approximately t - 4 nm in diameter (the upper size limit is yet to be determined, but on the basis ofl ferritin exc lusion , is probab ]y aJ-ways less than 10 nm) penetrate junctions between endothelial cells (Wissig and V{illiams., I974; Casley-5mith et â1. , I975; Gosselin and Stibitz, I977; Renkin, I977 , I979, 1980) . The li,kely morphological correlates of the physiologically determined permeability of capillary walls has been recently reviewed (Palade et al. , 1979). There is some evidence that trans- endothelial channels or fused vesicles also contribute to a continuous r wâter-fi lled channeling system (Simionescu et el., I975, I978) although the existence of sutrstantial numbers of such channel-s is disputed by Cas ley-Smith (198lb), both experimentally and in view of the experÍence of many microvascu lar researchers. The hypothetÌcal 'sma11 porer system postulated to exist on early physiological- evidence (reviewed Renkin, I978) uppears to correspond well wil-h the size and number of close junctions, i. e. junctions where the opposing plasma membranes are approxÍmately 6 nm apart (Casley-Smith et a1. , 1975; cosse-l-in and Stibitz, 1977) . Sma-Ller molecules rvhich are hydraulically conducted across 63 capillary endothelium certainly have very much higher permeabiJ.ity rates than the bigger molecules postulated to move along a 'large pore' permeability pathway (Renkin, 1978) . The ' large pore, system is thought to be represented in the main by vesicles (Renkin, I977, 1978). The random, diffusive movements of these vesicles (CasÌey-Smith et at. I975; Nakamura and Wayland, I975) account for the slower permeability rates.

4.3 ,3 Permeab i litv of 0cto pus microvesse ls : Ferritin is found outside vessel walls of 0ctopus within I - 2 minutes of being introduced. The permeability of 0ctopus vessel-s to macromolecul-es up to the síze of ferritin is therefore comparab le with the mamma lian system. This wou ld be difficult to quantify since the electron microscope has, as noted by Renkin (I978), proved useful in Ídentification of transport pathways but is of less use in nìeasuring rates of perrneabi lity. However, the appearance of extravascular ferritin in Octgpgg, at least initially, is quite conclusively the result of direct movement in between pericytes. The invarying width of the pericyte junction indicates that the conductivity of molecules of less than approx. 12 nm in diameter Ís potentia lly comparable with the very rapÍd rates of conductivity of smaller molecules which pass down endothelÍal junctions in mammals, particularly in view of the flact that perhaps only 5% of these junctions are close, the rest containÍng tight 64 regions where opposíng Plasma membranes are fused (Casley-Smith et al., I975).

There are two major factors influencing the amount of convective flow that may occur through a junction differences in blood and tissue fluid pressures, which will be mentioned later, and the possible retardatory effects of materials existing within junctions. Knowledge as to the existence or composition of an intercellular cement binding endothe lia L ce lls in mamma ls is obvious ly scarce; most discussions on junctional morphology barely nrention it. Luft (I973 ) postu lates that a residue of mucopolysaccharides exists between cells in close junctional regions. The general opinion appears to be that diffusion is to some extent restricted by other than stearÍc hinderance imposed by narrow junctional diameters. More recently, Curry (1980) has postulated the exÍstence of a fibre-network about a 11 surfaces ofl endothelial cells (including the junctional region), and it is thought that this wi 1l s low down the passage of large molecules. It should be noted that the approx. 6 nm wide region of a close junction is usually only a constriction in an otherwise appreciab ly wider junction (see Fig. 9 , Cas ley-Smith et â1. , I975) . Tlre basement membrane in 0ctopus retards the movement of ferritin and the junctional l-umen between pericytes is filled with an amorphous, electron-dense material whÍch closely resembles 65 the basement membrane and which appears to be more substantial than the intercellular matrix observed between mammalian endothelial cells. This amorphous material must act as an intercel-lular connective tissue, for allhough pericytes normally interdigitate to a Large extent, they do not have desmosomes or any other type of Íntimate eontact between their opposing cel-l membranes. That the basement membrane of mammalian vessels retards movement of macromoLecules is well- documented (see for e.g. Caufield and Farquhar, I974; CasJ-ey-Smith, I976), although permeation of water and similarly sized molecules is probably Iittle effected by it (Casley-Smith et âf., I975), The fate of intravascuLar carbon and ferritin has also been investigated in vessels of the crab central nervous system (Abbott, I97O). l-lere tlre vasculature is apparently closed, a lthough in many regions it is defined only by a basement membrane. The basement membrane, simiJ-arly, was found permeabLe to ferritin, but not carbon.

The usua 1J.y low extravascu lar concentrations of ferritin in 0ctopus perhaps then reflects a low junctional permeabi macromo lecu Hovrever J.ity, ât least to larger les. ' ferritin is seen in quite large amounts in ob lique sections through junctions. Alternatively, the low tissue concentrations may be the result ol an efficient extravascular circulat,ion, although in later studies, no evidence could be found of an extensive sytem of Lissue 66 channels (Section IV). Whatever the real permeability of the pericyte junction, the higher concentrations about many of the junctional openings indicates that ferritin moves through the junctions as a resul-t of fLuid flow, and not merely by diffusion (although the effects of molecular sieving cannot be discounted).

FerritÍn may a Lso be transported across 0ctopus pericytes in vesicLes, and apparently freely in the pericyte cytoplasm. While the real importance of these non-junctional permeabi lities remains undecided, they wou ld not appear to be comparab le to the extensive vesicular transport typically seen in mammalian capillaries. These endothelial vesicles are about 60 nm across (Casley-Smith, 1969). However, the necks of vesicLes open at the endothelial surface have a mean diameter of only about 10 nm (Casl-ey-Smith, 1969; simionescu et âf., r978), and physiological. evidence suggests that the J.argest pore size is general1y Less than 12 nm in diameter (Renkin, I97g) . Therefore, although 0ctopus pericytes are comparatively free of vesicles, the constraints on permeabirity in regard to the upper size limil" of permeants is litt le di fferent to the mamma lian condition. Ferritin approaches this limit in both groups and colloidal carbon (hydrodynamic radius approx. iO nm), which is impermeab le to the pericyte wall, does not penetrate the mammalian endothel_ium (Renkin, I977). 67

4.4 ,4 Comparisons with the physioloqical data on

Octo pus : The only previous permeability study of 0ctopus microvasculature, the physiologicaL undertaking of Smith (1962a), was not corlelated with ultrastructural evidence. Smith ( 1962a) states that 0ctopus capillaries are readily permeable to sodium thiocyanate, slightly permeable to inulin (ggt mo1. wt. ), and totally impermeable to antipyrine, while aLl three substances readily leave mammalian capillaries. Inulin is however suflficiently permeable to be employed in determÍning the size of the tissue fluid compartment in 0ctopus (Martin et â1., 1958). The impermeability to antipyrine is puzzling for it Ís a small molecule with a degree of lipid solubility. The exper iments o f Smi th (1962a) were necessari 1y performed on isolated arms which had been arnputated two hours previous ly and le ft at room temperature for this period. These result,s are not in accord with the ultrastructural evidence presented here, which show a vascular permeability to ferritin, a protein of 500'000 mo1. wt. and a diameter of 11 nm. Smith (1962a) surmises that the sma 11 increases in arm weight whÍch fo llowed large increases in venous pressure were due to distention of bLood vessels, and not oedema. Any filtration that occurred was said to be sharply limited by the in-elastic tissue sheath and low permeabi lity of the capi llary wall-s. The i.mplied impermeability of the vessel wall to water cannot be supported. 68

The presence of a lymphatic system in ôephalopods has been reported by Ando-Yoshia (1953 ) and Nisimaru (1969). These reports are far from conclusive and SmÍth (1963) could not find any evidence of a lymphatic system in 0ctopus , nor can any suggestion of one be found in more recent ultrastructural observations. Smith ( 1962a) questions the probable need for lymphatics in cephalopods sínce his results show that the capi llaries are comparatively impermeable and that hydrostatic pressures are such that l.ittle protein is likely to be lost from the blood. However, the pressure determinations are questionable. A midpoÍnt capi 1lary hydrostatic pressure (CmHP) of I.6 cm nrO is referred to by Smith (1962b). This figure is the mean of only three estimations gaÍned when Octopus arms were perfused with saline (Smith, 1962a). A perfusant of saline and dextran with a colloid osmotic pressure close to that of reaL blood was aLso used. The mean CmHP here varied from 1.1 to 6.0 cm H2O, with a mean of t.5 cm HZO from five estimations (Smith, 1962a) . Smith perfused j.solated and quiescent arms. Johansen and Martin (1962) state that octopods are able to regulate peripheraJ- resistances and peripheral blood pressure extensively, and they share the opinion of Chapman (.1958) that muscular tension is important in increasing both of these. Arterial- pressures generated in cephaJ-opods are comparable to those of vertebrates (see Tables 154 and 2O3, Altman and Dittmer, I97L). For these 69 reasons it seems likely that the value of I.6 cm rrO CmHP has not been estabLished with any certainty, and the indications are that the CmHP ffiâY, at least periodically' be substantially higher than this. It is 1ikely, in view of the results presented here and pleceding disCussion on hydrostatic blood pressule, that filtration acloss cephalopod vessel walls occurs to a greater extent than indicated by Smith (I962a). There arises then the question of possible leakage of macromolecul.es from cephalopod vesse ls.

The blood o f 0ctoous contains 9 - 10 g/100m1 haemocyanin, this beÍng about 98% of the total blood protein (Ghiretti, 1966). The haemocyanin is dÍssoJved in the plasma, existing in aggregates with a diameter and length of approximate ly 70 and 14 nm respective ly ( van Bruggen and Wiebenga , 1962). Haemocyanin is thus restricted by its size from entering pericyte junctions. The concentration and mo.l-ecular weight of other proteins in cephalopod blood have not,. to my knowledge, been ( 1966) reference investigated. Ghiretti states, without ' that the remaining few percent are of low mo lecu lar weight. Decleir et a1. ( 1971) 1'ound that blood of Sepia officinalis (aduIt) contains onl- y one type of haemocynin and a single glycoprotein. The concentration and sj.ze of the latter were not determined. A small amount ofl low molecular weight protein in moll-uscan blood can exert a 70 comparatively large osmotic pressure ( Adair and Elliott, 1968). Proteins of less than 500,000 moI. wt. almost certainly penetrate pericyte junctions in Octopus. It is important to determine the size and concentration of non-haemocyanin proteins in cephalopod blood - the Ioss of such proteins from the blood to tissue fluids might resull- in the similar types of problems (particularly osmotic prob.l-ems ) which necessitated the development of a lymphatic system in vertebrates. Perhaps proteolysis by the extravascular amoebocy tes found in 0ctopus tissues is important fclr removing such spilled protein.

4.4.5 Soecu Iations on the evo lution of microvascu lar structure: Fina11y, it is interesting to specu late as to possible reasons for some of the morphological differences seen to occur between the microvasculatures of vertebrates and cephalopods. A fundamenta.l- dif ference in the circulatory systems is the absence Ín cephalopods of anything resembling the vertebrate erythrocyte. To have the respiratory protein in plasma sol-ution must place a very different emphasis on evolution of vessel structure in a c losed b lood system. Mamma lian capi llaries , for example, have a minimum diameter defined by the erythrocyte which contains the respiratory protein haemoglobin (HenqueJ.l et al., I976). The upper limit on capillary diameter is probably also related to erythrocyte size. Increased mixing of both the plasma and 7I intracellular contents of the erythrocytes, and thus increased efficiency of metabolic exchange, occurs when the vesseLs are small enough to enSUIe deformation of the erythrocytes which pass along them (Prothero and Burton, 196I; B1och, 1962). Such restrictions on vessel size must be Less important to cephalopods. The light microscope observatÍon of Smith ( 196l) that tlrin-wall-ed vessels vary greatly in lumenal diameter has now been confirmed at the ultrastructural level (see fig. 4-4). It is worth mentioning here that various regions of the mamma lian microvascu lature are invo lved in exchange ( Way land and Silberberg, I978) and the term capillary, used to describe an Íncreasingly wide variety of vessels, is Lrecoming more and more to be a statistical concept. For this reason it is felt that the use of the term capillary j.n describing cephalopod vessel-s (see Smith, 1963i Gray, 1969) is to some extent unjustÍ fied and that the term 'exchange vesse1'shouLd be used to denote vesseLs thought to be primarily functioning as such.

Constructiona 1 di fferences in the vascu l-ar wa lls would al-so seem to be related to the method by which the respiratory protein is circu lated. Mamma lian erythrocytes are usua lly many orders of magnitude larger than the wÍdest gaps normally found in capillary wa1ls. For example, the large fenestrae of sinusoidal rat liver capillaries are between 100 and 3OA nm wide (Naito and 72

Wisse , I978) and readily retain the erythrocytes which are approximately 2 x 7.5 um. 0n the other hand there is a requirement in cephalopods to keep haemocyanin within the vascular system, and the width of the pericyte junction is such that haemocyanin wí11 not be lost through it. Moreover, fenestrae do not occur in the pericytes. Vesicles, which move materials down a concentration gradient, are rare. The need for intimate contact between blood and visceral tissues is alternatively solved il 0ctopus b y the formation of sinuses in, for example, the salivary ifanCs (shown in fig. 4-Ð, digestive g1and, branchial gJ.and (Di1ly and Messenger, I972) and excretory organs (Schipp and Boletzky, I975). Also, where neurosecretory release into the blood stream is requ.ired, the pericyte layer Ís done away with, and the neurosecretory boundary is then separated from the blood only by a basement membrane (Section 4.1). It has aLso been reported that other neura I e lements are very occasionally directly bathed in blood (Barber and

Graziadei, 1967a) ,

The anal-ogies drawn between the circuJ.atory syst,em of vertebrates and cephalopods have some justification. A c losed b lood system is obvious ly high 1y des irab le , i f not totally necessary, f or deveJ-opment of an active existence in a large animaf. However, from the available evidence, it is apparent that sÍgni fícant and interesting 7t differences occur in their microcirculatory systems where the singularly most important function of the entire b lood system, that of metabo-Lic exchange, is carried out. 74

SECTION V: THE DIMENSIONS AND DENSITY OF EXCHANGE

VESSELS IN THE BRACHIAL TISSUES OF

OCTOPUS

5.1 I N TRODUCT IO N Oxygen consumption- rates in cephalopods are high. A resting rate of 0.151 m1/g.hr is recorded for a 22 g 0cto pus (Maginniss and We lls , 1969) , and a rate of 0.6 m1lg.hr is reported for active squid (converted from values determÍned by Redfiel-d and Goodkind, 1929). Both these res ting and active rates are in many instances higher than those recorded for the poikÍ liothermic vertebrates (see Table 175, Altman and Dittmer, I97I). As mentioned previously, comparisons are most usefully drawn with the vertebrates. Oxygen consumption rates in most other invertebrates (see Tab le I93, Altman and Dittmer, I97 I) and in the archaic cephalopod, Nautilus pompilius (Johansen et a1. , I97B), are appreciably lower.

The respiratory protein haemocyanin, although found in greatest concentration in cephalopod blood (Cniretti, 1966), confers only a comparatively lorv oxygen carrying capaclty. Squid and 0ctopus_ blood contains maximally about 4.0 vols % of oxygen (RedfieId and Goodkind, 1929; Johansen and Lenfant, L966) while representative vaLues for arterial blood in the lower vertebrates are twice this (see Table 177, Altman and 75

Dittmer , I97 I) . In view of the high oxygen consumption rates and Ìow carrying capacity of the blood ' the microvascuLar density in cephalopods may be expected to be comparatively high. 0n the contrârV, a low microvascular density has been previously noted (Smith, 1963; Young, I97 I) , although this was not described quantÍtatively. Until such information is available, little can be said about the actual distributive potential of the vasculature. As a result, a quantÍtative study was undertaken of the microvas cu l-atu re in 0ctoous pallidus. This involved analysis, at the ultrastructural leve1, of random Sections from both brachia I muscle and neuropi I tissues . The vesseL density, lumenal perimeters r minimum wall thickrress, and other observations were recorded. It vtas found that the vascularity of the tissues studied is indeed Iow, and extrapolation of the data indicated that the tissues are likely to begin to suffer oxygen deprivation as soon as metabolic activity moves upward of the restÍng rate.

5.2 MATERIALS AND METHODS 0ctopus pa 1lidus were anaesthet ized and tissues were fixed as described previous ly ( Section I I ) . Five randon sections from both the muscu Lar and central neuropil tissues of the arms were obtained for each of four anima l-s . The sections '¿lere stained with bot.h Lead 76 citrate and urany I acetate, and viewed on supported grids.

Under the electron microscope, the first grid square which was entire 1y covered by the section was scanned for vessels, and micrographs were taken of those present. One half of the vessels which intersected with the grid bars tvere included in the vessel count. Where possible, the remaining portions of these vessel-s were recorded from adjacent sections. Accurate magnifÍcations were determined with a grating replica. The area of any grid square, given to be 40,000 u*2 ( 100 mesh, VECS specÍmen grid catalogue, I978), was checked and found to vary maximally by less than 5%. Together with the vessel density, specific records were taken (for each vesse.l-) of the length of the lumenal perimeter, the minimum pericyte width, the width of the basement membrane at the site of the previous measurement, the open or closed state of the lumen, the presence or otherwise of obvious myofibrils' the percentage of the Lumen bound by endothelium, and the presence or otherwise of amoebocytes.

0nly the minimum wall- width was recorded; 1f each vessel resembles a tube with a wa11 of approximately constant vrÍdth, then the true cross-sectional view of the vessel wall (i.e. the mj-nimum wall width) wiIl be encountered in any random section through that tube. 77

The number of vesse.Ls per cross-sectional area of tissue was calculated. It was assumed that the vessels were equidistant, and the distance between vessels was then estimated as by Krogh ( 1919b). Each vessel was assigned the average area of tissue and this was assumed t,o approximate a circular section. The radius (R) of the latter was derived. .The perimeters bf the vessels were measured along the lumenal aspect of the basement membrane, and not the lumenaÌ pericyte wa11, although any difference was slight. Functionally, the basement membrane 'serves as the primary blood-barrier (Section VI). The perimeters were assumed to describe circular cross-sections, âñd the corresponding radii were ca l-cu lated. Because random sections through a tube were obtained, a final correction factor was applied (x I//2, Casley-Smith et â1., I979) and the average corrected vessel radii (r) were then estimated.

5.2,I The Krogh-Er J-ang equation: The results of this study were incorporated into the Krogh-Erlang equation ( Krogh, 19 19b ) so that some conclusion could be drawn about the like1y efficiency of the microvasculature, at least in regard to the distribution of oxygen. 78

The Krogh-Erlang equation is:

2 2 2 to T 15R R 1. logIR/r-(R -r ) /4lp/d

T (atmos. o = oxygen tension in microvessels ) T (atmos. ) R = oxygen tension in the tiss.ues mid-way' between microvessel-s

R = radius (cm) of cylinder of tissue supplied by each microvessel r = raclius (cm) of microvessels p = gas exchange, O2 absorbed cc/cn2.min d = rate of diffusion of Or/nin through I c^2 and the distance of I cm of tíssue

Shoul_d the calculated val-ue of TO - TR prove greater than the actual venous oxygen tension (Ty), the indication is that some tissues, and at least those about the more venous vessels, are suffering from oxygen deprivation. Va-Lues f or R and I wele derived from the present study. Values for the otlrer components were incorporated from the literature. The rationale for val-ues chosen and some of the probLems in their applicatÍon are discussed later. 79

5.7 RESULTS The details of 79 vessels were recorded from the muscle and neuropil tissues studied. All vessels with a minimum wa11 width greater than O.25 um contaÍned myofibrils (figs. 4-2, 4-7), while very few of the thinner walled vesse.Ls did so (approximate ly. four percent ) . 0n the basis that the myofibrillation indicates a specialized role apart from an exchange funct,ion, and because a convenient division was necessârY, it was decided that only those vessels with a minimum vessel wall width equal to, or Less than, O.25 um were to be classified as exchange vessels (figs. 4-6, 4-7). Six percent of the vessels in the muscle and nineteen percent in the neuropil could not be included in the count of exchange vessels, and were disregarded. (The higher proportion of thick-wa1led vessels in the neuropi I might be expected primary branches Smith since the arterial supply here; ' 1963; Section III ) . Quant-i.tative information on the densities and dimensions of the remaining vessels is tabulated (Table 5- 1). Estimates of both the vessel density and average vessel- diameter prove very similar for the muscle and neuropil tissue.

0f the vessels which intersected grid-bars and were to be recorded, eight could not be found in compLete view. 0f these, only three could not be included in the count of exchange vessels, although sbservation of the Table 1. Densities and dirnensions of exchange vessels in the brachial muscle and neuropil tissues of 9. Þallidusx

Number of Estimated distance Av. measured Estirnted av. Av. minirrum vessels/nm2 between vessels vessel perimeter vessel diarieter; walI width (um) (um) corrected (um) (um)

Brachial 44 17I 38.O I 6 0.09 o@ muscle (6) (5.1) (0.01)

Braehial 45 167 7I.t 7.O o.12 neuropil ( 11) (3.6) (0.02)

x Figures in brackets one Standard Error 8I incomplete portions may have led to their inclusÍon. This was high ly un likely in two instances and thus any associated error in the vessel count is s light. The possibitity that some vessels went, unnoticed is potentially a greater source of error. However the sections were heavily stained to heighten contrast and photomicrographs were surveyed of all tissues suspected to con tain vas cu l-ar tissue .

The ratio of open to closed vessels was ) approximately one to one. The number of open vesseLs in situ is, with little doubt, greater than found here because the contractile elements appear to react adversely to the primary fixative (SectÍon IV) . As a result, all- but one of the vessels with myofibrÍls were closed, as were most of the thin-walled vessels surrounded by muscle tissue (fig. 4-6). 0n the other hand, very few of those vessels well isolated from acljoining muscle tissue and without myofibrils, 'lrere closed.

The estimated average diameter of the exchange vessels is about I u. Although it is diff icult to decide accurately how any individual vessel has been sectioned (thus makÍng it diff icult to gain cross-sectional measurements direct ly ) , it was obvious that thin-walled vesseLs varied quite markedly in diameter. A range of t to 12 um is estÍmated f rom the vessel-s observed here. 82

It is worth noting that the thicker walled vessels tended to contain a more complete endothe.lium. The pericyte layer r^ras occasionally very thin (approximately O.02 um) but the vessels were nevertheless continuous. BLood borne cells were encountered in only eight percent of the vessels. The thickness of the basement membrane at the same site as the minimum pericyte width, varied little in exchange vessels, being about 0.08 um across. The basement membrane lvas invariabJ"y more substantial in the thicker walled vessels.

5.t, I Application of the Kroqh-Erlanq equation: The results vlere incorporated into the Krogh-Erhlang equation (described previous ly ) . The capi llaries in the muscle Krogh ( 1919b) investigated, were regularly arranged in parallel arrays. Krogh assumed that each capillary could then be reasonably associated with a cyJ-inder of tissue of radius R. Such arrays are not evident in the 0ctopus tÍssues studied here (Section III). However, the exchange vessels, at least in the muscle, are quite even ly spaced ( tnis is indicated by the comparative 1y sma l1 standard error for the estimated vessel density), and the derived values of R for q. palli_dqe are similarly incorporated into the equation. Krogh thought it necessary to limit his analysis to the orderly capillary beds of vertebrate skeletal muscle. No reason is immediately apparent u/hy use of the R estimates in the present analys is should 83 present any appreciab ly greater error, and it seems reasonable to use it, at least as a.first approximation. Although thin-wa-l-l-ed vesseLs in 0ctopus tissues may vary widely in lumenal diameter, most appeared to be about the estimated average, and the derÍved values of r are felt to be reasonab 1e. Values fsr the other components of the equation were necessarily extrapolated from the literature.

The di ffusion constant (d ) was assumed to be O.I33 x lO-4 min; this is Krogh's vaLue ) "^2/atm. (l(rogh 19I9a) for cold-blooded organisms. Gas exchange (p) was assigned a value of 0.0017 OZ cc/g. min, this being estimated from the resting rates for q. cynea of about equal weight (Maginniss and We lls , 1969) . The 9.. cynea had been subjected to water temperatures of approximate ly 25oc, whi le the maximum environmenta I temperature experienced by the 0 . pallidus used here is in V icinty of 15 zOoC. The assigned value of p for q . pa l- li dus is there f ore J.ike 1y to be s light lY overestimated. The estimates of to - TR for the muscle and neuropil, converted from the atmospheric units given by the equation, were 8.6 and 8.9 mm Hg respectively. The Tv pallidus pressures Y_ for 9. r or'ì the basis of known in other octopuses (¡onansen and Lenfant, 1966), is likety to be about 10 mm Hg or perhaps a little higher. 84

5.4 D ISCUSS ION 5 .4,I What constitutes an Octoous exchanqe vesse l? Early ínvest,igators reported the existence of capillari,es in cephalopods (eg. Langer, 1850, cited in Smith, 1963; Williams, I9O2; Isgrove, 1908). More recently, but sti.LI at the leveL of the f-ight microscope, Smith (L963) described the circulatory sy stem in 0ctopus arms and states that the capillaries varied between 5 and 35 um in diameter. Ultrastructural studies have been undertaken of the microvascular in the central tissues of 0cto US (Gray, 1969) and in the peripheral tissues, both muscular and neural, of 0ctopus and cuttlefish (Barber and Graziadei, 1965, 1967a). Gray, with qualification, used the term capi Ilary, while Barber and Graziadei did not. For reasons argued in Section IV, the term exchange vessel is thought preferab le to capi 1J-ary.

It is not €âs¡r to decide, from quaJ-itative observation a10ne, which vessels will be involved primarily in exchange. It has been shown in associated studies (Sections IV, VI ) that water soluble substances (proteins and Íons) penetrate the pericyte junction, but that passage occurs through the junctions in aLl vessels, both large and sma 11. Apart from the variation in thickness of the pericyte wa11, and less obviously in the basement membrane, there is little, if âoyr ultrastructural 85 evidênce to suggest where an exchange function may predonrinate. Nevertheless, some. delineation was necessâry, at least for the purposes of this study. In view of the resul-ts and factors mentioned elsewhere, the suggested criteria for an 0ctopus exchange vessel are that, it has a minimum wa11 width equal to, or less than, O.25 um and a lumena I diameter less than L5 um. The considerable majority of vessels encountered in the tissues are then Íncluded, and for this reason alone, j.t may be justifiably presumed that they encompass t,he major exchange cbmponent of the system.

Barber and Graziade i (1965, 1967a), in the first attempt to classify the cephalopod microvascul-ature, described four vessel types. Type I would Ínclude those discarded from the exchange vessel count by virtue o f their thick walls and obvious myofibrillation. Types II, III and IV are described as variant of the one form, and nearly all of those j.Llustrated could be carled exchange vessels according to the criteria proposed above. similarly, most of the vessels described by Gray (1969) as capillarÍes, would be included. Gray noted that the smaller vessels ranged in diameter between 0.5 and 6 - g um. It Ís timely to note here that the reported studies, and cursory observations of other Australian species, indicate that microvascul.ar structure and density'does not vary substantially between the kinds of octopuses normally 86 involved in laboratory studies. While structurally very similar, the microvasculature of the pelagic squid, as yet not quantified, might be expecLed to be flìore dense.

It is concluded that the broader interpretation of the classi fication of Barber and Graziadei ( which is essentially tlre one used here) is the more useful. That is, there are the Type I vessels, which are probably all arterial, and the generally smaller Type II or exchange vessel-s. Further division of the smaLler Type II vessels is not tírought profitable. Most dif f iculty exists in classification of the wide, yet thin-walled vessels which are occasionally encountered. Tlrese include the Type IV vessel-s of Barber and Graziadei ( 1965), the smalLer of which would be called exchange vessel.s on the basis of their wa11 width, whi le the J.arger of which, because of their lumenal. diameter, would not. Gray (1969) describes collecting vessel-s of 30 um diameter which, judging by the illustrations, may have regions of the pericyte wall approximately 0.1 um wide. Such thin walls surely allow appreciab le exchange. Nevertheless, the wide Ìumens and lov¡ frequency of the vessels indicate that the exchange function is not of primary concern. These vessels most probably have a venous function as previous Iy suggested. They are certain ly more common in the outer connective tissues of the arms, where the venous return is known to aggregate (Smith, 1963; Section III, fig. 4-4). 87

In terms of general morphology, little can be added to existing accounts, althou.gh the proportional endothelial cover and frequency of blood borne cells had not been measured before. The blood in the peripheraJ. tissues obviously carries few celLs. Endothelial cells in these tissues have been shown to phagocytose carbon and ferritin (SectÍon IV). However, the principal function of the endothelium remaíns obscure. The thicker walled vessels, and more particularly those known to be arterial (Barber and Graziadei, 1967b), contain a more substantial endothelial layer. PerhâpS, then, the endothelium gives necessary strength to the vessel wall and/or is involved with synthesis of the basement membrane for the basement membrane is also thicker in these vesseLs (Kawaguti , I97O; Barber and Graziadei, 1965, 1967b).

5.4,2 The comDarative vascularitv of Octoous tissues: Exchange vessels in both tlre brachial muscl-e and neuropil were estimated to average about I um across. The diameters of the capi llaries in the lower vertebrates are equal to, or are a little smaller, than this (tabte 17I, Altman and Dittmer , I97 I) . The comparatively wide range in diameters of the exchange vessels in 0ctopus is probably related to the conveyance of the respiratory protein, being not in specia lized erthrocytes, but in extracellular sol-ution (Section IV). An accurate estimat.e of the average width of the perÍcytes which line the 88 exchange was not readily obtainable. The average minimum width , however , is sma I1 (approximate Iy 0. I um) . In drawing comparisons, it is sufficient to note that mucil of the pericyte wall is as thin as the capi llary endothe-lium of the vertebrates. The presence of open necked ves-ic-les and varying types of endothelia 1 junctions in the latter , and even fenestrae in some, make more detailed comparisons di fficu lt .

It is immediate ly obvious that the microvascu lar density in' 0ctopus is low ; there are about 45 vessels per tt2 of tissue cross section in both the neural- and muscle tissues of the arms r âs opposed to a representative figure of about IOO per ^^2 for the lower vertebrates ( Tab le 172, Altman and Dittmer , I97 L) . Some tissues in the higher mamma ls may contain in excess of 5000 capillaries per ( may noted ^*2 . tn this regard, it be that while a large sampJ.e for 0ctopus was not presented, the study involved the time-consuming task of scannirrg a comparatÍvely large area of tissue. A similar area of cat muscJ.e, for example, would have revealed between 500 and 1000 capillaries ). The average maximal diffusion dis tan ces in the 0ct,o pus tissues studied Ís, as a resu.lt of the lc¡w vesseL density, comparatively large. Furthermore, because the vesseLs are not perfectly evenly spaced, this diffusion distance must sometimes be even greater than the B5 um estimated. In comparison, Krogh 89

(19f9b) estimated a diffusion distance of 28 um for both frog and cod muscle, while in mamrnalian tissues this may be less than 10 um (rable I7I, Altman and Dittmer, I97I). If the Octopus microvascul.ature is to be considered as a random array of vessels, then a better estimate of the vessel density may be the,tota1 Ìength of the microvessels per unit volume ( J), which is twice the number per unit area cross sectíon ( 0) (weibe l, 1969) . Fo 11owÍng Krogh's CIethodology, B r,las calculated as I//¡Q. If L/lr J is considered more applicable, then the values of R would be ,reduced bi a f actor of /2, and the partia 1 pressure estimates would be approximately halved. The microvasculature density in 0ctopus wou ld remaln, nevertheless, very much lower and the necessary partial plessures much higher than estimated for the vertebrate tissues. Furthermore, it is arguable that Krogh's figures should aLso be reduced to some extent, for the capillaries used in his calculations show a degree of tortuosity and are not perfect-ly para11e1. The magnitude of the di fferences between the 0ctopus and vertebrate s ys tems would remain similar under these circumstances. Similar comparisons cannot be drawn with other invertebrates as there is a lack of in formation about the few c tosed systems which exj.st outside the cephalopod group. The vasculature in oJ.igochaetes (Hama, 1960) proves remarkable like that of the cephaJ.opods in cross sectÍonal morphology, but the microvascuLar density is likely to be 90 lower: the o ligochaetes consume oxygen at a Lower rate ( Tab le I93 , Altman and Dittmer , I97 I) and, un like the cephalopods, cannot be described to be ana logous in general development with the vertebrates.

5.4,7 The dÍstributive- potential of the 0ctopus vascuLature: It has often been presumed that tissue demand for oxygen determines the upper limits on microvascular density, while energetic costs and spatial f actors presumab ly prevent the growth of f urther vascu.l-ar tissue. This proposal is contentious. Perhaps the need for effecíent removaL of toxic wastes (Schmidt-Nielsen and Pennycuik, 196I), or the distribution of heat, is the more important, ât least in mammals. Duling and Berne ( 1970) report that in the hamster cheek pouch, most of t,he oxygen has left the vasculature before the capi-llaries are reached. The diffusion estimates of Krogh, made some sixty years ago and based on a simplistic modeJ., are stj.11 those usually referred to, while the real diffusion geometry o f b lood and tissue exchange remains obscure (Scfrmidt-Nielsen and Pennycuik, 196I). So, in addition to the previously mentioned problems encountered with use the-of Krogh-Erlang equation in this study, there are some general uncertainties about the relevance of associating microvascular density with the tissuesr needs for oxygen. 9I

Nevertheless, not withstanding these uncertainties, it can be said that oxygen consumption, transport, and diffusion rates are perhaps the best understood aspects of circulatory function. The Krogh-Erlang equation certainly gives some idea as to the distributive potential of the vasculature, and because it has been applied to a comparatively wide range of animals, direct comparisons can be drawn with any resul-ts obtained. The results for 0ctopus indicate that the partial pressure of oxygen, necessary to allow tissues to respire at the resting rate, Ís comparatively hÍgh (about I mm Hg), and varies .littIe from the level of the venous partial pressure. Krogh (1919b) reported necessary partial pressures of only 0.4 and 0.1 mm Hg for cod and horse respectively, while the corresponding venous partiaJ. pressures are an order o f magnitude , or more , greater ( Tab le I77 , Altman and Dittmer, I97I). Whatever the limitations of the analysis, it is very obvious that the distributive potential of the vascul.ature is f ar lower in 0ctopus. The evidence suggests that the demands of resting tissues for oxygen closely match the delivery capabilities of the vasculature, there being littLe reserve capacity to meet the demands of more active tj.ssues. The assumption made for the purpose of the analysis, that all vessels would be perf used simultaneous ly , strengtl-rens the argument; recruitment under these ci.rcumstances could not occur. 92

Jones (I972), working from the data of Redfield and Goodkind (1929), shows that . a much Íncreased circuLation rate would be required to account for the high oxygen consumption rates of active squíd, because the oxygen capacity of the blood is so 1ow. The low vascular density further limits the availability of oxygen to the tissues. Furthermore, there is no evidence Ín 0ctopus of an extensive back-up system of tissue channel-s (Section VI). As noted in the Íntroduction, these factors are not reflected in the oxygen consumption rates, which are generally higher than measured in the poikilothermÍc vertebrates. The need for a high and consistent uptake of oxygen has been shown critical for both octopuses (MagÍnniss and We1ls, 1969) and squid (Dykens and Mangum, I979; Redfield and Goodkind, 1929) Cephalopods appear t,o uti lize a comparative 1y limited circu latory system with high efficiency and to good effect, judging by their mode of 1Ífe and collectivety large biomass (Packard, I972). 97

SECTION VI: TISSUE CHANNEL MORPHOL0GY IN 0CTOPUS

6.0 INTRODUCT ION The general convergence between cephalopods and vertebrates o ften draws comment, and in organization and complexity their circu latory systems are certain ly comparable. However the structure of the microcirculation in the two groups is appreciably different (Section IV) and the density of exchange-vesse ls in cephalopods is quite low (Sectíon V). Previous observation of connective tissue in both neural tissues (Gray, Lg69) ancl body musculature (Section IV) of 0c'topus suggested the presence of an extensive tissue channel network, which could be expected to augment the distributive function of the microvasculature. Smith (Igeza) concluded from his physiological studies that filtratÍon through the vessel wa l1 in 0ctof:us arms was sharply limited by a high vascular impermeability to water (this is now thought improbable; - Section IV) and by the presence of a fibrous connective tissue sheath which prevents any distention of the muscular tissues. The low fÍltration rate could also be related to the nature of extracel-Iular connective tissues which may offer littte avenue for movement of water. An extensive channel network would not be expected on this basis. This section reports an attempt to gain some idea as to what extent the refined, closed mícrovasculature of 0ctopus somatic tissues is associated 94 wÍth eÌements of the more primitive, "open", system characteristic of less advanced anima Ls , and to compare this tÍssue channel organization with that of the higher vertebrates.

While the elecron-microscope has not proved very useful in direct visualization of water-fi1led spaces in the connective tissues, morphologÍca1 information can be obtained by a technique which involves the formation of electron-dense deposits of molecules known to be largely associated'with the tissue channels on the basis of their small size and high charge density. Commonly the cl interst.itium is permeated by innocuous ferrocVþiOe ions in animals while they are stil1 alive (Chase, 1959; Dennis, 1959; Rodbard, I975i Casley-Smith and Vincent, l97B). The ions are then fixed by reacting them with a precipitating saLt, which is inc luded with the prÍmary fixative. In these experiments, as rvith others, the ferrocyanide ions were Íntroduced into the circu lation from where they leaked into the surrounding tissues. The precipitating ferric salts normally usecJ are quite acidic. In place ofl these, tris (ethylenediamine) - cobaLt (frr) chloride was employed. Use of this salt in mammalian studies produced resu lts consistent with previous determinations whi l-e aJ.lowing for better fíxatÍon. This procedure is an improvement on earLier experiments, which involved penetrat.ion of test-ions into tissue blocks during primary 95 fixation, followed by appJ-ication of an acidic precipitating solutiorr (after Casley-Smith et âf., I979). The investigation of mammalian tissues was undertaken to test the validity of the technique, details of which were later published. As the investigation is not central to the thesis topic, further descriptions will not be made here and a copy of the paper is attached as an appendix (Browning, 1980) . In order to quantitate the data, morphological information was collated with the aid of a Quantimet.

6.2 MATERIALS AND METHODS 0ctopus pa.Llidus were used, âhd anaethes Ísed as previousj.y described (Section 1I), and perfused via the anterior aorta. The perfusant contained per 100m1, I.O25g

Nac1, 0.49 vgs04, o.77g Mgclr, 0 .24g cac1r, o .0759 (À KCI and 3.5g sod-ium f errocy/nÍde. This so.l-ution is a basic octopus rirrger (Smith, 1962a) but where half of the NaCl has been replaced with an approximately osmotically equitabLe amount crfl sodium ferrocyanide (see Table D-255, Weast, I975) . For control experiments the perfusant contained 2.O59 NaCÌ and no sodium ferrocyanide. The addÍtion of a little carbon (pelikan cII/ I471., O.2nI/ 100m1 perfusant ) allowed the arrival of the perfusant in the tissues to be easi ly detected. The primary fixative contained per 10oml, 2.5% glutaraldehyde, 96

2% paraformaldehyde, 2.I49 sodium cacodylate (see Glauert, I975), 5g glucose and 119 tris (ethylenediamine) cobalt ( rrr) chloride (see appendix; Browning, 1980). The osmotic contribution of the cobalt salt, coupì.ed with the glucose and other constituents, âPpeared to give good perservation of tissues. In control experiments the cobalt salt was omitted from the fixative and the ot,her constituents were disso.l-ved in octopus ringer . The washing so l-ution contained the same concentration o f sodium cacody late and g lucose disso lved in the washing solution. The pH of alI solutions was 7.O.

Tissue samples were subjected to primary fixation for 1.0 - 1.5 hours . This included a renewal of the fÍxative at about 30 min; a continued excess of precipítating ions !.ras thus ensured. Samples were dehydrated and embedded as described previous ly (Sectlon I I ) . Sections were viewed unstained, to avoid confusion with stain precipitates.

6.2, I Quantitative methods: In addition to obtainirrg general information from the e -lectron micrographs , quantitative data vlere co llated with the aid o f a Quantimel 720 (Cambridge InsLrument Co. ). The procedure, which is simi 1ar to that described by Cas1ey-Smith et aL. (I979), involved analysis of random micrographs - in this case with 25 micrograph negatives, at a magnification of 97

14,500 times, for each of 5 anÍmals. Provided a tissue/resin interface was included in the block face, problems with finding precipitate bearing regions prior to random selection were rarely encountered. Images transmitted from negatives in an epidiascope were edited on the Quantimet screen, and the sÍzes and total area of the precipitate deposits in the inter!titial matlix were automatically computed.

The area of the deposits withÍn each of seven size classes wás obtained directly from the Quantimet. All deposits in the random survey fe l1 within this range , although some slightly larger deposits were occasionally found in non-random observations. The number of deposits within each size class, and the total area of the extracellular matrix in which the deposits rvere scattered, was also obtained directly. Some difficulties were encountered with use of the Quantimet. Determination ofl correct grey- leve ls , i . e . what was to be inc luded on the basis of its electron-density, had to be decided arbitrarily. Also, editing of tlre unstained negatives, So that onJ.y interstitiaL areas were scanned, could not be done wi th great accuracy. Neverthe less , these are minor consÍderations compared to the large informational gain. The limit of the discriminatory abi J-ity of the Quantimet vlas reached in the smaÌlest size class and the information gained about the deposits here is limited. However, these 98 small deposits did not occur in great numbers and did not constitute an important component of any of the summed derivatives.

The average areas of each size class were taken to approxÍmate random sections through tubular channeLs. The radii of the areas lvere calculated and multiplied by I/lZ to give final corrected vaLues (Cas ley-Smith and Vincent, I978). All radiÍ discussed henceforth ale corrected estimates.

Diffusional and hydraulic conductivities of the channe ls rvere estimated. To faci litate ready comparison , units are the same as those used by Cas ley-Smith et a l. (I979), Diffusional conductivity is calculated from

Fickrs Law of Di ffus ion ,

_^g J -DA dL where J, the solute flux moved per unit time, is related to the diffusion coefficient (D) and concentration differencu (¡c) of that solute, the distance over which the concentration difference occurs ( L) , and the cross-sectional area (A) available for diffusj.on.

Diffusional conductivity (Y) equals J/LC. Thus for a tube of radius I

2 lTT Y -D tr 99

The tabulated values of Y in each size cl-ass are the sum of all the channels (number of observations/cn2) in that size class. The D component, which is solute and so.Lvent specific, is incorporated into the final statement.

Hydraulic conductivity is calcu lated from

PoÍseui lÌers equation ,

4 TT 0 = BzL AP

where the rate of fl-ow (0) of a fluid in a tube is relaLed to the radius (r) of the tube, the hydraulic pressure gradient (AP) the viscosity of the fluid (z), and the ' length of the tube (L). Hydraulic conductivity (lV) is Q/^P, thus

nr4 w BzL

Simi lar ly, the summed hydrau lic conductivit-Ìes f or eaclr size class are tabulated, with L arbítrarily given a va-Lue of 1 cm. The model, then, is simplified to a I cm cube of tissue with n channeÌs traversing it from one side to the other. The number of channels leading away from this pathway are presummed equal to the num[¡er contributing to it. It is assumed that functionally the channels can be lrepresented by a straight pathway, which is I cm long and normal to the surl'ace ofl the cube r âñd t,hat the ef f ective radius of each channel does not vary along this length. 100

Thus, once the concentration dÍfference and diffusion coefficient, or the pressure gradient are known the amount of diffusion of a substancer or flow of a fIuÍd, through a I cm cube of tissue can be arrived at. The assumpti"on that each cross-sectional area represents the effective radius of the channel over a I cm pa1-hway may be invalid to a greal-er or lesser extent. Herein lies a potentia.L source of signif icant error in the calculation of conductivities vrhich is going to be very difficult to investigate, and for the moment must be ignored.

6.3 RESULTS Few vesse ls were found in precipitate-bearing regions because vessel density is l"ol in cephalopods; any vesse Is present r^/ere di fficu 1t to identi fy , since the sections were unstained and therefore of low contrast. Hence a comprehensive study of the immediate extravascuLar matrix was not undertaken, but apprecÌable deposits utere seen in the surrounding matrix of those vesse ls observed (figs. 6-I, 6-2). Electron-dense deposíts situated extravascu lar 1y cou ld be ident.i fied as ferrocyanide precipitates with confidence, since carbon is who11y retained by the vessels (Section IV) and is of a distinctive appearance (fig. 6-2). Further, the artifactual precipitates which inevitably occur with additional staining, were avoided. 101

Precipitates tvere rare ly found within the pericytes, although often ali.gned with their outer cell membranes. Some disp lacement of the deposits will probably have resulted from knife-drag in the cutting of the sections. llowever, the position of the bulk of the deposits may be expected to be relatively unchanged. The vesse I l-umens vvere never f i 1led with precipitate a J-tl-tough they carried large amounts of test-ion. Precipitates rvere sometimes seen in pericyte junctions ( fig . 6-3) indicating, that test-ions do indeed pass out of the circulat.Íon through this junctional pathway. It is probable that precipitate-induced occ lusion of the smalI junctional gap will prevent further penetration of large amounts of ion-bearing fixative. This argument, aPPlied to all small pathways, would also help explai.n the observation made here and previous ly ( Cas ley-Srnith and Vincent, 1978; Casley-Smith et g1.' 1979) tnat the heaviest precipitate leve ls occur in a re lative ly sma ll peripheral region of a tissue-b1ock, in spite of an excess of precipitating ions.

In tissues viewed, Precipitates were largely limited to the extracellular matrÍx, including ceIl junctions, with occasional deposits being found associated with mitochondrial compl-exes in muscle (fig. 6-4) and elsewhere. Some of the precipitates near the mitochondria appeared to be intrace 1lu lar, a lthough it was o ften IO2 difficult to determine the spatial relationships of the mitochondria, the precipitates , and . any confining ce 1l- membranes. Test-ions readily penetrated the waLls of the thick-wa 1led anterior aotta , being limited Ínitia lIy to cell junctíons in the lumenal wa11 and then to the extracellular matrix (fig._ 6-5). Figs. 6-6, 6-7 and 6-B have been se lected to show tissues which bear comparatively large numbers of deposits; the tissues are otherwise typical of the random micrographs analysed in the quantitative study. Blocks of connective tissue and the narroner paths of ground substance that run between muscle tissue usually contained many deposits. The clear spaces indicated in fig. 6-7 are like those labelled as elements of the sarcoplasmic reticulum in squid cardiac musc le by Dykens and Mangum ( 1979) , These spaces , whi J.e often large, rvere almost always devoid of precipitate and thus rarely directly linked with the interstitial channels. It is likely that the spaces are to some extent artifactual, perhaps as a result of adverse reaction of the muscle to the fixative, or respresenting material lost during fixation. 0ften precipitates would be aggregated about ce llu lar e lements of the extrace 1lu lar matrix ( figs . 6-I, 6-9), particu lar ly in the large areas of the interstitial matrix which contained litt le muscle tissue and where precipitate was otherwise scarce.

In addition, neural tissues of the brachial IO7 neuropil and brain were investigated, although these were not ineluded in the quarìtitative study. The neural tÍssues are of particu lar interest , because previous observations had suggested they may contain an extensive system ofl interstítia1 channels (tfris is discussed later) . However, precipitates here were never very extensive (figs. 6-10, 6-II, 6-12); tissues in fig. 6-10 are an example of those with the largest amounts of precipitate. As with other t.issues , precipitates vvere most ly limited to interstitia 1 regions , including connective tissues ( fig. 6- l0) and junctions between cellular elements (fig. 6-11) and, again, sometimes assoeiated with the mitochondria ( tig. 6- 1.0) . In immersion r âs opposed to perfusion r €Xperiments with the toad retina, Lasansky and Wald (pAZ) found similarly that ferrocyanide precipitates were confined to the extracellular matrix, often within the narrolv gaps between the ce1ls, indicating the passage of water and Íons in these regÍons. Lasansky and Wald (neZ) found no evidence that ferrocyanide ions bind to any of the tissues of toad retina.

Cont.rol experiments showed that the precipitating salt introduced with the pri.mary fixative could form precipitates in tissues not perfused with test-ions. However these prec.ipitates have a characteristically spikey appearance ( fig . 6-13) and on ly occurred to any I04 noticeable extent in tissues not washed after the primary fixation process.

6.3,1 Quantitati ve Resu lts : The quantitative results are tabulated (Table 6-l). The total percentage area of the channels was O.44% of the interstitÍal tissue, with a standard error of O.O3%. This is directly proportional to the percentage volume of the interstitium that is occupied by channeLs. The average channel radius was approx. lB nm. The total, diffusional conductivity was estimated to be approx . 22.O x IO-/+ /D ( cm ) , and the hydrau l-ic conductivity to be approx. 0.30 x 1o-12 (cm4ldyne sec).

6.4 DISCUSSION a 6.4 1 Visualization of tissue channeÌs: The conclusion can be drawn from recent reviews of advances and prob.l-ems in microvascular research, like that by Diana and FJ.eming (1979), that transport through the interstitium is perhaps the least understood aspect of the mammalian cÍrculatory system, a system to which the majorÍty of research effort has been directed. Direct ultrastructural observation of the tissue channel network, which links the blood vessels to other tissues and the lymphal-ics, has not been readily achieved. Important advances in visualizatíon of the fine-morphology of tissue channels have resulted from use Table 6-I. Numbers, radii and conductivities of channels in each of the seven, predetermined size classes.

2 -2 Area size classes, nm x 10 048 I6 32 64 I28 256

Average îffi 2 -2 area, (x10 ) 4.0 7.6 11.9 2I.2 47.O 7 6.6 209.5 (Se in brackets) ( 1) (.e4) (1.5) (2.5) (6.8) ( 15. 1) (25 .8)

Average radius, nm 7 .9 I1.0 lt. 8 18 . 4 27 .2 34.9 57 .7

Number of observalions, (N ) 91 408 423 I52 57 13 t- i 325 o \'l

Nr/tOO unZ of interstitial tissue 13.6 60.6 63.0 48.3 22.6 8.5 r.9

Diffusíona1 conductivity (x 104/O;cm) O.27 Z.30 3.77 5.15 5.25 7.25 2.OO

Hydrau lic conductivity 0.000 0.004 0.012 0.031 0.069 0.071 0.116

( x rol2; cn4 /dyne sec ) 106

Fig. 6-I Cross-section of a blood vessel in brachial tissue,5 - l0 min after introduction of the test ion. The vessel lumen, characteristÍca1ly, , is bound by a discontiunous endothelial layer (E), a substantial basement membrane (B) and a continuous pericyte layer (P). A portion of a pericyte junction appears to be fi 1led with precipitate (star). Deposits are found extravascularly (arrows). (Unstained, Bar = 0.5 um)

Fig. 6-2 Cross*section of a vessel wa11 in the brachial neuropil, 5 - 10 min after introduction of the test ion. Carbon (C) is contained within the lumen and is readily distinguished from the extravascular deposits (arrowheads), many of which are aligned with outer membranes of the pericyte nucleus (N) and cytoplasm. (Unstained, Bar = O.i um) F \ n9 -'.t.1::- II I

;: f:

>

¡ b/ , 107

Fig.6-3 Preci pit ate de pos it s (arrowheads ) within the pericyte junction of a vessel in brachial- musculature. (UnstaÍned, Bar = 0.1 um)

Flg.6-4

Precipitates (arrowheads ) associated wÍth part of a mitochondrial complex which Ís embedded in muscular t issue (M). (Unstained, Bar = O.2 um)

Fig. 6-5 TÍssues of the wa11 of anterior aorta close to the ablumenal surface of the vessel. PrecipÍtates (stars) are limited to the ground substance. (Unstained, Bar = O.2 um) t ,#,0\ ¡ ,! ù :*\. '

I

t

I 108

Fig.6-6 Mantle tissue, 5 - 10 min after introduction of the test ion. Precipitates a,re almost totally excluded from the muscle (M), being limited to the extracell-ular matrix. The wide bed of connective tissue contains collagen fibres (co). (Unstained, Bar = O.2 um)

FÍ9. 6-7 Mantle tissue, 5 10 mÍn after Íntroduction of the test íon. Precipitates are clearly associated with the narrow, and somewhat indistinct, extracellular path that separates the muscular tÍssue (M). Precipitate is notably absent from the clear regions indicated (stars). (Unstained, Bar = O.3 um) fi'"r . e'ó ..y+ ç +-; t4- I ù |fr I I O ra # t a ' :t a*'' -r. .a (}Q

. î'{' 109

Fig.6-8 Precipitates associated with what appears to be elastic tissue embedded in a coarsly reticular ground substance. Again the muscular tissue (M) is quite free of precipitate. (Unstained¡ Bar = O.2 um)

Fig. 6-9 Preci pi t ates associated with ce 1lu lar e.lements of the extrace ll-u lar matrix (arrows ). (Unstained, Bar = 0 4 um) 4f' ,' ''

I ' i '.r;i a t t .rÈ

.\: J

^ ..1.-.. ¡^ .t.-. ,: _ :' ¡l

I f,ri I,r1l

tl t 1s- I É. ¡ i.'i " a. þ f: Ít ry- Íe;e .t' ¡rË llo

FÍ9. 6-10 Brain tissue, fixed 10 - ß min. after introduction of the test ion. Precip-itates are largly conf ined to the block of connective tissue (CT). Some precipitate Ís associated with the mitochondria (arrow), but 1Íttle or none has

penetrated the neural tissue (NT) . (Unstained, Bar = O.3 um)

Fig.6-11 Precipitates (arrows ) confined to junctional pathways between neural elements of the brain. (Unstained, Bar = O.2 um)

Fig.6-12 Brachial neuropil tÍssue, fixed 10 - 15 min after introduction of the test ion. Precipitates (arrows ) are in the extracellul-ar matrix and in close to celluLar elements of the neuropil. (Unstained, Bar = O.7 um) j..

t I ../$>

r¡- I

I* ¡' lLr

Fig.6-7t Brachial epidermal tÍssue exposed to the precÍpitating Íon alone, and left unwashed between primary and secondary fixation. The charaeteristically spikey precipitate is found outside the epidermis, between the microvillÍ (MV), and wíthin the junctions of the epiderma t ce lls (arrow-heads). (Unstained, Bar = O.Z um) ,'å !,; þ

I ) I12 of the precipitation technique reported here. Previous ly the position and sÍze of tissue channels were largely guessed at f rom e.lectron micrographs, f or any clear areas simply represented regions of l-ow electron densÍ.ty and little further could be inferred. Attempts to stain the connective tissues have not proved very satisfactory and the difficulty mentioned above still exists. The evidence for the presence of tissue channels, the difficulties in obtainÍng a morphological- picture of the network and usefulness, of the precipitation technique are discussed in detail by Casley-Smith ( L98tc). The precipitation technique was used here for the first time in an investigation of the tissues of an invertebrate. Whi Ie the convergence between cephalopods and marine fish is the more natural, most of the fi.ndings of this study of

0ct o pus tissue channe 1s are discussed Ín re lation to the mammalian system, since comparative information from other groups is scarce or non-existent.

There are some obvious prob lems in interpreting the resu lts of these precipitation experiments, and untÍ I additional techniques are avai lable to further describe the ultrastructure of the channel system, they must stand in relative isolation. It is not known how accurately the precipitates describe tlre position and dimensions of the tissue channe l-s . It can be assumed that the high ly charged ferrocyanide Íons will remain, to a large extent, II3 in the water-fi1led or water-rich regions ofl the interstitium and therefore those precipitates found can be viewed as representing the actual position of channels with some confidence. How many channel_s are left unobserved or have only been partially outlined because of a lack of test ions or inadequate exposure to precipitating ions (a possible reason for'the latter was discussed ear lier ) remains unanswered. Nevert,he less , vessels in mammalian tissues appear leaky enough to ensure that most channels, ât least Ín the immediate extravas.rî", matrix, will contain large quantities of test ion, and exposure to the precipitati.ng ions is probabJ-y adequate in the periphery of the tissue blocks.

6,4,2 Permeation of the test ions in Octoous tissues: The density of microvessels is lower in 0ctopus and the on ly avenue for appreciab le movements of hydrophi lic materials appears to be the pericyte junction. As noted previ.ously (Section IV) the sut¡stance within the pericyte junction is likely to retard the rate of movement of .l_arge mo l-ecu les , a lthough ferritin permeates Ít readi ly and was found extravascularly within 2 - 7 min of being introduced. The junctions are permeable to the ferrocyanide ions, but the hydrau l-ic conductivity is not known. Some evidence for bulk flow through the junctions was gained previous ly ( Section I V) . Neverthe less , it is probably not high because the microvascular blood pressures, while un likely Lt4 to be as low as suggested by Smith (I962b), are almost certain 1y lower than occur in mamma ls . The low vesse L densit¡, can only act to lower the number of distribution sites of ion permeation. However, these lÍmitations are partially offset by the much higher concentrations of ferrocyanide and precipitating ions in the perfusant and primary fixative respectively, allowable for osmotic reasons (Octopus body fluids are virtually isosmotic to seawater). In addition, all tissues involved in the quantitative analysis were perfused with the test ion for at l-east 10 minutes be f ore being f ixed .

To summarize, although it is probable that permeatÍon of tissues by ferrocyanide ions does not occur as readi ly in 0ctopus as it does in mamma ls, conditions were such that inadequate exposure to test ions in 0ctopus vlas not thought likely to constitute a gross experimental deficÍency. Furthermore, the channels directly connected with tlre outflow regions of the blood vessel wall (that is, adjacent to the pericyte junctÍons ) are the most 1ikely to be de lineated . Presumab 1y these are the most important channels for extending the distributive funct,ion of the blood vessels, particularly with regard to transfer of hydrophilic metabolites.

6.4.3 Re o iona I di fferences i.n 0ctcr D us tissue channe l-s : The distributive function of a tissue channel system, 115 should it exist, would be best directed towards metabolicaLly active eLements of the extravascular tissues, and the position of the channe ls in 0ctopus appears to be so related. In the larger blocks of connective tissue, where a protective and/or supportive function is Iikely to be important, channels between cells are generalJ-y infrequent, although .oru are found near the embedded interstitia 1 ce lls . By contrast, many more channels are found scal-terecJ in the smal"l blocks of collagen bearing connective tissue and tracts of ground substance *nicn separate muscle tissue. Precipitates were a lso found associated with some o f the mitochondria 1 comp Lexes.

0bservation of channels ín the neural tissues of 0c top us is of particul-ar interest. Young (I97I) and Gray (Lgeg ) Oescribe an extensive connective tissue matrix which largely surroundsr âñd is often continuous between, the neural cells and the blood vessels. Terms such as I'extracellular tunnefs" or ttglio-vascu.l-ar channeLstt are invoked, and a probable ro.l-e of this arrangement in the distribution of nutrients is suggested. Young (I97I), for instance, notes that while the blood supply to 0ctopus neural tissues is not rich, this does not mean that tissues are remote from the circulation, since they are served by these communicating spaces. The results here show that the glio-vascular tunnels, which are mainly 116

filled with connective tissue, do contain tissue channels, and these will have a role in the transfer of metabolites. However the brain and brachial neuropil in

0ctopus a ppear not to be as highly vascularized as other somatic tissues, and this would have influenced the distrÍbution of test ions,. The results, while on this basis may have signÍficantly underestimated the channel organization, nevertheless give no indicaion of an extensive extravascular circulation. Small deposits rvere found within the narrow intercellular clefts in the neuraL tissues, âs shown in fig. 6- 11. Nicho lls and Kuffler (1964) report that similar clefts provide pathways for the rapid movements of small hydrophilic solutes in the avascular central nervous system of the Leech.

A very great dea I is unders tood about the structure and function of the advanced neural system of cephalopods (see, e. g. r Young , I97 Ii We lls , I978) , but the importance and degree of homeostasis conferred to this neura-l- environment by the circulatory system remains obscure. It is knov¿n that 0ctopus (l¡a ginnis and lt/e J.ls, 1969) and squid (Dykens and Mangum, I978), while alert and active, maintain a relatively high and constant O, consumption when faced with decreasing amounts of environmental oxygen, until a critical lower level- is reached, beyond which compensatory mechanisms can no longer supp ly enough OZ to the tissues, âñd the anima ls 117 quickly malfunction. However, heart s lices of 0ctopus only show a depressed ability to resume normal OZ uptake after 4g or more hours of total anoxia (pritchard et A!, 1967), and I have found that anaesthetized anÍmals may be revived after 2 hours of being out of water ( I did not attempt to find how far this tinre could be extended). The low vascularity and apparent low tissue channel- density suggests that the neural tissues of Octopus do not require a high rate of metabolic exchange. Further physiological investigations which may prove useful, could include for example, selective interference of blood flow to the brain of active animals to test for the dependence of neural- function on blood flow, and determination of the ranges ín various blood parameters within which neural tissues function, to gain some idea as to how necessary and how de licate the homeostatic function is of the blood. The possib le importance of direct neuro- and g lio-vascu lar contacts is discussed later.

6.4,4 Comparisons between channel orqanization in octopuses and mammal-s: The volume of channels in 0ctopus was estimated to be approximate ly O.44% of the interstitial vo.Lume and the average channel- radius to be 18 nm. These are both within the ranges of most mammalian estimates, although the latter is towards the lower limits (Casley-Smith et â1., I979; Casley-Smith, 198Ìc). Estimates of the diffusional and hydraulic conductivities 118 of the tissue channe ls in rat connective tissues were arrÍved at from analysis of precipitate deposits by casley-smith et a1. (1979). (rney determined the channel volume to be only o.16% of the interstitial vorume in norma I rat tissues ) . In simi lar derivatÍons, the 0ctopus channels show a hydraulic- conductivity which is r/j of that reported for rat tissues, while the greater number of, albeit smaller, channels will produce â diffusional conductivity which is twice that of the rat tissues. For reasons discussed earJ-ier, the determinations for 0ctopus are probably underestimated to some extent, andr âs they stand, are broadry comparable with the mammalian system. No evidence rvas gained to indicate the presence in 0c topus tissues of a notably more extensive tissue channel network.

The extrace llu lar compartments in Octopus and mamma -Ls contain very simi lar amounts of water with estimated vo.l-umes of about 22% of body volume for 0ctopus (Martin et a1., 1958) compared with about 17% for man (see for e.9., Guyton et â1., I97I). Val_ues for marine vertebrates are also eomparable (see table 7-II, Gordon,

1972) . That is, if 0ctopus tissues were to contain a notably larger channel syste.m, then significantly less water would have to be intimately bound in a tight protein network, to leave more at high concentration in observal¡le channels. This does not appear to be the case. Diffusive and to a greater extent, hydrauric conduetivities within a 119 tube, increase disproportionately with tube dÍameter (as 12 and ,4, respectiveJ.y). Therefore, for a fixed amount of water, a smaller number of larger channels would confer a greater distributive potential. Again, thÍs appears not to be the case fqr 0ctopus; indeed a greater number of smaller channels were observed.

6.4 .5 What ro les are tissue channels likelv to olav in 0_g!opus? 0f particular interest here is metabolic exchange, for the investigation was initiated by the possibility that an extensive extravascular channel system au gmen ted the vasculature in 0ctopus. In general introductory statements on blood-tissue exchange in higher vertebrates, diffusion of hydrophilic metabolites and of the more lipid soluble respiratory gases is said to occur through the tissue fluids of the interstitium. Any fluid confined largely to an urrimpeded channel is J.ikely to facilitate such transfers, increasing the extent of both díffusive and hydraulic conductivities. The passage of extravascular proteins within restricted regions in the interstitium is well documented for mammals (see t e. 9.r Witte , I975) . These channe ls may drain back to more venous aspects of the vasculature, or penetrate the tissues and eventually lead to the lymphatics. Especially J.ong and obvious prelymphatic channeLs (reviewed by Cas 1ey -Smith, 198 la ) drain f luid and protein in those regions not immediate ly served by the lymphatic system. 120

It may be stated then, oñ the basis of this protein movement, that the channels in mammals must be involved in transfer of water-borne substances generally, and the same can be presumed true in 0ctopus . However, shou ld the movement of extra-ce llular proteins have been primary consideration in development of the channel system in mammals, then a simil.arly structured system would not be exp ect ed in 0ctoous , where leakage of vascu lar protein, while possibily of some consequence, must be comparatively slight (Section IV). The apparent low diameter of 0ctopus channels {uite possibly reflects a smaller size of the bulk of the substances transferred within them.

In studies of vertebrate tissues ' both Longmuir and Bourke (lgeO) and McDougaJ-1 and McCabe (1967) found that 0 di ffused into tissue at a faster rate than 2 reported by Krogh (19L9a), and both groups mention intra- cellular mixíng due to protoplasmic streaming and,/or mitochondrial pulsations as probably being at least partially responsible for this. It should be noted that any net movement of fluid wi 1I result in a convective component to metabolic transfer, and to describe the overalL process as diffusion is to use the term J.oosely. Loca lized all-erations in tissue-f lui.d pressure due to eompression and expansion of the channels is likely to occur when there is adjacent muscular activity. This potentially greater, extracellular, source of hydraulic 12I conductivity will also increase the rates of soLute transfer above that attainable by diffusion alone (Parsons and McMaster, 1938). Gray (1969) described the presence of muscle fibres within the extracellular gIÍo-vascular tunnels in 0ctopus brain, and suggested that contraction of these fibres possibily acts to speed up metaboli c exchange . Pu lsating gang lia in 0ctopus are also mentioned by Gray (1969 ) in this regard. The presence of ferrocyanide precipitates near mitochondria could be a result of mitochondrial pulsations which are said to be associated with the movement of water ( Longmuir and

Bou rke , 1960 ) .

It is pertinent to note here that the Ínterstitial region in mammals is understood to readily accept or donate fluid following alterations in blood volumes, pressures or osmolarity. The larger the channel.s the more easily would the fluÍd be mobilized. Indeed the bulk of the interstitial fluid is not in channels and is thought to be intimate ly bound within the interstitia I protein network (Guyton et a!, I97I), much of it ef f ectively trf rozen'r. The need for an easily accessible, internal reservoir would appear less important in an animal surrounded externally by an aqueous, isosmotic environment. This factor may influence the number and size of tissue channels in Octopus being another reason why there is not more extracell-ular fluid, and what there 122

is, is not kept in wider channels.

The perivascular region is understood to affect capillary permeability in mammals, and indeed largely defines the permeabÍlity of fenestrated capÍ rlaries (Intaglietta and de P1omb, I973; Casley-Smith et â1., I975). In Octopus the permeability of the pericyte watl to water and water-borne substances is very much a function of the pericyte junction, which is of invariable width and composition throughout the microvasculature. Fenestrae'have not been found and vesicles are uncommon (Section IV). Therefore, ffiorphologicaJ. specia lization of the vessel wall (not withstanding differences in the structure of the basement membrane) whÍch would allow di fferences in permeabi lity appear LÍmited to variations j.n junctionaJ. length associated with variations in the thickness of the vessel wall. under these circumstances variation in vessel permeability to smal1 molecules is potentialy determined to a great extent by the immediate extravascu lar matrix, governed by the number, size and position of the tissue channels.

The vesse 1-tissue barrier in the non-neura I somatic tissues of Octopus and mammals appears basj.cally similar, i.e. while a vesse.L or portions of a vessel may be closely apposed to muscle, there is al-most invariab ly an interposing and isolating connective tissue component. 123

Far fewer 0ctopus vessels have been observed, but cellular bridges, if they occur, are certainly. not common. Indeed, the layer of connective tissue is often extensive (Barber and Graziadei, 1965; section rv). sometimes extravascular cells are observed to be closely associated with portions of pericyte layer. However these generally look very like pericytes and are themse lves iso lated from non-vascu lar cells by connective tissue. This isolating layer of connective tissue is probably necessary to give vessels some structural independence from physical changes in the musc.Le tissues.

In neural tissues of Octopus r orì the other hand, neuraL and glial processes have been observed to directly abut portions of the outer pericyte surface (Barber and Graziadei, 1967ai Gray, 1969;). The interposing clefts are about 10 - 15 nm wide. specialized membrane contacts with the pericytes are said not to occur (Barber and Graziadei, 1967a). Analogies v/ere drawn by Bart¡er and Graziadei between these relationships and those known to occur in mammals, although in the latter a substantial, interposing basement membrane is almost invariably found (tVolff, I977), A possj.ble nutritive role is suggested by Young (I97 I) and Gray (1969 ) for the glio-vascular contacts in cephalopods. Barber and Graziade i (1967a) discuss these contacts in terms of added barr-iers to transfer of water-borne solutes. However, in view of the 124 obvious compJ.exity of cephalopod neural function and comparative ly avascu Iar neural environment there is a distinct possibility the appreciable flows of O, and C0 occur within an almost continuous membranous 2 pathway. It should be noted here that direct replacement of parts of the pericyte wall by neural elements has also been reported in a number of cephalopod tissues (Martin, 1968; Berry and Cottrell, I97O, Froesch, I974; Froesch and Mangold, I976a), although the extent to which the lumenal surface area is made up of other than pericyte cells was not been ..timated. Barber and Graziadei (1967a) and Gray (ßeg) record that neural vessels are most commonly surrounded by connectÍve tissue. This indicates that much metabolic-exchange occurs, as elswhere, via the tissue channe ls.

Thus it can be concluded that the tissue channel network in 0ctopus is not comparable to the open system which is found in protochordates and most other Ínvertebrates, including all the other molluscs (reviewed Casley-Smith, 198Ìa). The cephalopods have not only evo lved a c l-osed b lood system but a 1so organized and reduced the quantity of extravascu lar f lu-id to further resemble the vertebrate condition. The reduction in tÍssue fluÍd is undoubtly correlated with the need for cephalopod somatic tissues to be not spongy and vulnerable, but tough, capable of fast and strong muscul_ar 125 activity, and mobi le , unrestricted by the presence of a protective calcareous covering. I have used the term cephalopod to include only the commonly studied octopods, squid and cuttlefish. A comparatÍve investÍgation of the circulatory system of the slow and prÍmitÍve shelled Nauti lis wou l-d be interesting. 126

SECTION VII: CONCLUDING REMARKS

In this final section, brief comments are made on broader and more speculative aspects of the structure and development ofl the cephalopod microcirculatory çystem. A specific summary of the resufts is presented at the beginning of this thesís.

The wide phyJ.ogenetic divergence of the mo llusc and vertebrate groups indicates that the vascu lar systems ) are not, to any extent, homologous. However, many of the same deve lopments occurred in response to the prob lems encountered with evolution of a circulatory system which ís highly pressurized and in which blood flow is confined to a system of cLosed tubes. Further evidence was shown of the gross-structura I specÍa lization of the cepha lopod circulatory system (Section III), âñd the remarkable analogy with the vertebrate system is extended. It is particularly notable that the arterial and venous casts differ similarly Ín the two groups. Additionally, some strong simi larities are seen in the arrangments of the microvasculatures in cepha-lopods and vertebrates at the ultrastructural- level.

The wa l1s of the exchange vesse.l"s are a single ce 1I layer thick and are of simi lar average diameter. Furthermore, they occur, at l-east in muscle and neural 127 tissue, within collagen-bearing sheaths of connective tissue. Indeed, in respect to the appearance of the immediate extravascular surroundings, the two systems are virtuaJ-ly identical. Evidence was also presented that the simi larÍty extends into the arrangement of the tissue channels (Section VI). This channel system comprises the fÍna1 extracellular exchange network between blood and celIs, ât least for the meovement of hydrophilic substances. The similarity in tissue channel organization between cephalopods and vertebrates, which includes a reduction in volume of extravascular fluids, is undoubtedly correlated with their mode of life. Like the te leosts , cepha lopods require a muscu lar and mobi le body to pursue an active predatory exístence. Most other molluscs, in contrast, have more spongy tissues enclosed within rigid , câ lcareous coveri-ngs .

That some prob.l-ems wi 1l necessari ly be so l-ved the same way by divergent groups of animals is an interesting proposition, but one which is not easi ly defended. Leaving aside any question of inevitability, it can be fairly stated that the vascular systems in cephalopods and vertebrates are essentially similar in design.

Neverthe less the 0ctopus microvascu.l-ar s ys tem differs from that found in vertebrates in two major ways in the fine structure o f the exchange v esse L wall 128

(Section IV), and Ín the microvessel density (Section V). The major components of the blood barrier, the basement membrane and encirc ling ce Ll layer, are arranged differently in cephalopods. Apart from this it is very obvious that the ce11 wall is structurally less specialized. There are no fenestrae or complex junctional arrangements, nor many vesicles in the pericyte laye::. It tvas argued that this lack of specia Lization and wide variation in diameter of the exchang e vesse ls in 0ctopus can be direct Iy related to the mode by which the respiratory protein is transported.

A fundamenta 1 di fference between the cepha lopod and the vertebrate circulatory systems is the presence of red blood cells in the latter. Cephalopods have haemocyanin in solution Ín the bl-ood. The gaps in the pericyte waIl need to be smaller than the haemocyanin aggregates if constant loss of the protein to the interstitial space is to be avoided. The red ce11, oñ the other hand, allows the presence of a variety of comparatively large gaps (e.9. fenestrae) in the vessel wal-l without Íncurring ttre loss of the haemoglobin. The hypothesÍs that the red celLs all-ow transport of haemoglobin in quantÍties that would otherwise render the blood too vÍscous ( Lehmann and Huntsman , 196I) has been shown falacious (Schmidt-Nielson and Taylor, 196B). Indeed, Charm and Kurland (I974) conclude that transport 129 of haemoglobin would be more efficient if Ít were in solution. However apart from ensuring retention of the respiratory protein, red cells ensure close packaging of the haemoglobin with various materials such as allostearic modu lators ( e . g. ATP) and enzymes ( e . g. carbonic anhydrase ) . This wi 11 a-llow for far more control over the protein's function.

The compromise which must occur in the development of a closed btood system was mentioned earlier (Section I). There is fnu need for a relatively tight vasculature in order that b lood and tissue f luid wi l1 remain distinguishab 1e , and the need for permeab le vesse I wa 1Is in order that the exchange function of the blood can be realized. In regard to the latter requirement, it would appear that the possession of the red ce ll conferred an important advantage to the vertebrates. Furthermore vertebrates developecl additional drainage vesseLs - the lymphatics. In essence, the lymphatics allow the continued function of a microvasculature which is so permeable that it constantly leaks significant quantities of fluids and so lutes , inc luding the b lood proteins ( a lthough no.L the red cells). No good evidence has been presented for the existence of a lymphatic system in cephalopods. When comparisons are drawn vrith the microvascu lar system of vertebrates , one can on ly conc lude that the cepha lopod system has not developed to the same extent. 110

Active cephalopods show comparat,Ívely high oxygen consumption rates, achieved in spite . of the low carrying capacity of the blood. The microvascular density, on this account, may reasonably be expected to be comparatively high. Indeed , it Ís exceptiona 11y 1ow, and there is no evidence of an especia lly extensive system of extravascular tissue channels. Further study of oxygen uptake, blood flows and tissue demands (amongst other parameters) may help explain this apparent anomaly. Certainly the Krogh-Erlang analysis suggests that the paucity of vesse ls will mean actÍve tissue wi L1 not receive sufficient oxygen to function aerobically. It is generally presumed that the major resistance to the transfer of the resp-iratory gases is the water-rich compartment vrithin the interstitium. The rate of di ffusion of oxygen through connective tissues is an important component of the Krogh-Erlang analysis.

However, are the respiratory gases, with their low water and high lipid so lubi lity r eXchanged across the water-rich, lipid-poor medium of the interstitia I tÍssues? Any ceI.l-ular contacts between the ablumenal vessel wall and the active tissues might be expected to form an avenue for more rapid gaseous diffusion. Such contacts are rarely described in the literature, but neither have they , to my know ledge, been speci fica 11y looked for. Scow et â1. , (1976) proposed an 73I intramembranous pathway for lipid transport from blood to tissues; the ultrastructural evidence presented is, unfortunately, somewhat unconvincing. Perhaps only very close contacts are required for lipid-phase diffusion to be of greater importance. Neural tissues in 0ctopus are as avascular as the muscular tissues, but there are regions in the former, where the pericyte and neural cells are in very close contact. This arrangement may be important in the transfer of oxygen. The possibi J.ity of intramembranous pathways, coupled with some of the uncertaintîes mentioned prevÍously concerning capillary function in vertebrates (Section V), suggests that the disadvantag es 0ctopus tissues su ffer r âs a result of the relative paucity of exchange vessels, may not be as severe as first appeared.

0nce the closed system had evolved in cephalopods, it is difficult to understand how selection for an increased density in the cl-osed microvasculature could not have occurred, shou ld it have proved advantageous. Perhaps the limits of the present vascular system are rarely stretched. Alternatively the economic cost of producing and maintaining additional vascular elements may be prohibitive. Economic constraints associated with development of a more extensive channel system must be less severe. Whatever the limitations in the cephalopod circulatory system, a remarkably complex physiology and I72 successful predatory mode of existence are due in, large part to the efficíency of exchange. between the active tÍssues and the blood. This is imparted by the closed vasculature, albeit of 1ow density, by the tissue channel sys tem, which is apparent ly not particu lar ly extensive , and by other possible faclors such as metabolite transfer via membranous pathways. 133

REFERENCES

ABB0TT, N. J. (1970). Absence of blood-brain bamier in a

crustacean, .Carcinus maenas L. Nature, 225, 29I-293.

ADAIR, G. S., and ELLIOTT, F. G, (1968). Measurements of very small osrmtic pressures of the haemocynin of Pila leopoldvi llensis. Nature, 2I9, 81-82.

ALTMAN, P. L. and DITTMER, D. S. (1971). I'Respiration and Circulation". Federation of American Societies for Experimental Biology.

ANDO-YOSHIA. (I95t). Studies on the lymphatics of the octopus, mollusca. J. Jap. physiol. Soc. 15 , 16-19.

BARBER, V. C. and GRAZIADEI, P. (1965). The fine structure of cephaJ-opod blood vessel-s. I. Some smaller peripheral vessels . Z. Zellforsch. , 66, 765-78I.

BARBER, V. C. and GRAZIADEI, P. (I967a). The fine structure of cephalopod blood vessels. II. The vessels of the nervous system. 7. Zellforsch. , 77, 147-16I.

BARBER, V. C. and GRAZIADEI, P. (I967b). The fine structure of cephalopod blood vessels. III. Vessel innervation. Z. Zellforsch. , 77, 162-174. 134

BARNES, R. D. (1968). rrlnvertebrate Zoologyl'. Vl. B. Saunders Co.,

Lond.

BERRY, C. F. and COTTRELL, G. A. (1970), Neurosecretion in the vena cava of the cephalopod Eledone cirrosa. Z. Zellforsch. 104,107-tt5.

BLOCtl, E. H. (1962). A quantitative study of the hemodynamics in the llving microvascular system. Am. J. Anat. , L.l_o, I25-I45.

BRUGGEN, van, E. F. J. and WIEBENC,A, E. H. (1962). Structure and properties of hernocyanins. II. Electron micrographs of the

henocyaníns of Sepia officinalis, Octopus vulgaris and Cancer pagu!!19. J. Mol. Biol., 4, B-9.

BRUNS, R. R. and PALADE, G. E. (1968). Studies on blood capillaries. II. Transport of ferritin molecules across the wall of rruscle capi llaries. J. Cell Biol. , 37, 277-299.

CASLEY-SMITH, J. R. (f962). The displacement of ferritin molecules by the microtome knife. J. Microscopie , 1, 335-342.

CASLEY-SMITH, J. R. (1969). The dÍmenslons and numbers of small vesicles in cells, endothelial and rnesothelial and the significance of these for endothelÌal permeability. J. Microsco , 90, 25I-268. It5

CASLEY-SMITH, J. R. (I97I). The fine structure of the vascular system of amphioxus: implications in the development of lymphatics and fenestrated blood capillaries. Lvmoholoqv 4, 79-94.

CASLEY-SMITH, J. R. (1976). Calculations relating to the passage of fluid and protein out of arterial-limb fenestrae, through basenent nembranes and connective tissue channels, and into venous-limb fenestrae and lympha tics. Microvascular Res. 12, It-34.

CASLEY-SMITH, J. R. (1980). Comparative fine stucture of the

microvasculature. In'rVascuLar Endothelium and Basement Membranes" (ed. B.M. Altura), Karger, Basel, N.Y., Advances in Microcirculation 9 , I-44

CASLEY-SMITH, J. R. (198la). The phylogeny of the lymphatic system. In I'Lymphology" (ed. M. Földi), Thieme, Stuttgart - in press.

CASLEY-SMITH, J. R. (198]b). Freeze-substitution observations on the endothelium and passage of ions. MicrocirculatÍon - Ín press.

CASLEY-SMITH, J. R. (198lc). The fine structure of tissues and

tissue channels. In ilTissue Fluid Pressure and Compositionr' (ed. A. R. Hargens), pp. 99-II2. V/averly Press, Baltimore. I36

CASLEY-SMITH, J. R. and CASLEY-SMITH, J. (1975). The fine structure of the blood capillaries of some endocrine glands of the hagfish, Eptatretus stouti: implicat.ions for the evolution of blood and lymph vessels. Revue suisse Zool.

82, 35-¿tÐ.

CASLEY-SMITH, J. R. and VINCENT, A. H. (1978). The quantitative morphology of interstitial channels in some tissues of the rat and rabbit. Tissue and Cell f0, 57I-584.

CASLEY-SMITH, J. R., GREEN, H. S., HARRIS, J. L. and WADEY, P. J. (1975). The quantitative norphology of skeletal muscle capillaries in relation to permeability . Microvasc. Res. 10, 43-64. cASLEy-SMrrH, J. R., F0LDI-BöRCSöK, E. and röLDr, t"r. (1979).

A fine structural study of the tissue channelsr numbers and dinnnsions in normal and lymphoedematous tissues. Z. Lvmpholoqie. 3 , 49-58.

CAULFIELD, J. P. and FARQUHAR, M. c. (1974). The permeability of glomerular capillaries to graded dextrans. J. CeLl Biol. 67, 883-903.

CHAPT\4AN G. (1958). The hydrostatic skeleton in the invertebrates. Biol. Rev. , 7V, 778-37I. 737

rrThe CIIAPMAN, R. F. (1969). Insects: Structure and Function'r. The English Univ. Press Ltd.

CllARM, S. E. and KURLAND, G. S. (1974). "Blood Flow and Mícrocirculation". John Wiley and Sons., Lond.

CHASE, vl. H. (1959). Extracellular distribution ofl ferrocyanide in m.rscle. Arch. Path., 67, 525-532.

CLIFF, Vr/. J. (1976). "Blood Vessels". Cambridge Unív. Press. Lond.

CoTToN, B. C. and GoDFREY, F. K. (1940). rrThe l'4ol.I-uscs of South Australia". Part II. Scaphopoda, Cephalopoda, Aplacophora and Crepípoda. Govern. Printer, Adelaide.

COUTRICE, F. C. (1981). The endothelium: some historical milestones in our understanding of its structure and function. In: 'rProgtess in Microcirculation Research", (ed. D. GarLick) Univ. New South lVa-les. p. I-37.

üJRRY, F. E., (1980). Is the transport of hydrophilic substances across the capillary wa11 determined by a network of fibrous molecules? The Physiol. , 23, gO-91.

DALES, R. P. (1967). 'rThe Annelids". Hutchinson Univ. Library.

Lond 138

DAVIS, lV. C., DOUGLUS, S. D., PE'IZ, L. D. and FUNDENBERG, H. H. (1968). Femitin antibody localization of erythrocyte antigenic sites in immunohemolytic anemias. J. Immun., 101, 62I-627

DECLEIR, W., LEMAIRE, J. and RTCHARD, A. (1971). The differentiation of blood proteins during ontogeny in Sep.þ officinalis L. Comp. Biochem. Physiol., 40, 923-930.

DENNIS, J. B. (1959). Effects of various factors on the distribution of ferrocyanide in ground substance. Arch. Path. , 67, 533-549.

DIANA, J. N. and FLEMING, B. P. (1979). Some current problems in microvascular research. Microvasc. Res. , 18, I44-I52.

DILLY, P. N. and MESSËNGER, J. B. (1972). The branchial gland: A site of haernocyanin synthesis in Octopus. Z. Ze11forsch., I32, I93-nI.

DRINKER, C. K. (1942). The lymphatic system: it's part in regulating cornposition and volume of tissue fluid. Lane MedicaJ. Lectures, Vol- 4. Stanford University Press,

U. S. A. 139

UJLING, B. R. and BERNE, R. M. ( 1970). Longitudinal gradients in periarteriolar oxygen tension. A possible nechanism for the participation of oxygen in local regulation of blood flow. Circ. Res., 27, 669-678.

DYKENS, J. A. and MANGUM, C. P. (1979). The design of cardiac muscle and the mode of metabolism in molluscs. Comp. Biochem. Physiol. , 62, 549-554.

FROESCH, D. (1974). The subpedunculate lobe of the octopus brain: Evidence for dual function. Brain Res. , 75, 277-295.

FRoESCH, D. and MANGoLD, K. (I976a). 0n the structure and function of a neurohemal organ in the eye cavÍty of EJ-edone cirrosa (Cephalopoda ). Brain Res. , 111, 297-297.

FROESCI-|, D. and MANGOLD, K. (I976b). Uptake of ferritin by the cephalopod optic gland. Cell Tiss. Res. , 170, 55I-549.

GANNON, B. J. (1978). VascuLar casting. In 'rPrinciples and Techniques of Scanning Electron Mic::oscopy'r. Vol. 6. (ed. M. A. Hayat), ch. 7. Van Nostrand Reinhold Co. Ltd. 140

GANN0N, B. J., CAMPBELL, G. and RANDALL, D. J: (1973). Scanning electron microscopy of vascular casts for the study of vessel connections Ín a complex vascular bed - the trout gil1. In.rrProceedings, 31st. Ann. Meeting, Electron Microsc. Soc. Am." (ed.-C. J. Arceneaux), pp. 442-44I. Claitor's Publishing Division.

GHIRETTI, F. (1962). Hemerythrin and hernocyanin. In "Oxygenases". (ed. 0. Hayaishi), ch. 12. Academic Press, N.Y.

GHIRETTI, F. (1966). Molluscan hemocyanins. In "Physiology of Mol-lusca" Vol-. II. (eds. K. M. tilÍlbur and C. M. Yonge), ch. 7. Academic Press, N.Y.

GLAUERT, A. M. (1975). Part I: Fixation, dehydration and embedding of biological specimens. In "Practical Methods in Electron Microscopy", Vol. IlI. North-Holland Publishing Co.,

Amsterdam.

GoRDON, M. S. (1972). 'rAnimaL Physiology: Principles and AdaptatÍons". The Macmillan Co., London.

GOSSELIN, R. E. and STIBITT, G. R. (1977). The diffusíve conductance of slits between endothelial cells Ín muscle

capi llaries . Microvasc. Res. 14, 763-382. 141

GRAY, E. G. (1969). Electron microscopy of .the glio-vascular

organization of the brain of 0clppus. Phil. Trans. Rov. Soc. Lond. B. , 255, IJ-32.

GRIMMER, J. C. and HOLLAND, N. D. (1979). Haemal- and coelomic circulatory systems in the arms and pinnules of Florometra serratissima (Echj.nodermata: Crinoidea). Zogmorph., 94, 93-Iæ.

GRIMPE, c. (I9I3). Das Blutgefässsystem der dibranchiaten Cephalopoden. Tei-L I. Octopoda. Zeitschrift f. wissensch. Zooloqie_. , 104, 572-62I.

GUYTON, A. C., GRANGER, H. J. and TAYLOR, A. E. (1971). Interstitial fluid pressure. Physiol. Rev. , 5I, 527-563.

HAMA K. (1960). The fine structure of some blood vesse.Ls of the earthworm, Eisenia foet:Lgq. J. Biophysic. Biqqhern. Cyto1., 7, 717-724.

HAYAT, M. A. (1970). 'rPrinciptes and Techniques of Electron Microscopy", vol .l-. Van Nos.trand Reinhold, N.Y.

HËNQUELL, L., LACELLE, P. L. and FONIG, C. R. (1976). Capillary cliameter in rat heart In Si!u: Relation to erythrocyte deformabitity, 0, transport, and transmural 02 gradients. Microvasc. Res. , 12, 259-274. I42

INTAGLIETTA, M. and DE PLOMB, E. P. (1971). Fluid exchange in tunnel and tube capillaries. Microvasc. Res. , 6, L53-I69.

ISGR0VE, A. (1908). L.M.B.C. Memoirs. XVIII. Eledone. Liv. Biol. Soc. , 23, 469-573.

JOHANISEN, K. and l-llJSToN, M. J. (1962). Effects of some drugs on the circulatory system of the intact, non-anaesthetized cephalopod, Octpzus dof leini. Comp. Biochem. Phys-þ!., 5, I77-I94.

JOHANSEN, K. and LENFANT, C. (1966). Gas exchange in the cephalopod, 0ctopus dofleini. Am. J. Physiol. , 2IO, 910-919.

JOHANSEN, K. and MARTIN, A. Vl. (1962). Circulation in the cephalopod, Octopus dofleinÍ. Comp. Bioehem. Physiol. 5, 16I-176.

JOHANSEN, K. and MARTIN A. Vf. (1965). Comparative aspects of

cardiovascular function in vertebrates. In 'rHandbook of

Physiology"; Section 2, VoI. 3. (eds. tlll. F. Hamiliton and P. Dow) Am. Physiol. Soc., Washington.

JOHANSEN, K., REDM0IÐ, J. R. and BOURNE, G. B. (1978). Respiratory exchange and transport of oxygen in Nautilus pompi-lius. J. exp. ZooI. , 2O5, 27-76. I43

.!NES, J. D. (1972). "Comparative Physiology.of Respiration". Edward Arnold (Publishers) Ltd.

KAMPMEIER, 0. T. (1969). "The evolution and comparative morphology of the J.ymphatic system"- Charles C. Thomas, Publisher.

KAWAGUTI, S. (1970). Electron microscopy on muscLe fÍbers in blood

vessels and capíllaries of cephalopods. Biol. J. Okayama Univ., 76, 19-28.

KELLER, H. P., SCHAFER, D. and LUBBERS, D. Ìt/. (1972). The

structure of the network of the vessels of the carotid body of the cat and its evaluation by neans of the scannÍng electron microscope . Z. Naturforsh. , 27, 1118-1118a.

KR0GH, A. (1919a). The rate of diffusion of gases through animal tissues, with some remarks on the coefficÍent of invasion. J. Physiol. , 52, 3gI-4O9.

KROGH, A. (19I9b). The numt¡er and distribution of capillarÍes in

muscles with calculations of the oxygen pressure head necessary for supplying the tissue. J. Physiol. , 52, 409-415.

KROGH, A. (l9l9c). The supply of oxygen to the tissues and the regulation of the capiì-lary cÌrculation. J. Physiol. , 52, 457-474. 144

LAMETSCHWANDTNER, 4., ALBRECHT, U. and ADAM, H. (1978). The vascul-arization of the anuran brain. The choroid plexus of the fourth ventricle. A scanning electron microscopical

study of vascular corrosíon casts. Acta zoo-I.(Stockh.). 59 229-237.

LANDIS, E. M. and PAPPENHEIMER, J. R. (1963). Exchange of

substances through the capillary waJ.ls. In "Handbook of

Physiology"; Section 2, vo]., 2. (eds. $l. F. Hamiltion and P. Dow.) Am. Physiol. Soc., Waslrington.

LASANSKY, A. and WAI-D, F. (1962). The extracellular space j.n the toad retina as defined by the distribution of ferrocyanide. A light and electron microscope study . J. Cell BioL. , 15, 463-479.

LEl-fr\4ANN, H. and HUNTSMAN, R. G. (1961). lvhy are red cells the shape they they are? The evolution of the human red cell. In

I'Functions of the Bl-ood" (eds. R. G. MacFarlane and A. G. T. Robb-Smith), ch., 2. Academic Press, N.Y.

LONGI"|UIR, I, S. and BOURKE, A. (1960). The neasurement of the diffusion of oxygen through respiring tissue. Biochem. J. 76, 225-229. 145

LUFT, J. H. (1973). Capillary permeability. I. Structural

considerations. In "The Inflammatory Processrr Vol. II. (eds. B. ÌV. Zweifach, L. Grant and R. T. McCluskey), ch. 2.

Academic Press. N.Y.

MacDUC,ALL, J. D. B. and McCABE, M. (1967). Diffusion coefficient of oxygen through tissues. Nature, 2I5, II77-II74.

MAGINNISS, L. A. and WELLS, M. J. (1969). The oxygen consumption of Octopus cyanea. J. exp. Biol. , 5r, 607-613.

MARTIN, A. lrtt., HARRISON, F. M., HIJSTON, M. J. and STEWART, D. M. (1958). The blood of some representative moll-uscs. J. exp. Biol. , 75, 260-279.

MARTIN, R. (1968). Fine structure of the neurosecretory system of the vena cava in Octopus. Prain Reg., 8, 2OI-2O5.

MmT0N, J. E. (1967). "Molluscs". Hutchinson Univ. Press.

MURAKAMI, T. (Þ7f). Application of the scannÍng electron microscope to the study of the fine distribution of the blood vessels. Arch. histol. 32, 445-454. 746

MURAKAMI, T. (1975). Pliable methacrylate casts of blood vessels: Use in a scanning electron microscope study of the microcirculation in rat hypophysis. Arch. histol.'iap. 78, 151-168.

MURAKAMI, T., UNEHIRA, M.,' KAWAKAMI, H. and KUBoTSU, A. (1973). 0smium impregrration of methyl melhacrylate vascular casts for scanníng electron microscopy. Arch. histol. jap. , 76, II9-,I24.

NAITo, M. and WISSE, E. (1978). Filtration effect of endothelial fenestrations on chylomicron transport. in neonatal rat lÍver sinusoids. Cell Tiss. Res. , 190, 37I-382.

NAKAMURA, Y. and WAYLAND, H. (1975). Macromolecular transport in the cat nesentery. MÍcrovasc. Res. ,9, I-2I.

NICII0LLS, J. G. and KUFFLER, S. \¡J. (1964). Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the Leech. J. Neurophysiol. , 27, 645-67 I.

NTSIMARU, Y. (1969). Studies on comparatíve physÍology of body fluid flow. Hirosh. J. Med. Sc. , f8,95-118. I47

PACXARD A. (1972). Cephal-opods and fish: The limits of convelgence. Biol. Rev. , 47, 24I-3O7.

PALADE, G. E. (1953). FÍne structure of blood capillaries. J. Appl. Phys., 24, 1424.

PALADE, G. E., SIMIONESCU, M. and SIMIONESCU, N. (1979). Structrual aspects of the pernreability of the microvascular endothelium. Acta Physiol., Scand. , suppl. 463, II-32.

PAPPENHEIMER, J. R. (I95t). The passage of mo.l-ecules through capillary walls. Physío1. Rev. , 33, 187-427.

PARSONS, R. J. and McMASTER, P. D. (f918). Normal and pathological factors influencing the spread of a vital dye in the connective tissue. J. Exp. Med. , 68, 869-890.

POTTS, Ìtl. T. t.f. (1965). Ammonia excretion in Octop-us dofleini. Comp. Biochem. Physiol. , 14, t39-355.

POTTS, Vll. T. td. and TODD, M. (1965). Kidney function in the Octopus. . Biochem. io1. , 16, 479-489.

PRITCHARD, A. Ìtl., HUSTON, M. J. and MARTIN, A. tÍ. (I96t).

Effects of in vitro anoxia on metaboLism of Oc_topus heart tissue. Proc. Soc. Exp. Biol. Med. , II2, 27-29. 148

PROSSER, c. L. (1973). rrComparative Animal Physiology". !tl. B. Saunders, Lond.

PROTHERO, J. and BURTON, A. C. (1961). The physics of blood flow. Biophys. J. , I, 565-579.

REDFIELD, A. C. and G0ODKIND, R. (1929). The signiflicance of the Bohr effect in the respiratÍon and asphyxiation of the squid Lo li_go pea lii . J. exp . Bio l. , 6 , 340-349 .

REI{(IN, E. M. (1977). Multiple pathways of capillary permeabilÍty. Circ. Res. , 4I, 775-743.

RE[\ü(IN, E. M. (1978). Transport pathways through capiJ.lary endothelium. Microvasc. Res , 15, 123-135.

RENKIN, E. M. (1979). Relation of capillary morphology to transport of fluid and large molecules: a review. Acta sio 1. Scand., suppl. 467, BL-91.

RENKIN, E. M. (1980). Transport of proteins by diffusion, bulk flow and vesicular mechanisms. The Physiol. , 23, 57-6I.

RENKIN, E. M. and CURRY, F. E. (1978). Transport, of water and solutes across capillary endothe-Lium. In t'Handbook of Biological Membranes't (eds. G. Giebisch and D. C. Tosteson) Springer Verlag, Berlin. I49

REYNOLDS, E. S. (1967). The use of lead citrate at high pH as an

el-ectron opaque stain in electron microscopy. J. Ce11 Biol., 23, 2O8-2I2.

RODBARD, S. (1975). Selective staining of fibrous connective tissue capsules and lymphatics. Rn evaiuation of "interstitiaL" fluids. Lymphology, I, I42-I48.

SATCIIELL, G. H. (I97D. "Circulation in Fishes". Camúridge Univ. Press.

SCtIIPP, R. and BOLETZKY, von, S. (1975). Morphology and function of the excretory organs in dibranchiate cephalopods. Sond. Fortschritte Zool. , 23, 89-111.

SCIIMIDT-NIELSEN, K. and PENNYCTJIK, P. (1961). Capillary density in mammals i-n relation to body size and oxygen consumption. Am. J. Physiol-. , 36, 787-794.

SCIIMIDT-NIELSEN, K. and TAYLOR, C. R. (1968). Red blood cel1s: Why or why not. ScÍence, 162, 274-275.

SCÐW, R. 0. , BLANCIIETTE-MACXIE, E. J. and SMITH, L. C. (1976). Role of capillary endothelium in the clearance of chylomÍcrons. Circ. Res_. , 79, 149-162. 150

SIMIoNESCU, N., STMTONESCU, M. and PALADE, G. E. (197t). Perneability of muscle capillaries to exogenous myoglobin. J. Cell Biol. , 57, 424-452.

SIMIONESCU, N., SIMIONESCIJ, M. and PALADE, G. I. Q975). Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels. J. Ce]l Biol. , 64, 586-607.

SIMIONESCU, N., SIMI0I.ESCU, M. and PALADE, G. E. (1978). Structural basis of permeability in sequential segments of the microbasculature of the diaphragm. II. Pathways followed by micrope roxidase across the endothelium. Microvasc. Res. 15, 17-76.

SMITH, L. S. (I962a). Some aspects of perÍpheraL circulation in Octopus !_qüeinr. Doctoral Dissertation, Univ. Washington, Seattle, Washington.

SMITH, L. S. (I962b). The role of venous peristalsis in the arm circulation of Oótopus dofleini. Comp. Bioclrem. Physiol, 7,269-275.

SMITH, L. S. (1963). Circulatory anatomy of the octopus arm. J. Morph. , ID, 26I-266. 151

STARLING, E. H. (1897). Some poÍnts in the pathology of heart disease. Lancet I, 727-726.

WAYLAND, H. and SILBERBERG, A. (1978). The microvascular wal1. MÍcrovasc. Res. , 15, 367,-374.

WEAST, R. C. (1975). "Handbook of Chemisty and Physicsrr C. R. C. Press, Inc. Ohio.

WEIBEL, E. á. Q969). Stereological princíples for rmrphometry in efectron microscopic cytology. Int. Rev. Cytol., 26,

275-302.

WELLS, M. J. (1978). "octopus". Chapman and Ha]l, Lond.

WILLIA|4S, L. lV. (I9O2). The vascular system of the common squid Loligo pealii. Am. Nat., 36, 787-794.

WISSIG, ttl. L. and WILLIAMS, M. C. (1974). Passage of microperoxidase across the endothelium of capillaries of the diaphragm. J. Cell Biol. 631 375a.

WITTE, S. (1975). Microscopic techniques for the in situ characterization of concentration of tissue components and penetrating nnlecules. Bior[., 12, I73-78O. 752

W0LFF, J. R. (1977). Uttrastructure of the terminal vascular bed as related to function. In "Microcirculation" (eds. G. Kaley and B. M. Altura. ) vol. 1. Univ. Park Press, Balt., U.S.A..

YAMAM0To, T., TASAKI, R., SUGAWARE, Y. and TON0SAKI, A. (1965). Fine structure of the octopus retina. J. Cell Biol. ,25, 345-759.

Y0UNG, J. Z. (1971). 'rThe Anatomy of the Nervous System of 0ctcpus vulgaris". Clarendon Press, Oxford.

YOUNG, J. Z. (1976). The nervoLrs system of Loligo. II. Suboesophagea 1 centres. Philo. Trans. Roy. Soc. B , 274, 101-Ì67.

ZWEIFACI-|, B. !V. (1977). Perspectives in microcirculation. In "Microcirculation" (eds. G. Kaley and B. M, Altura.), vol. l. Univ. Park Press, Balt., U.S.A. APPEND ICES

Browning, J. (1979) 0ctopus microvasculature: PermeabiLit to ferritin and carbon. Tissue Cell, II; 3 Y I-383.

Browning, J. (1980) oemarcation o ft issue channels by ferrocyanide deposits: Us eo f an alternative precipitant. Microvasc. Res 19; 380-384. Browning, J . ( 1980) fne vaseulature of 0ctopus arms: A scanning electron microscope study of corrosion casts. Zoomorph. , 96i 24t-253. Browning, J. and CasIey-Smith, J. R. (f981) Tissue channel mor 1o ov in Octopus. Ce 1I Tiss. Res. 2I5; bt- 17 6ho Browning, J. ( f981) The densíty and dimensions of exchange vessels in 0ctopus pallidus (Hoyle). (aecepted for publication; J. Zooloov Lond. )

J. Browning (1979) Octopus microvasculature: Permeability to ferritin and carbon. Tissue and Cell, v. 11 (2), pp. 371–383, 1979

NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library.

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http://dx.doi.org/10.1016/0040-8166(79)90050-8

J. Browning (1980) Demarcation of tissue channels by ferrocyanide deposits: Use of an alternative precipitant. Microvascular Research, v. 19 (3), pp. 380–384, May 1980

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J. Browning (1980) The vasculature of Octopus arms: A scanning electron microscope study of corrosion casts. Zoomorphology, v. 96 (3), pp. 243-253, December 1980

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J. Browning and J. R. Casley-Smith (1981) Tissue channel morphology in Octopus. Cell and Tissue Research, v. 215 (1), pp. 153-170, February 1981

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J. Browning (1982) The density and dimensions of exchange vessels in Octopus pallidus. Journal of Zoology, v. 196 (4), pp. 569–579, April 1982

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