J. Electron Microsc, Vol. 32, No.

1, 33-44, 1983

Tridimensional Architecture of Elastic Tissue in the Rat Aorta and Femoral ArteryA Scanning Electron Microscope Study Kojiro
WASANO

and Torao

YAMAMOTO

Department of Anatomy, Faculty of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812 Japan (Received January 6, 1983; accepted March 10, 1983) Overall tridimensional architecture of the elastic tissue in the rat aorta (elastic type artery) and femoral artery (muscular type artery) has been studied by scanning electron microscopy after hot-formic acid extraction followed by freeze-drying method. In the aorta the elastic tissue is composed to 6-7 concentric plate-like laminae interconnected by radially oriented interlaminar elastic fibers, whereas in the femoral artery it is composed of two distinct inner and outer sheet-like laminae bridged by a dense continuous interlaminar network of elastic fibers. The internal elastic lamina has numerous fenestrations which considerably differ in size, shape and structure between the two types of arteries. Tunnel-like compartments free of elastic tissue extend helically into the medial wall of both types of arteries. These findings are discussed in relation to conventional transmission electron microscopic information and some new functional roles of arterial elastic tissue are proposed. Key words = scanning electron microscopy (SEM): elastic tissue: aorta and femoral artery: rat: formic acid INTRODUCTION
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Elastin is the major structural component of the medial connective tissue at various levels of arteries. Measurements of the elasticity and tensile strength of whole arterial tissue1"41 and of pure elastic tissue isolated from the arterial tissue 5 " 8 ' have indicated that the elastic tissue is involved in the static and dynamic mechanical properties of arterial walls. For a better understanding of its mechanical behavior in response to external stresses applied to arterial walls, it seems essential to know the overall tridimensional architecture of the elastic tissue near its in vivo form. Conventional light and transmission electron microscopy (LM and TEM) are of little use for this purpose, since these methods permit no estimation of the spatial relationships of such an intricate structure due to the thinness of the sections. Scanning electron microscopy (SEM), however, is eminently suited for visualizing tridimensional images, since it

provides a much larger focal depth than LM and TEM. To visualize arterial elastic tissue under SEM, it is necessary to remove selectively other tissue components in arterial walls, including collagen fibers, ground substances, smooth muscle cells and other cell components. Several techniques have been developed for isolating pure elastin in past studies.9"14' Among these techniques, hot-formic acid extraction method9' was used in this study, since the method has several advantages in the following points. (1) It has been shown that perfusion-fixation of arterial tissues at a physiological pressure is necessary to preserve the structural organization of the elastic tissue near its in vivo condition.3' Only hot-formic acid can be used for the isolation of elastic tissue from fixed tissue materials.15' (2) It is a single-step extraction method which can dissolve all other tissue components in arterial walls except the elastic tissue.7'9' This can

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minimize mechanical damages on the delicate network of the isolated elastic tissue which may be unavoidable in other multi-step extraction techniques. (3) The structural integrity of elastic tissue has been shown to be preserved during long extraction periods up to 400 hr.7'9' This slow extraction rate permits the delicate regulation of extraction duration necessary for the degradation of only nonelastic tissue components. Removal of water from the isolated elastic tissue is the second critical step necessary for SEM examination, since the structural integrity as well as the mechanical property of elastin is known to largely depend on its water content.16' In the present study, freezedrying method was used, instead of critical point drying method, to avoid distortion and shrinkage artefacts due to the interaction between purified elastin molecule and organic solvents during dehydration process.17' Using these techniques, the present study demonstrates the overall architecture of the elastic tissue in two different types of arteries of the rat.
MATERIALS AND METHODS

Adult male WKA rats, four months of age and weighing about 250 g, were used. All the animals were sacrificed by intraperitoneal injection of pentobarbital (30 mg/kg) and perfused from the left ventricle with fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at a constant pressure (120 mmHg) for about 20 min. After the perfusion fixation, the segments of the abdominal aorta between the levels of the renal and ileolumbar arteries, and the femoral arteries between the branching points of the superficial circumflex iliac and superficial epigastric arteries were dissected out and immersed in the same fixative for 24 hr at 4C. After careful removal of extraneous adventitial tissue with a fine forceps under a biocular microscope, the tissue specimens were processed for subsequent transmission and

scanning electron microscopic preparations. Transmission electron microscopy. To examine the general structure of intact arterial walls, some segments of the aorta and femoral artery were cut into small rings and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 hr. To determine the least extraction period, the segments of the aorta and femoral artery were cut transversely with razor blades into two hundred cylindrical rings of about 1 mm in length and divided into ten groups respectively. Each group was separately incubated in glass-stoppered vessels containing 5 ml of 88% formic acid at 45C for various periods of time of 24, 36, 48, 60, 72, 84, 96, 120, 168 and 216 hr. In proportion to the incubation time, the arterial rings become transparent and swollen. After the incubation, five rings were washed in 0 . 1 M phosphate buffer (pH 7.4) and immersed in fixative solution containing 3% glutaraldehyde and 2% tannic acid in the same buffer for 30 min. The other fifteen rings were processed for subsequent scanning electron microscopic preparations. After a brief rinse in 0.1 M phosphate buffer (pH 7.4), the tissue specimens were postfixed in 1 % osmium tetroxide in the same buffer for 1 hr, dehydrated in graded ethanols and embedded in Epoxy resin. Ultrathin sections were stained with 2% uranyl acetate in 50% ethanol and lead hydroxide with or without prior staining in 2% aqueous tannic acid solution, filtrated through a Sartorius membrane filter of 5 nm pore (Zeiss, West Germany) before use, for 15 min and examined in a Hitachi Hu-I2A transmission electron microscope. Scanning electron microscopy. The isolated elastic tissue were carefully washed in several changes of 0.002 N HC1 until they returned to their original dimensions according to the method of Kuhn,15) since washing in neutral buffer results in a shrinkage of the tissue to a considerable extent. The tissue specimens were rapidly frozen in liquid nitrogen and freeze-dried in a JEE-5S vacuum evaporator

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Tridimensional Architecture of Arterial Elastic Tissue

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Fig. 1. (A) A cross-sectional view of an intact aortic wall. The elastic tissue (stained black with tannic acid) is composed of 6-7 concentric elastic laminae and fragmental cords of interlaminar elastins. The internal and external elastic laminae are thinner and more discontinuous than the other medial elastic laminae between them. L: vascular lumen, A: adventitia. (B) A transmission electron micrograph showing the structure of an aortic wall incubated in hot-formic acid for 96 hr. All the structural components of the vascular wall except the elastic tissue (stained black with tannic acid) are completely removed. L: vascular lumen, A: adventitia. x2,100, Scale bar=5 /im.

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under a vacuum of 10 5 Torr in the presence of P2O6 as a water trap. The dried specimens were carefully affixed on aluminum stubs with double-sided sticky tape, coated with about 100A thick gold-palladium alloy in an Eiko IB-5 ion sputter coater and examined in a Hitachi S-430 scanning electron microscope. Stereo-pair photographs were taken at 7 tilt angles.
RESULTS

Transmission electron microscopy In thin sections routinely stained with uranyl acetate and lead hydroxide, the discernment of the amorphous elastin from other extracellular tissue components is difficult, since it remains unstained with these two metal salts as an electron-lucent homogeneous structure. Prior staining with tannic acid, however, considerably enhances its electron-density, making it possible to survey the overall morphology of the elastic tissue easily. Thus, the following findings were obtained from thin sections stained with tannic acid, uranyl acetate and lead. General structure of intact arterial walls. In the aorta, the elastic tissue is composed of 6-7 concentric laminae oriented in roughly parallel direction (Fig. 1A). Each lamina is 1-2.5 /*m thick and disposed 5-20 /<m apart. The most

internal and external elastic laminae (IEL and EEL) are thinner and have much larger fenestrations, up to 200 ,um in width, than the other elastic laminae that appear as almost thick continuous sheets (Figs. 1A and 3B). Through the fenestrations collagen fiber bundles pass. Between these laminae fine branching elastic fibers occur intertwined with medial smooth muscle cells and randomly oriented collagen fiber bundles (Fig. 1A). In an adequate plane of section, some interlaminar elastic fibers can be traced for long distance, arising from the surface of the laminae and extending into the interlaminar space. Most of the medial smooth muscle cells are arranged obliquely to the plane of section, but in the outermost one to two layers transversely cut profiles of smooth muscle cells occur (Fig. 1A). The elastic tissue in the femoral artery is poorly developed as compared with that in the aorta and is basically composed of two distinct 1EL and EEL (Fig. 6A). The IEL is about 2 fim thick and has numerous small gaps of about 1-1.5 /xm in width (Fig. 8B). Endothelial cells frequently give rise to slender processes through the gaps to form myoendothelial junctions with the innermost smooth muscle cells (Fig. 8B). The EEL is about 1 /<m thick and has small gaps of about 3 ^m in width, through which collagen fiber

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Fig. 2. (A) Low power stereo scanning electron micrographs showing the overall tridimensional architecture of the elastic tissue in the formic acid-treated aorta. The elastic tissue consists of 6-7 concentric plate-like laminae interconnected with each other by radially oriented fibrous or fenestrated septum-like elastins. Note that tunnel-like compartments, surrounded by the elastic tissue, extend obliquely into the vascular wall from right to left direction. L: vascular lumen, A: adventitia. (B) Higher magnification view of the luminal half of Fig. 2A. The internal elastic lamina (small arrows) is thinner than the other medial elastic laminae and has large fenestrations (large arrow) irregularly crossed by fine elastic fibers. (C) Close up view of the adventitial half of Fig. 2A. The external elastic lamina (asterisk) does not show continuous sheetlike structures, but consists of fenestrated disk-like plates giving off numerous elastic fibers from their margins. Only sparse fine elastic fibers (arrowheads) interconnect the external elastic lamina and the adjacent medial elastic lamina. (A) x400, Scale bar= 20 /<m, (B, C) x 1,100, Scale bar=10//m.

Tridimensional Architecture of Arterial Elastic Tissue

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bundles pass. The medial wall between the two elastic laminae is predominantly occupied by closely packed smooth muscle cell layers with isolated fragmental elastic fibers (Fig. 6A). Most of the smooth muscle cells are arranged obliquely to the plane of section

except the outer few smooth muscle cell layers running in parallel direction to the longitudinal axis of the artery (Fig. 6A). In both types of arteries, the adventitial elastic tissues show similar organization, consisting of sparsely disposed elastic fibers.

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K. WASANO and T. YAMAMOTO

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Fig. 3. (A) A scanning electron micrograph showing the luminal surface of the aortic internal elastic lamina. The internal elastic lamina has numerous large fenestrations irregularly crossed by fine branching anastomosing elastic fibers. (B) A transmission electron microscopic image of the aortic internal elastic lamina (IEL). The internal elastic lamina has large fenestrations interrupted by only dot-like or fibrous elastins. Formation of myoendothelial junctions through the fenestrations can hardly be seen. E: endothelial cells, S: medial smooth muscle cell, L: vascular lumen. (A) X880, Scale bar = 10 //m, (B) x 4,200, Scale bar = 2 /im. Fig. 4. A scanning electron micrograph showing the adventitial half of the aortic elastic tissue viewed from oblique adventitial direction. The medial elastic laminae have small round fenestrations (arrows) with various sizes, asterisk: external elastic lamina, x 760, Scale bar = 10 //m. Fig. 5. A scanning electron micrograph showing the adventitial surface of the aortic elastic tissue. The adventitial elastic tissue consists of a randomly tangled network of fine elastic fibers giving off branches that are directly joined with the surface of the subjacent external elastic lamina (asterisk), x 1,000, Scale bar= 10 pm.

Tridimensional Architecture of Arterial Elastic Tissue

IS

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Fig. 6. (A) A cross-sectional view of the wall of an intact femoral artery. The elastic tissue (stained black with tannic acid) is poorly developed as compared with that in the aorta shown in Fig. 1A. Jn this type of artery, however, the internal elastic lamina is remarkably developed and appears as an almost thick continuous sheet. The medial wall is predominantly occupied by closely packed smooth muscle cells layers interspersed with isolated fragments of interlaminar elastins. L: vascular lumen, A: adventitia. (B) A transmission electron micrograph showing the structure of the wall of a femoral artery treated in hot-formic acid for 72 hr. All the non-elastic tissue components are completely digested. L: vascular lumen, A: adventitia. x2,100, Scale bar=5 fim.

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They are closely associated with long slender processes of adventitial fibrocytes that are widely separated by wavy thick adventitial collagen fiber bundles (Figs. 1A and 6A). Structure of formic acid-treated arterial walls. In both types of arteries, the amorphous ground substances and all the cell components were completely dissolved after initial 36 hr of extraction in hot-formic acid. Collagen fibers are the most resistant component against formic acid and persist for a longer time than the other non-elastic tissue components. The persistence appears to vary with the thickness of the arterial wall and the compactness of its structure. In the femoral artery all the collagen fibers disappeared between 60 and 72 hr (Fig. 6B), while in the aorta 84 to 96 hr of extraction was necessary for complete removal of the collagen fibers (Fig. 1B). No detectable change of the organization of the elastic tissue occurred for further extraction period until the end of 120hr, but more prolonged extraction resulted in the partial enlargement

of the spaces between adjacent elastic laminae probably due to the degradation of fine elastic fibers interconnecting them. The elastic tissue, however, fully retained its affinity for tannic acid and appeared homogeneous or almost amorphous in texture after prolonged extraction time up to 216 hr. Scanning electron microscopy The preliminary TEM study has revealed that the least extraction time is 72 and 96 hr in the femoral artery and aorta respectively. Thus, the following SEM observations were made on these materials. The removal of non-elastic tissue components with formic acid treatment unveiled with great clarity the structure of the arterial elastic tissue. The morphology of the elastic tissue is considerably different between the aorta and femoral artery. In the aorta, the elastic tissue is composed of 6-7 concentric laminae interconnected with each other by interlaminar elastic fibers (Fig.

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Fig. 7. Stereo scanning electron micrographs showing the tridimensional architecture of the elastic tissue in the formic acid-treated femoral artery. The elastic tissue is composed of two distinct internal and external elastic laminae interconnected with each other by a dense continuous network of branching anastomosing elastic fibers. Note that tunnel-like compartments extend obliquely into the arterial wall from left to right direction. The external elastic lamina splits into double layers, surrounding an elastinpoor compartment (asterisk) which extends into the arterial wall in parallel to its longitudinal axis. L: vascular lumen, A: adventitia. x 1,050, Scale bar= 10 //m. Fig. 8. (A) A scanning electron micrograph showing the luminal surface of the internal elastic lamina of the femoral artery. The internal elastic lamina has numerous small round fenestrations traversed by a few crosscut elastic fibers. (B) A transmission electron microscopic image of the internal elastic lamina (IEL) of the femoral artery. The internal elastic lamina has simple narrow gaps through which endothelial cells (E) frequently give rise to slender processes to form myoendothelial junctions with the underlying medial smooth muscle cells (S). L: vascular lumen. (A) x87O, Scale bar=10/<m, (B) x4,200, Scale bar = 2 //m. Fig. 9. A scanning electron micrograph showing the adventitial surface of the elastic tissue in the femoral artery. The adventitial elastic tissue consists of sparse curly elastic fiber network through which the subjacent external elastic lamina (asterisk) can easily be seen. The adventitial elastic fibers are evidently joined with the surface of the external elastic lamina. x900, Scale bar= 10 /<m.

Tridimensional Architecture of Arterial Elastic Tissue

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2A). The laminae appear as solid sheet-like structures, about 1-2.5 jum in thickness, that are arranged in almost parallel, although somewhat irregularly undulated, at intervals of about 5-20 fim (Fig. 2A, B and C). The IEL has numerous large oval fenestrations of varying diameter from 50 up to 200 /xm (Figs. 2B and 3A). The fenestrations are irregularly crossed by fine branching anastomosing elastic

fibers, being divided into small pores with various sizes and shapes (Fig. 3A). The fenestrations are randomly distributed, with their long axes running in different directions, indicating that they have no special relationship to other non-elastic components, especially endothelial cells which closely covered the surface of the IEL before the extraction. The EEL does not form a continuous sheet, but
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consists of extensively fenestrated disk-like plates and branching anastomosing network of elastic fibers arising from their margins (Figs. 2C, 4 and 5). The medial elastic laminae, although having round fenestrations of about 5-10 fim in diameter, appear as almost thick continuous sheets (Figs. 2B, C and 4). All these elastic laminae are interconnected with each other by radially oriented interlaminar fibrous or fenestrated septum-like elastins. They are often arranged in parallel rows just like roadside trees, resulting in the formation of tunnel-like compartments free of elastic tissue which extend into the aortic wall in the same helical direction (Fig. 2A, B and C). In the outermost interlaminar space, however, only sparse fine elastic fibers bridge between the adjacent laminae without making obvious compartments as mentioned above (Fig. 2C). The adventitial elastic tissue is composed of a complicated network of randomly tangled fine elastic fibers of about 1 fim in diameter. There fibers are evidently linked with the disklike plates or fibrous elastins constituting the aortic EEL (Figs. 2C, 4 and 5). In the femoral artery, the elastic tissue is basically composed of two distinct 1EL and EEL that are interconnected with each other by a complicated interlaminar elastic fiber network (Fig. 7). The IEL appears as a slightly undulated thick plate-like structure, about 2 fim in thickness, and has numerous small round fenestrations, varying in size from 1 to 5 fim (Fig. 8A). The fenestrations are uniformly distributed throughout the IEL and are sometimes divided by a few crosscut elastic fibers into a few partitions. The EEL shows a slightly undulated sheet-like structure, about 1 fim in thickness, having numerous fenestrations similar to those seen in the IEL (Figs. 7 and 9). In some places, the EEL is split into double layers, surrounding an elastin-poor compartment which extends into the arterial wall parallel to its longitudinal axis (Fig. 7). Although the IEL and EEL are disposed in an almost parallel fashion, about 30-35 fim apart, their undulation phases are

not necessarily synchronized with each other (Fig. 7). Interconnecting these two laminae are a dense continuous network of branching anastomosing elastic fibers, in which incomplete fragmental elastic laminae occasionally occur (Fig. 7). Tunnel-like compartments, although not as clearly seen as those in the aorta, extend into the arterial wall in the same helical direction (Fig. 7). The adventitial elastic tissue is poorly developed as compared with that in the aorta and consists of a sparse network of fine curly elastic fibers, through which the subjacent EEL can be easily seen (Fig. 9). The fibers are clearly joined with the surface of the EEL.
DISCUSSION

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The overall tridimensional architecture of the elastic tissue in the rat aorta (elastic type artery) and femoral artery (muscular type artery) has been studied by scanning electron microscopy after hot-formic acid extraction followed by freeze-drying method. The general organization of the elastic tissue was not considerably altered by the formic acid treatment, as confirmed by the comparison on thin sections from intact and extracted tissues. Previous TEM studies18'101 have suggested that the arterial elastic tissue may have a continuous network throughout the vascular walls. This suggestion remains speculative, however, since one can hardly know the overall architecture of such a spatially intricate structure as the elastic tissue only by a two-dimensional plane of section in which it often appears in the form of isolated fragments. The present SEM study has clearly demonstrated that the arterial elastic tissue actually makes a completely continuous network throughout the vascular walls from its intimal to adventitial element, regardless of the considerable differences in its overall structural organization between the two distinct types of arteries examined. This finding strongly supports the idea13'191 that such a completely continuous network of the arterial elastic tissue, surround-

Tridimensional Architecture of Arterial Elastic Tissue

43

ing compartments in which medial smooth leaving it entirely electron-lucent, while readily muscle cells and collagen fibers are enclosed, filling the extracellular matrix of the vascular can function as a unit, distributing the intra- media after passing through the fenestrations luminar distension pressure uniformly around of the IEL, it might be expected that the the whole circumference of the vascular walls arterial elastic laminae act as a diffusion and thereby counterbalancing the large pulsatile barrier to some extent large molecules probaintraluminar pressure of the vessels. bly due to the inner compact structure. If By tridimensional observations, tunnel-like this speculation is correct, the second intercompartments, extending into the vascular pretation seems more likely and may well walls in the same helical direction, have for the explain the structural diversity of the IELs first time been demonstrated clearly in the between the aorta and femoral artery, since aorta and less clearly in the femoral artery. the former has a thicker wall as well as a much When correlating the SEM images with TEM more compact medial elastic tissue than the micrographs in which the elastic tissue often latter. seems to encircle the individual smooth muscle Ayer et al.7) examined the light microscopic cell, it is obvious that these compartments appearance of the elastic tissue in the dog probably correspond to the burrows of the aorta after hot-formic acid extraction and medial smooth muscle cells. This finding reported that the elastic laminae are not solid indicates that the medial smooth muscle cells sheet-like structures, but are composed of are aligned in parallel to one another and are multiple fine elastic bands which run in arranged helically around the arterial walls. different directions without order and repeatedThe internal elastic laminae have numerous ly split and fuse with each other, resulting in fenestrations which are considerably different the formation of tightly weaved mesh-like in size, shape and structure between the two elastic laminae. A similar fibrous substructypes of arteries examined. These structures ture of aortic elastic laminae has been demondo not seem to be artificial products, since strated by Hart et al.n) who examined the the correlative TEM examinations of intact SEM appearance of the elastic tissue in the tissues have also shown the presence of com- young swine aorta using guanidine-NaOH parable large fenestrations, interrupted only by extraction technique. According to these dot-like elastins, in the aortic IEL and simple authors, the aortic elastic laminae consist of small fenestrations in the IEL of the femoral completely fibrous elastins running alternately artery. This finding raises the question of in different directions in successive laminae. whether these fenestrations have any signifi- In the present study no such fibrous subcant functional meaning and, if so, what func- strcture could be demonstrated in the rat tional role(s) they play. One interpretation aortic elastic laminae. It is not possible at is that the fenestrations are the pathways present to determine whether these structural through which cellular elements and/or inter- differences between the aortic elastic laminae cellular connective tissue components can pass of various animals examined to date is atto form cell to cell junction and/or to establish tributed to the interspecies variations, difdirect connections between the connective ferent developmental stages of animals extissues in adjacent compartments. They may amined or different extraction procedures used. also serve as pathways through which nutrient In view of the fact, however, that Carnes et substances can diffuse from the vascular al.14) have demonstrated, using the same exlumen into the avascular media. Considering traction technique as used by Hart et al.,22) the previous TEM findings20-211 that an elec- that the aortic elastic laminae of the human tron-dense tracer (horseradish peroxidase) adult consist of solid sheet-like structure which cannot penetrate into the arterial elastin matrix is very similar to those of the rat described in

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this study, it might be proposed that the generalization about structural details of the arterial elastic tissue cannot be made without regard for its animal source rather than the extraction technique employed.
Acknowledgment. This work was supported by Grants (No. 107003, No. 57770022) from the Ministry of Education, Science and Culture, Japan.

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