A B S T R A C T An in vitro preparation of rabbit aortic "intima-media" previously shown to exhibit stable rates of respiration and glucose metabolism and the high rate of aerobic glycolysis considered characteristic of the metabolism of this tissue was subjected to electron microscopic examination. In samples examined immediately after the aortae were dissected free of adipose tissue and adventitia, under conditions similar to those now in common use, marked and widespread alterations in endothelial cell structure were present, including loss of cell integrity. The vascular smooth muscle cells retained a normal electron microscopic (EM) appearance. During subsequent incubation with 5 mM glucose in KrebsRinger bicarbonate (KRB), pH 7.4, under the conditions usually employed in studies of this preparation, large zones of the luminal surface were rapidly denuded of endothelium, and the remaining endothelial cells exhibited a wide range of ultrastructural alterations. The smooth muscle cells, however, continued to maintain a normal EM appearance.A method was developed to prepare segments of rabbit aortic intima-media which retained an intact layer of endothelium resembling that observed in tissue fixed in situ. During a 1-h incubation with 5 mM glucose in KRB, pH 7.4, gas phase 5% C02/95% 02, containing 6% bovine serum albumin, the intact aortic intima-media preparation retains an essentially unmodified EM appearance and exhibits linear rates of respiration. Under these conditions the intact aortic intima-media preparation exhibits significantly higher rates of 02 uptake and glucose uptake than those observed in our previous prep- aration or in other reported aortic intima-media preparations. The intact aortic intima-media does not exhibit the high rate of aerobic glycolysis during in vitro incubation that has been considered characteristic of the metabolism of rabbit, rat, and swine aortic intima-media. In addition, the magnitude of the Pasteur effect was far greater than that observed in other aortic intima-media preparations. The data suggest that component cells of the aortic intima-media may derive a major fraction of their energy requirements from respiration; they raise further questions concerning the significance of the high rate of aerobic glycolysis observed when aortic intima-media preparations are incubated in vitro, and they suggest that documentation of the EM appearance of the endothelium in such preparations is desirable.

The fcasibility of using the Halc stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Thrcc kinds of mouse ascites tumor cclls were fixed in neutral buffered formalin, exposed to fresh colloidal fcrric oxide, treated with potassium fcrrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acctate and potassium pcrmanganatc was done aftcr sectioning in order to increase contrast. Those cells known to be coated with sialomucin showcd deposits of clectron-opaquc ferric ferrocyanidc crystals in the areas where sialomucin concentrations were expected. When these cells were treated with ncuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with clcctron microscopy to identify sialomucin.

A B S T R A C T Paired samples ofan intact rabbit aortic intima-media preparation were incubated for short periods under aerobic or anoxic conditions in Krebsbicarbonate buffer containing 6% albumin and 5 mM glucose. During aerobic incubation for as long as 1 h the preparation retained an electron microscopic (EM) appearance similar to that of tissue fixed in situ, and scanning EM confirmed the presence of an uninterrupted endothelial surface. After 2.5 min of anoxia there was widespread endothelial swelling, but the alterations in the EM appearance of these cells were not striking and did not progress during a subsequent 30 min aerobic incubation in fresh medium. After 10 min of anoxia there were marked and widespread alterations in endothelial cell structure, including loss of cell integrity, and numerous discrete interruptions in the endothelium were'consistently observed on both trnpsnmission and scanning EM. After a subsequent 30 min aerobic incubation in fresh buffer, a major fraction of the luminal surface was denuded of endo--thelium. The aortic vascular smooth muscle cells did not exhibit evidence of irreversible anoxic injury after 2.5 or 10 min of anoxia or after subsequent aerobic incubation for 30 min. Exposure to anoxia for 10 min induced persistent alterations in the composite metabolism of the preparation during subsequent aerobic incubation in fresh medium; 02 uptake was reduced, and the fraction of the glucose uptake that A portion of this work was presented at the Annual Meeting of the Association of American Physicians at Atlantic City., N

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