Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy

Cell Tiss. Res. 148, 111--125 (1974) �9 by Springer-Verlag 1974

Structure of Rat Liver Sinusoids and Associated Tissue Spaces as Revealed by Scanning Electron Microscopy*

Pietro Motta and Kei th i~. Porter

Department of Anatomy University of Rome, Rome, Italy Department of Molecular, Cellular and Developmental Biology

University of Colorado, Boulder, Colorado, USA

Received November 28, 1973

Summary. The inner surface of sinusoids and adjacent hepatoeytes have been examined by scanning electron microscopy. The endothelial cells lining the sinusoids show large numbers of fenestrations which vary greatly in size and arrangement. Some are very small (0.1 ~m) and arranged in clusters; others that are much larger (~1.0 ~m) are subdivided by slender strands of cytoplasm. At sites where the larger fenestrae are present it is evident that the endothelial lining of the sinusoid is double. This may represent a kind of structural assurance against complete breakdown of what seems to be a very thin and fragile endothelial wall. Junctions between adjacent endothelial cells have not been found in these preparations.

The open continuity of the sinusoid is occasionally interrupted by slender extensions of cells morphologically distinct from the thin fenestrated endothelial cells. These possess a characteristically textured surface and are thought to represent stellate Kupffer cells.

The SEM images describe the subendothelial Spaces of Disse as being larger and as having more extensive ramifications than is generally evident from transmission micrographs. The space, limited on one side by the hepatocyte with numerous microvilli and on the other by endothelial cells, appears actually to be only part of an extensive labyrinth of intercellular channels. These connect the more discrete Spaces of Disse and extend into the narrower spaces between the hepatocytes. The total effect of this system is to expose the greater part of the liver cell surface to the blood filtrate. Microvilli populate the hepatocyte surfaces except for narrow margins which border the bile canaliculi. Whether their presence coincides with the adsorbing surfaces and their absence with secreting surfaces can be decided best by experimental studies.

Key words: Liver - - Sinusoids - - Kupffer cells - - Spaces of Disse - - Scanning electron microscopy.

Introduction

Studies, uti l izing t ransmission electronic microscopy (TEM), of h u m a n liver as well as other mammals , have clarified several s t ructural problems posed by early light microscopists (Fawcett , 1955; Novikoff and Essner, 1960; Rouiller and Jeze- quel, 1963; Cossel, 1964; David, 1964; Brun i and Porter, 1965; Jezequel and Or- landi, 1972). I n part icular sinusoids and their relationship with liver cells have been examined extensively both at the light and u l t ras t ruc tura l levels because of their physiological and pathological importance in metabolic exchanges with the

* This work was supported in part by a contract from the Special Virus Cancer Program, National Cancer Institute. - - The study was made while Dr. Motta was a guest investigator and Fulbright Scholar in the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado.

112 P. Motta and K. R. Porter

bloodstream (Aterman, 1963; Wassermann, 1958; Wood, 1963; Burkel and Low, 1966; Nicolescu and Rouiller, 1967; Wisse, 1970).

Although still largely disputed and somewhat incomplete, the picture of the sinusoidal space and surfaces as revealed by TEM is substantial ly clearer than tha t resolved by using conventional light optics. According to these studies the liver sinusoid is characterized by an endothelial wall tha t is interrupted by m a n y fenes- trat ions and by the complete absence of a typical basal lamina. The structure, number and size of these openings is a problem yet to be resolved satisfactorily because of the different observations made by various investigators (see review of Wisse, 1970). Addit ionally the picture includes numerous microvilli projecting from the hepatic cells, a very limited amount of collagen, as well as "fat-storing cells" in the pericapillary Spaces of Disse (Ito and Nemoto, 1952 ; Yamagishi, 1959). Whether or not the Kupffer cells comprise an integral par t of the sinusoidal wall is not clear, but the consensus is tha t they do contribute over small areas with most of the lining being formed from thin endothelial cells (see reviews of Aterman, 1963; Cossel, 1964; David, 1964; Tanikawa, 1968; Carr, 1970; Jezequel and Or- landi, 1972).

During the course of a scanning electron microscope (SEM) s tudy of rat liver, some new and seemingly valuable details concerning the sinusoids and their relationship with the Spaces of Disse were revealed. To our knowledge these same observations have not been reported elsewhere and since they contribute to a bet ter unders tanding of the structure and topography of these unusual blood vessels and associated tissue spaces, the findings are reported and discussed here.

Material and Method

Liver tissues from five healthy, young, adult albino rats were perfused with a Tyrode solution, through the left ventricle. The perfusion solution was used at room temperature and contained oxygen, 0.1% procaine and heparin. Gravity maintained a perfusion flow of about 60 ml/min. After about 30 sec. the flow of Tyrode solution was interrupted and 2.5% glutar- aldehyde in caeodylate buffer solution (Sabatini et al. , 1963) (0.18 M; pH 7.3) was perfused for about 10 min.

Small pieces of liver were subsequently excised an immersed in the same fixative. After a period of time between one and five days the tissues were washed for 3/4 hrs. in buffer and cut into small blocks with a razor blade or carefully fragmented with jeweler's forceps. De- hydration was carried out rapidly in a graded series of acetone and the specimens were trans- ferred to liquid CO 2 for critical point drying (Porter et al. , 1972). The dried samples were mount- ed on aluminium studs using conducting silver paint, and coated with a thin layer of carbon and gold in a high vacuum evaporator (DV-502, Denton Vacuum) under continuous rotation and with an appropriate tilt of the stud.

All the specimens were examined and photographed using a Cambridge Stereoscan Model $4 operated at 10 or 20 kV.

Observations

Scanning microscopy of these preparations provides a readily interpretable image of the sinusoids and hepatocytes even though the aspect is new. Thus it is easy to recognize the individual liver cell, the open (1/2) bile canaliculi running along their sides and the sinusoids adjacent to other surfaces (Fig. 1). Prepared

Liver Sinusoids and Associated Tissue 113

Fig. 1. Topographical relationship among sinusoids (S), hepatocytes (E), and bile canaliculi (B). Small intercellular spaces (arrows) occasionally extend (*) through a sheet of parenchymal cells andconnect adjacen~ Spaces of Disse. Blood cells (R) are shown in the lumina of some capil-

laries. • 3570

b y these technics the l iver cells seem to separa te along thei r surfaces r a the r t han break ing open (but see Fig. 2). I t follows t h a t t hey are only loosely a t t a c h e d to one ano ther and then, as is well known, along the margins of the bile canaliculi .

8 Cell Tiss. l~es. 148


Fig. 2. Portion of a liver lobule showing plates of hepatocytes (E) and sinusoids (S). The liver cells form a continuous system of anastomosing sheets. The perisinusoidal Spaces of Disse intercommunicate through channels present in the laminae of liver cells (arrows). Small bundles

of "retieulin" fibers (*) populate the subendothelial spaces. •

Where the image does include m a n y cells (Fig. 2) i t is appa ren t t h a t a few are broken open. The contents of these are f rac tu red i r regular ly and seem no t in th is form to give any useful informat ion. F r equen t l y the plane of f rac ture coincides wi th the or ien ta t ion of several sinusoids as though these made the t issue more suscept ible to f rac ture in t h a t plane. Where this is the aspect as in Fig. 3 (stereo) the observer obta ins a good view of the in terna l faces of several sinusoids and the impress ion t ha t those sinusoids, no t in the plane of f racture , are or iented roughly no rma l to those t h a t are.

The Sinusoidal Endothelium and Related Cells. The sinusoid t h a t runs d iagona l ly across the low center of Fig. 3 shows the centra l nuc lea ted pa r t of an endo theha l cell (5-6 ~m in d iameter ) and several p rominen t arms ex tend ing from it. These arms immed ia t e ly give way to smal ler branches and the spaces between those are occupied b y fine nets of p ro top lasmic s t rands . This image wi thout the cell body is r epea ted in o ther sinusoids in the micrograph and seems indeed to be typical . The majo r pa r t of the cell t akes on the form of a net.

This p ic ture of the endothel ia l cell is r epea ted in Fig. 4 wi th grea te r detai l . The nucleus-conta ining pa r t of the cell is a t the left and is t i pped up so as to appea r nea r ly on edge. Here again p rominen t arms reach out f rom the centra l mass in to


Fig. 3. This stereo pair helps to clarify the relationship between liver cells, sinusoids and bile canaliculi. In the sinusoid at X with its branches and perforated margins an endothelial cell

spreads out over the underlying hepatocyte. X 19801

the ve ry thin extensions of the cell. The perfora t ions or fenestrae in the th inner pa r t s of the cell v a r y t r emendous ly in size. Some are ex t r eme ly small and seem to be in clusters. Others are larger (as much as 2 ~m in d iameter) . Through these l a t t e r i t is possible to view the microvil l i on the hepa tocy te surface across the peri- vascu la r Space of Disse. The s t rands of p ro top lasm which t ransec t some of the larger fenest rae are ex t r eme ly s lender and in life mus t represent the more fragile por t ions of the net. I n several places wi th in Figs. 4, 5 and 7 and genera l ly where the fenest rae are the largest i t is appa ren t t ha t the endothe l ia l l ining is double as though one cell overlies another . This m a y represent a kind of double assurance agains t complete b reakdown of this th in sieve-like bar r ie r between the c i rculat ing blood and the per ivascular space.

Lines of fusion between ad jacen t or over lapping endothel ia l cells have not been ident i f ied in these p repara t ions by scanning microscopy even though endothel ia l cells in vitro or in situ have been observed by SEM to show them (Por ter et al., 1973 ; Smi th et al., 1971).

1 The stereo view pictures may be studies using a folding pocket. CF-8-stereoscope, made by Abrams Instrument Corp., Lansing, Michigan and available from Ernest F. Fullam Inc., Schenectady, New York (or with any stereo glasses).

8*


Fig. 4. In this stereo pair the body of an endothelial cell is shown in 3-dimensional view. Large, laminar extensions prominently fenestrated arise from the cell body. Microvilli, present in the subjacent Space of Disse, are evident through larger fenestrations. Small pits and mierovilli

are distributed over the perisinusoidal surfaces of adjacent hepatocytes. • 4400

Small pi ts are present in the surface of the larger a rms and most p rominen t ly in the surface of the centra l mass. They m a y represent openings in to cort ical vesicles which are fa i r ly numerous in these cells (Karnovsky , 1967).

Add i t iona l fea tures of these endothe l ia l cells are dep ic ted in Figs. 6 and 7. These micrographs provide more de ta i led in format ion on the form and size var ia- t ion shown b y the fenestrae. The clustering of the smal ler perfora t ions is par t i - cu lar ly well i l lus t ra ted in Fig. 6. The presence of a double endothel ia l l ayer is pa r t i cu l a r ly well shown in Fig. 7. Whe the r the small p i ts are incipient perfora t ions canno t be suggested b y the images.

Occasionally, as in Figs. 8 and 9, the open con t inu i ty of the sinusoid is inter- r up t ed b y cell extensions. These seem to ex tend from one side of the sinusoid to the o ther in a s tel late fashion and seem to belong to a cell body t h a t is not so i n t ima t e ly in t eg ra ted into the endothe l ium as the cells descr ibed above. Only a t thei r d is ta l ends do the arms of these cells sp lay out to occupy a place in the endo- the l ium (Figs. 9, 10). These are t hough t to be Kupf fe r cells.

Other cells associated with the sinusoids bu t on the t issue side of the endothe- l ium have been inf requent ly encountered. These have re la t ive ly smooth surfaces and slender a rms t h a t p ro jec t away from the cell body and into the spaces be tween the hapa tocy t e s (Fig. 11). Slender " re t icu l in" fibres seem to associate wi th the i r surfaces so we assume t h a t these represent f ibrocytes or poss ibly per icytes of the sinusoids.


Fig. 5. View of another endothelial surface in which there are numerous fenestrations of a size smaller than those in Fig. 4. Microvilli (m) are evident through the larger gaps (arrows)

and on the adjacent surfaces of the hepatocytes. • 12750

The Sur]ace~' o/the Hepatocytes and the Space o/Disse. Transmission microscopy of liver has provided detailed profiles of the microvi]]i that cover the hepatocyte where it faces the sub endothelial Space of Disse. These are short (0.5 ~m or less), of uniform diameter (0.1 ~m) and fairly numerous. In these scanning images these microvilli show through the endothelial perforations (Figs. 4, 5, 7, 8).

I t is also apparent in these images that microvilli project prominently from other surfaces of the hepatocyte. Where for example the Space of Disse extends into the space between adjacent liver cells the microvilli are also present in large numbers (Figs. 1, 4, 6, 8, 9, 11).

The full extent of this expansion on the Space of Disse has not perhaps been fully appreciated from earlier observations on liver ultrastructure. A part of it can be seen in profile in Fig. 1 (arrows mark limits). Here and elsewhere it is evident that the limits coincide with the limits of intimate contact between adjacent hepatocytes, which contacts in turn are the smoothest parts of the cell surface. I t is a zone about 1 ~m wide on either side of the bile canaliculus. Even here within this zone the surface is not absolutely smooth; it is simply the region where the microvilli are relatively short and scarce (Fig. 9). Where, along the Space of Disse side o2 this zone, the hepatocyte begins to round toward the Space and the


Figs. 6 and 7


distance between the cells increases (arrows Fig. t) the microvilli increase in length and number. Obviously these latter extensions contr ibute great ly to the volume of the perivascular space in the liver and great ly enhance the total surface of the liver cell in contact with the blood filtrate. The transit ion between space associated surface with long and more numerous microvilli and the smooth contact zone is part icularly well shown in Figs. 8 and 11.

Obviously the extensions of the Space of Disse m a y occasionally extend through a sheet or cord of parenchymal cells so as to connect adjacent Spaces (as at as- terisk ill Fig. 1). This confirms what one would readily infer, which is t ha t the subendothelial spaces are all par t of a continuous labyr inth of spaces in which the filtrate moves toward the lymphat ic channels.

The surfaces of the hepatocytes, especially along the zone of more int imate contact show numerous small holes or pits. Some of these are about 100 nm in diameter others are smaller and are essentially at the limits of SEM resolution (Figs. 9, 11). Then also there are larger holes which are extensions of the bile canaliculus and will be described more fully in another paper (Motta, in preparation).

The nature of these pits or holes has not been determined. Some could represent the openings in coated pits t ha t are common on the surfaces of liver cells; others may be sites of secretion where a lbumen or lipoproteins are leaving the cell (Bruni and Porter , 1965; Stein and Stein, 1967; Parks, 1967).

Discussion

By scanning electron microscopy the endothelial wall of rat liver sinusoids has been shown to comprise laminar extensions of thin cell bodies. The laminar processes are fenestrated and sometimes overlap to form a double net-like structure. Typical contacts between these endothelial cells have not been noted in these views of the sinusoids even though characteristic contacts between endothelial cells have been observed in other situations (Smith et al., 1971 ; Por ter et al., 1973) and axe described as well in transmission images of thin sections (Wisse, 1970). I t is easier with these SEM images in mind to unders tand the numerous published transmission images which apparent ly show a double layer of cell profiles limiting the sinusoid (Majno, 1965; Carr, 1970). This morphology is curiously similar to t ha t shown by lymphat ic capillaries (Leak and Burke, 1966; 1968).

The number of fenestrations seen over the length of sinusoids appears to be higher than tha t reported from light and TEM studies (Fawcett, 1955; Hampton , 1960; Aterman, 1963; Rouiller and Jezequel, 1963; Wood, 1963; David, 1964; Tanikawa, 1968). The diameter of the openings appears also extremely variable

Fig. 6. Endothelial wall of a sinusoid showing numerous, very small fenestrae arranged in clusters. A large number of microvilli are present (m) on the adjacent surfaces of the hepato-

cytes bordering the Spaces of I)isse. • 6562

Fig. 7. Sinusoidal wall in which overlapping of laminar extensions of endothelial cells is well illustrated. A number of small pits (arrows) and occasional short microvilli (m) are present on the surfaces of the endothelial cells and adjacent hepatocyte surfaces. Through the larger openings microvilli (m) of hepatocytes project into the Space of Disse. Small pits (*) are also

evident on the surface of the liver cell facing the intercellular space. • 12750


Figs, 8 and 9


Fig. 10. Stereo view of a Kupffer cell bulging into the lumen of a sinusoid. Note the rough surface of the cell and the branching arms connected with the endothelial wall. •

wi th measurements r epor ted ranging from 0.01 micron to 2 micra (Roull ier and Jezequel , 1963 ; Ashwor th and Sanders, 1960; Cossel, 1964 ; David , 1964 ; Baronc et al., 1967; Bloom and Fawce t t , 1968; Wisse, 1970; Brooks and Haggis, 1973). I n our observa t ions the larger openings are in m a n y ins tances subdiv ided into several smal ler fenes t ra t ions b y th in connect ing s t rands of cy toplasm. These m a y correspond to the small openings of the sieve pla tes r epor ted by Wisse (1970) and Brooks and Haggis (1973). All the endothe l ia l fenest rae observed are ac tua l holes th rough the cells. No t included in this general izat ion, however, are the few smal l "p i t s " in the cell surface which m a y be inc ip ient perfora t ions or m a y correspond to the occasional p i ts closed b y d i aphragms r epor t ed in some studies (Laschi and Casanova, 1969). The large number of fenes t ra t ions wi th large d iameters , revea led b y SEM, expla ins the resul ts of well known exper iments showing t h a t pa r t i cu la te

Fig. 8. Hepatocyte (E) with bile canaliculi (B) and sinusoids (S). The lumen of one sinusoid is straddled by two arms of a cell at K which is interpreted as a Kupffer cell. The distal ends of the arms seem to blend into the endothelial lining of the sinusoid arrows. (See Fig. 10) Other

cells in the sinusoid (L) are probably leukocytes. • 2500 Fig. 9. Enlargement of a part of Fig. 9. I t shows more clearly the association of the distal end of the Kupffer cell pseudopodium with the endothelial cell of the sinusoid. The various pits in the surface of the hepatocyte, some existing as extensions of the bile canaliculus, are particu-

larly well shown. • 6250


Fig. 11. This stereo view shows the relation between the relatively smooth area lateral to the attachment zones of the bile canaliculi (low center) and the more peripheral areas (basal surfaces) of the hepatocyte covered with numerous microvilli. A perieyte is present in the sinus-

oidal space. • 5400

material injected in the animal very rapidly leaves the blood and enters the Spaces of Disse as well as the liver cells and Knpffer cells (Dogliotti, 1929; Weatherford, 1932; Fawcet t , 1955; Hampton , 1960; Wisse, 1970). Fur thermore the extreme susceptibility of the sinusoidal wall to apparent breakdown under various pathological and drug conditions in which free blood cells are found in the Space of Disse or within the liver cells (Faweett, 1955) is easily understood when one considers the thinness and apparent fragility of the endothelial net.

Wi th the above observations in mind it is obvious tha t the amount , size and shape of the liver sinusoid fenestrations are related to the capabil i ty of the liver to filter portal blood. I t seems probable moreover tha t the number and the diameter of the fenestrations is not static in living animals, but is altered by a monitoring system in the endothelial cells. Such changes would support the assumption tha t " the structure of the hepatic sinusoids varies with functional and nutri t ional state (Kuhn and Oliver, 1965) from one species to another (Wood, 1963) and from one point to another (Burkel and Low, 1965) in the liver lobule" (Carr, 1970). I n addition the intricate branching and especially the overlapping of the cells commonly observed in the endothelial wall, would seemingly enhance the control


possibilities and reduce the chances of blood cells traversing the endothelial barrier. The phagocytic cells of Kupffer and their relation to the sinusoidal wall may also contribute to the barrier function of this layer. However, despite these possibilities, it is difficult to conceive that selective absorption of material by the liver is solely regulated by sinusoidal barriers. On the contrary the liver cell "per se" is probably the most efficient and highly selective filter of material transported by the blood stream.

In the liver a distinct intralobular lymphatic system has not been reported although it is well known physiologically that a large volume of lymph flows from the liver (Brauer, 1963; Scott and Deane, 1966; Bloom and Fawcett, 1968; Ham, 1969). Since the work of Elias (1949), there is agreement in histological literature (Bloom and Fawcett, 1968; Ham, 1969) that the Spaces of Disse should not be considered a true lymph vessel because they lack an endothelial lining. On the other hand the morphological aspects of liver sinusoids as revclaed by SEM clearly demonstrates that the capillary wall (the endothelial net) really delimits two spaces: the lumen of the sinusoids and the Space of Disse. The former indeed represents the lumen of a "virtual blood capillary" because it is fenestrated and lacks a basement membrane. The Space of Dissc, on the other hand, could be regarded as a "virtual lymphatic lacuna" because it lacks a complete endothelial lining. Thus, there is reason to consider the Spaces of Disse as special lymphatic lacunae in which the lymph, or in this case the blood filtrate, flows from the central part to the periphery of the lobulc in the portal spaces (Brauer, 1963 ; Bloom and Fawcett, 1968).

In this regard it is also necessary to note tha t the SEM micrographs show the Spaces of Disse to be larger than one would gather from TEM pictures. In fact they extend among adjacent hepatocytes in numerous recesses limited by hepatocyte surfaces covered with microvilli. Considering that normally the liver cells have six or more sides and that half of these face adjacent hepatocytes and bile canaliculi (Bruni and Porter, 1965) it is rather logical to conclude that the total liver cell surface in contact with the blood filtrate is enormous. Actually the pcri- sinusoidal Spaces of Disse extend not infrequently into a labyrinthine system of intercellular channels. These channels, earlier recognized by Steiner (1961), are limited by the junctional complexes of the bile canaliculi but extend elsewhere in all directions clearly to connect adjacent subendothelial Spaces of Disse.

Therefore the topography of the bile canaliculi, sinusoids and Spaces of Disse permits one to distinguish in the hepatocyte three different faces. Some faces front the sinusoidal wall, some the bile canaliculi and others intercellular spaces. All of these surfaces are provided with different numbers and sizes of microvilli. The surfaces of the liver cells facing directly the sinusoidal wall may have a different functional significance from the surfaces facing the intercellular spaces. I t is possible that the former, because of the blood flow, serve mostly as absorbing surfaces and the latter, not so directly exposed to the blood stream, as "endocrine" secreting surfaces. There is in fact some evidence that the numerous pits or holes present on the lateral surfaces of the hepatocytes may be the sites of secretion of albumen of lipoprotein leaving the cell (Bruni and Porter, 1965; Parks, 1967; Stein and Stein, 1967). The demonstration of this assumption requires of course further experimental studies.


One final point to be considered concerns the na ture of the sinusoidal wall and the Kupffer cells. Some investigators describe the sinusoid as bounded only by one type of cells which assume various aspects with different funct ional states (Steiner, 1961; David, 1964; Barone et al., 1967; Nicolescu and Rouiller, 1967). According to other investigators, however, the endothelial cells do not t ransform into Kupffer cells and tha t these therefore represent an independent type of cell (Carsten, 1961; Wisse, 1972).

The results from the present s tudy do not contr ibute much to the solution of this problem. Nevertheless, if the irregularly shaped cells with the tex tured surfaces, which are observed in the sinusoids, correspond to Kupffer cells they mus t be regarded as morphologically different at the t ime of f ixat ion from the th in fenestrated endothelial cells. Considering the s tructure of the sinusoidal wall, one can readily imagine tha t Kupffer cells, as macrophages, could move quite freely through or among the endothelial cells and thus adopt a var ie ty of postural relationships to these la t ter cells and even part icipate in l imit ing the sinusoid.

Re~erenees

Aterman, K.: The structure of the liver sinusoids and the sinusoidal cell. In: The liver, vol. I (Ch. Rouiller, ed.), p. 61-136. New York: Academic Press 1963

Barone, P., Inferrera, C., Carrozza, G.: Aspetti ultrastrutturali dell' epatocita in rapporto alla secrezione biliare ed alle sue alterazioni. Relaz. Atti X Congr. Soc. Ital. Patologia, Messina-Taormina, p. 49-211 (1967)

Bloom, W., Fawcett, D. W.: A textbook of histology, 9th ed. Philadelphia: W. B. Saunders Co. 1968

Brauer, R. W.: Liver circulation and function. Physiol. Rev. 43, 115-213 (1963) Brooks, S.E.H., Haggis, G. H.: Scanning electron microscopy of rat's liver. Application of

freeze-fracture and freeze-drying techniques. Lab. Invest. 29, 60-64 (1973) Bruni, C., Porter, K. R.: The fine structure of the parenchymal cell of the normal rat liver.

I. General observations. Amer. J. Path. 46, 691-755 (1965) Burkel, W. E., Low, F. N.: The fine structure of rat liver sinusoids, Spaces of Disse and asso-

ciated tissue space. Amer. J. Anat. 11, 769-784 (1966) Carr, I.: The fine structure of the mammalian lymphoreticular system. Int. Rev. Cytol. 27,

283-338 (1970) Carsten, P. M.: Elektronenmikroskopische Untersuehungen an der Sinusoidwand mensch-

licher fetaler Lebern. Z. Zellforsch. 54, 252-261 (1961) Cossel, L.: Die menschliche Leber im Elektronenmikroskop. Jena: Gustav Fischer 1964 David, H.: Submikroskopische Ortho- und Pathomorphologie der Leber. Berlin: Akademie

Verlag 1964 Dogliotti, G. C.: Ricerche sull' assunzione di trypanblau da parte dellc cellule epatiche nei

ratti albini. Monit. zool. ital. 4@, 100-106 (1929) Elias, H.: A re-examination of the structure of the mammalian liver. II. The hepatic lobule

and its relation to the vascular and biliary systems. Amer. J. Anat. 85, 379465 (1949) Fawcett, D.W.: Observations on the cytology and electron microscopy of hepatic cells.

J. nat. Cancer Inst. 15, 1475-1502 (1955) Ham, A. W.: Histology, 6th ed. Philadelphia: J. B. Lippincott Co. 1969 Hampton, J.: A re-evahiation of the submicroscopic structure of liver. Tex. Rep. Biol. Med. 18,

602-611 (1960) Ito, T., :Nemoto, M.: ?~ber die Kupfferschen Sternzellen und die ,,Fettspeicherungszellen" in

tier Blutkapillarenwand der menschlichen Leber. Folia anat. jap. 24, 243-258 (1952) Jezequel, A. M., Orlandi, F.: Fine morphology of the human liver as a tool in clinical pharma-

cology. In: Liver and drugs (F. Orlandi and A. M. Jezequel, eds.), p. 145-188. New York: Academic Press 1972


Karnovsky, M. S.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967)

Kuhn, N., Olivier, M. L.: Ultrastructure of the hepatic sinusoid of the goat, Capra hircus. J. Cell Biol. 26, 977-979 (1965)

Laschi, R., Casanova, S.: Fenestrae closed by a diaphragm in the endothelium of liver sinusoids. J. Mierosc. 8, 1037-1040 (1969)

Leak, L. V., Burke, J. F.: Fine structure of lymphatic capillary and adjoining connective tissue area. Amer. J. Anat. llS, 785-810 {1966)

Leak, L.V., Burke, J. F.: Ultrastructural studies on the lymphatic anchoring filaments. J. Cell Biol. 36, 129-149 (1968)

Majno, G.: Ultrastructure of the vascular membrane. In: Handbook of physiology, vol. III/2 (W. F. Hamilton and P. Dow, eds.), p. 2293-2375. Baltimore: Williams-Wilkins Co. 1965

Nicolescu, P., Rouiller, C.: Beziehungen zwischen den Endothelzellen der Lebersinusoide und den yon Kupfferschen Sternzellen. Elektronenmikroskopisehe Untersuchung. Z. Zellforsch. 76, 313-338 (1967)

Novikoff, A., Essner, E.: The liver cell. Some new approaches to its study. Amer. J. Med. 29, 102-131 (1960)

Parks, H. F.: An experimental study of microscopic and submicroscopic lipid inclusions in hepatic cells of the mouse. Amer. J. Anat. 120, 253-280 (1967)

Porter, K. R., Kelley, D., Andrews, P. M.: The preparation of cultured cells and soft tissues for scanning electron microscopy. In: Proceedings of the 5th Annual Stereoscan Collo- quium, p. 1-19. Chicago: Kent Cambridge Scientific Co. 1972

Porter, K. R., Todaro, G.J. , Fonte, V.: A scanning electron microscope study of surface features of viral and spontaneous transformants of mouse balb/3T3 cells. J. Cell Biol. (1973) (in press)

Rouiller, C., Jezequel, A. M.: Electron microscopy of the liver. In: The liver, vol. I (Ch. Rouiller, ed.), p. 195-264. New York: Academic Press 1963

Sabatini, D. D., Bensch, K. G., Barrnett, R. J. : Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58 (1963)

Scott, J. F., Dearie, H. W.: Liver and gallbladder. In: Histology, 2nd ed. (R. 0. Greep, ed.), p. 531-553. New York: McGraw-Hill Book Co. 1966

Smith, U., Michie, D. D., Ryan, J. W., Smith, D. S.: Endothelial projections as revealed by scanning electron microscopy. Science. 173, 925 927 (1971)

Stein, 0., Stein, Y.: Lipid synthesis, intracellular transport, storage and secretion. I. Electron microscopic radioautographic study of liver after injection of tritiated palmitate or glycerol fasted and ethanol-treated rats. J. Cell Biol. 33, 319-339 (1967)

Steiner, J. W.: Investigations of allergic liver injury. I. Light, fluorescent and electron microscopic study of the effects of soluble immune aggregates. Amer. J. Path. 38, 411436 (1961)

Tanikawa, K.: Ultrastructural aspects of the liver and its disorders. Berlin-Heidelberg-New York: Springer 1968

Wassermann, F.: The structure of the wall of the hepatic sinusoids in the electron microscope. Z. Zellforsch. 49, 13-32 (1958)

Weatherford, H. W.: The Golgi apparatus and vital staining of the amphibian and reptilian liver. Z. Zellforsch. 15, 343 373 (1932)

Wisse, E.: An electron microscopic study of the fenestrated endothelium lining of rat liver sinusoids. J. Ultrastruct. Res. 31, 125-150 (1970)

Wisse, E.: An ultrastructural characterization of the endothelial cell in the rat sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells. J. Ultrastruct. Res. 38, 528-562 (1972)

Prof. Dr. Pietro Motta Istituto di Anatomia Umana Normale Universits di Roma Viale Regina Elena 289 1-00161 Roma Italia

Documents

Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy