463

Atherosclerosis, 28 (1977) 463-470 0 Elsevier/North-Holland Scientific

Publishers,

Ltd.

A SCANNING ELECTRON MICROSCOPIC STUDY OF ARTERIAL ENDOTHELIAL CELLS USING VASCULAR CASTS

M.A. REIDY * and M.J. LEVESQUE Department of Biophysics, Ont. N6A 5Cl (Canada)

Health Sciences Centre,

(Received 7 July, 1977) (Revised, received 12 September, (Accepted 14 September, 1977)

University

of Western Ontario, London,

1977)

summary Vascular casts were made of rabbit aortas by infusing Batson’s No. 17 anatomical corrosion compound into the artery at physiological pressure. The arterial tissue was then digested with sodium hydroxide and the cast viewed by scanning electron microscopy (SEM). Outlines of the endothelial cells and their silver stained boundaries were clearly visible. Cell nuclei and fine surface detail were also discernible. In EDTA damaged arteries, injured endothelial cells and platelets could also be observed in the vascular casts. Key words:

Endothelium

-Scanning

electron

microscopy

- Vascular casts

Introduction In recent years considerable attention has been focused on arterial endothelial cells since it has been shown that injury to this layer of cells may initiate the development of atherosclerosis [l-3]. Due to the location of these cells on the luminal surface of vessels, the endothelium can be studied with en face techniques and much information regarding cellular integrity and morphology has been gained using Hautchen [4,5] or whole thickness preparations [6]. More recently the scanning electron microscope (SEM) has been employed in morphological studies of arterial endothelium. Although this instrument has the advantage of high resolution and the ability to scan large areas, some doubt * Fellow of the Ontario Heart Foundation. Supported by Grant T3-14 from Ontario Heart Foundation awarded to Dr. Margot R. Roach.

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has been cast on the usefulness of these studies because of artifact formation. Recent advances in the fixation [ 7,8], staining [ 7,9] and drying [ 19,111 of arterial tissue for SEM, however, have greatly improved this technique for assessing endothelial morphology, although there is still some debate about both the choice of fixative and the most suitable drying procedure [ 121. In this study we report a method for preparing arterial endothelium for observation by SEM which overcomes many of the above problems. This involves making a plastic cast of the artery and then observing by SEM the impressions of the cellular features made in the cast surface. By this technique it is possible to observe the surface features of endothelial cells without fixing or drying the vessels and since there is no need to cut open the artery to expose the luminal surface, then the true in vivo dimensions should be preserved. Methods and materials Experimental design Six New Zealand White rabbits, aged 12-14 weeks, were used in this study and were subjected to the following procedure: Group 1: 6 animals were killed by an overdose of Nembutal and a plastic cast was made of the aortas as described below. In 2 animals the silver staining was omitted. Group 2: 2 animals were anaesthetised with Nembutal (i.v., 3 mg/kg body wt.) and their abdominal aortas exposed through a midline incision. The left femoral artery was cannulated, the aorta clamped just below the renals and blood was washed out of the abdominal aorta by infusing isotonic Tyrode’s solution (pH 7.4) into the vessel through the femoral catheter. The right femoral artery was immediately clamped and a 0.1% solution of ethylenediamine tetraacetate (EDTA) in saline was introduced into the abdominal aorta via the left femoral cannula for approximately 90 sec. The 2 clamps were then removed and the blood was allowed to flow through the abdominal aorta for 90-120 sec. The animals were then killed and a plastic cast taken of their aortas. Casting of arteries Each animal was heparinised (100 U/kg body wt.) and then killed with an excess of Nembutal. The rib cage was opened, the upper thoracic aorta exposed and a femoral artery transected to allow the blood to drain. A plastic cannula (0.4 cm diameter) was inserted and secured into the thoracic aorta through which isotonic Tyrode’s solution (pH 7.4) was introduced to wash out the blood. A solution of silver nitrate (0.25%) was introduced into the aorta for 30 set which was then washed out with more Tyrode’s solution. Batson’s No. 17 plastic diluted with methyl methacrylate was next infused into the vessel at a constant pressure of 110 mm Hg as shown in Fig. 1. All the solutions were held in reservoirs placed above the animal and were connected to the cannula by means of a 3-way tap. After the plastic had completely set, the aorta was excised from the animal and placed in a solution of approximately 7 M sodium hydroxide for at least 3 days or until the aortic tissue was completely digested. Occasionally a white

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Pressure

head

-

Fig. 1. Apparatus

used for infusion

of Batson’s

3-way

tap

into rabbit aorta.

residue remained on the plastic after this procedure and in these cases the cast was soaked in dilute hydrochloric acid for lo-12 h. The cast was then washed in distilled water and allowed to dry. Segments of the material were mounted onto a SEM stub with Epoxy resin and high conductivity silver paint and were coated with gold in a Technics Sputtering unit. Specimens were viewed in a Hitachi HHS-2R scanning electron microscope at a beam voltage to 15 kV. Preparation of casting material Batson’s No. 17 (Polysciences Inc., Paul Valley Industrial Park, Warrington, Pa. 18976) was used in this study and was diluted with methyl methacrylate (Eastman Co., Rochester, N.Y. 14650) in the following proportions: Batson’s Component A 100; Batson’s Component B 12; Batson’s Component C 1; methyl methacrylate 100. Components A and methyl methacrylate were mixed together and divided into 2 fractions of equal volume. Component B was added to one fraction and Component C to the other. Both these solutions were mixed well and then combined. The plastic now set in approximately 20 min. Air was removed from the liquid plastic by placing it under vacuum for 4 min. Results For all photographs, blood flow in the artery was from top to bottom. Group 1 - normal aortas The surface features of a plastic cast taken from a silver stained rabbit aorta are shown in Fig. 2. The cell boundaries of the endothelial cells are clearly visible and the cells form a single continuous layer over the luminal surface

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Fig. 2. Surface

features of a plastic cast taken from a silver stained rabbit aorta. X 250.

Fig. 3. Surface detail of silver stained aortic cast. Impression (arrowhead) on the cell surfaces are visible. X 600.

of cell nuclei

(N) and small indenta ltions

467

Fig. 4. Surface

features

of a plastic cast taken from an unstained

Fig. 5. Surface features of plastic cast frem EDTA-treated surface cephalad to the clamp are shown. X 250.

rabbit aorta.

rabbit.

X 500.

The site of clamping

and the aortic

468

Fig. 6. Surface

features

of plastic cast from EDTA-treated

aorta.

X 250.

Fig. 7. Surface detail of plastic cast from EDTA-treated platelets (P) and fibrin-like material are present: X 600.

aorta.

Two injured

endothelhl

cells 03). many

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with their long axis orientated in the direction of blood flow. A magnified view of these endothelial cells is presented in Fig. 3. The nuclei can be seen as darker regions in the centre of the cells and some small breaks or loops in the cell boundaries are present. Very small indentations covering the surface of the cells are also visible. A cast of an unstained rabbit aorta is presented in Fig. 4. Endothelial cell nuclei orientated along the length of the vessel are very prominent but no cell boundaries can be observed. Group 2 - EDTA-treated aortas The cast of the silver stained abdominal aorta immediately proximal to the aortic clamp is presented in Fig. 5. Above the line of the clamping, normal endothelial cells with prominent nuclei are visible whereas at the site of the clamp the regular pattern of cells is lost and no cellular structures are present. Below the clamp (EDTA-treated region) a cast of the aortic endothelial cells shows signs of injury and many cells appear to be curled up with no recognizable cell borders (Fig. 6), although a few normal endothelial cells are still visible at the left edge of this section. Elsewhere in this section the surface is covered with small bodies interdispersed amongst fibrillar material. These features can be seen clearly in Fig. 7 where 2 curled up endothelial cells are surrounded by long strands of fibrin-like material and many platelets. Discussion The results of this study demonstrate that nuclei and silver stained boundaries of endothelial cells can be clearly observed in the arterial vasculature casts. The outline and shape of the cell impressions are similar to those previously reported for actual endothelial cells [4,5,14,15], although they appear to be narrower and more elongated. This may be because there is minimal shrinkage of liquid Batson’s while setting [13,16], indeed we have measured a volume change of < 1% during its polymerization [ 171. Biological tissue, however, is known to undergo considerable shrinkage during dehydration and volume changes of up to 62% have been recorded [ 181 after various drying procedures. The dimensions of the cells observed in the vasculature casts may therefore represent the true in vivo shape of endothelial cells. The sensitivity of the casting technique can be judged by the results of the EDTA-treated aorta. Damaged endothelial cells, fibrin-like material and platelets are all seen in the casts. These findings are in agreement with other SEM studies where both similar platelets [ 191 and spindle-shaped endothelial cells [ 14,151 have been reported. Furthermore in normal untreated aortas very small impressions or holes are found covering the surface of many cells and these may reflect the presence of the microvilli which have been shown to cover the endothelial cell surface [ 20,211. Vascular casting has been extensively used in observing microvascular structures [ 13,22,23] including 3dimensional measurements of atherosclerotic lesions [ 241, but has not previously been used in morphological studies of vascular cells. The advantage of this technique, as compared to other more orthodox procedures for preparing arterial tissue for the scanning electron microscope, is that the vessel does not need to be fixed, dehydrated or dried

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and that the in vivo geometry is preserved. Thus by casting the luminal surface of arteries, possible artifact formation, caused by fixation or drying and mechanical distortion of the endothelial cells, can be avoided. Acknowledgements We are grateful to Dr. L. Langille and Dr. Margot Roach for their valuable advice and discussion and thank Mr. D. Ardiel of the electron microscopy unit, Department of Anatomy, University of Western Ontario. References 1 Ross, R. and Glomset, J.A., The pathogenesis of atherosclerosis, Part II. New Engl. J. Med., 295 (1976) 420. 2 Ross, R.. Glomset. J. and Harker. L.. Response to injury and atherogenesis, Amer. J. Path& 86 (1977) 675. 3 Wissler. R.W.. Vesselinovitch. D. and Gets, G.. Abnormalities of the arterial wall and its metabolism in atherogenesis. Progr. Cardiovasc. Dis.. 18 (1976) 341. 4 Silkworth. J.B.. McLean, B. and Stehbens, W., The effect of hypercholesterolemia on aortic endothelium studied en face, Atherosclerosis, 22 (1975) 335. 5 Poole. J.C.F.. Sanders, A.G. and Florey, H.W., The regeneration of aortic endothelium. J. Pathol. Bact., 75 (1958) 133. 6 Bjtirkerud, S., Preparative, staining and microscopic techniques for the study of whole artery segments, Atherosclerosis, 15 (1972) 147. 7 Davies, P.F. and Bowyer, D.E., Scanning electron microscopy: arterial endothelial integrity after fixation at physiological pressure, Atherosclerosis, 21 (1975) 463. 8 Clark, J.M. and Glagov. S., Luminsl surface of distended arteries by scanning electron microscopy: eliminating configurational and technical artifacts, Brit. J. Exp. Pathol.. 57 (1976) 129. 9 Christensen, B.C. and Garbarsch, C.. A scanning electron microscopic (SEM) study on the endothelium of the normal rabbit aorta, Angiologlca. 9 (1972) 15. 10 Davies, P.F.. Reidy. M.A., Goode, T.G. and Bowyer. D.E.. Scanning electron microscopy in the evaluation of end&he&l integrity of the fatty lesions of atherosclerosis, Atherosclerosis, 25 (1976) 125. 11 Goode, T.B., Davies, P.F., Reidy, M.A. and Bowyer, D.E., Aortic endothelisl cell morphology observed in situ by scanning electron microscopy during atherogenesis in rabbits, Atherosclerosjs. 27 (1977) 235. 12 Bowyer, D.E., Davies, P.F.. Reidy, M.A. and Goode, T.B., Scanning electron microscopy (SEM) of arterial end&helium. In: Proc. 1st Int. Atherosclerosis Conf., Progr. Biochem. Pharmacol.. Karger. Basel. in press. 13 Gannon, B J., Campbell, G. and Randall, D.J.. Scanning electron microscopy of vascular casts for the study of vessel connections in complex vascular bed. In: D.J. Aceneaux (Ed.). 31st Ann. Proc. Electron Microscope Sot. America, New Orleans, 1973. p. 442. 14 Reidy. M.A. and Bowyer, D.E.. Scanning electron microscopy: morphology of aortic endothelium following injury by endotoxin and during subsequent repair, Atherosclerosis. 26 (1977) 319. 15 Reidy. M.A. and Bowyer, D.E., Scanning electron microscopy of arteries: the morphology of aortic endothelium in haemodynamically stressed areas associated with branches, Atherosclerosis, 26 (19771 181. 16 Batson, O.V., Corrosion specimens prepared with a new material, Anat. Rec.. 121 (19551425. 17 Reidy. M.A. and Levesque, M.J.. Unpublished observations. 18 Boyde. A., Bailey, E.. Jones, S.J. and Tamarin, A., Dimensional changes during specimen PreParation for scanning electron microscopy, Proc. 10th Ann. SEM Symp. IITRI. Chicago, (19771607. 19 Sheppard. B.L.. Platelet adhesion in the rabbit abdominal aorta following the removal of endothelium with EDTA, Proc. Roy. Sot. Lond. B. 182 (1972) 103. 20 Hammersen. F.. Endothelial filaments and intercellular gaps -a sufficient evidence for contractihty?, Bibliotheca Anat.. 12 (1973) 159. 21 Hammersen, F.. Endothellal contractility - an undecided problem in vascular research, Beitr. Path.. 157 (1976) 327. 22 Murakami, T.. Application of the scanning electron microscope to the study of the fine distribution of the blood vessels, Arch. Hlstol. Jap.. 32 (1971) 446. 23 Murakami, T.. Miyoshi, M. and Fujita. T.. Glomerular vessels of the rat kidney with special reference to double efferent arterioles. A scanning electron microscope study of corrosion casts, Arch. Histol. Jap.. 33 (1971) 179. 24 Barndt. R. and Crawford, D.W.. A new three-dimensional post mortem method to study the toPography of atherosclerosis using profilometry. Atherosclerosis. 27 (1977) 121.

A scanning electron microscopic study of arterial endothelial cells using vascular casts.

463 Atherosclerosis, 28 (1977) 463-470 0 Elsevier/North-Holland Scientific Publishers, Ltd. A SCANNING ELECTRON MICROSCOPIC STUDY OF ARTERIAL ENDO...
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