PASSAGE OF L I P I D ACROSS VASCULAR E N D O T H E L I U M IN N E W B O R N RATS An Electron Microscopic Study

An electron microscopic study of the fine blood vessels in the skin and muscle of 95 newborn rats (sucklings, and therefore subject to physiologic lipemia) has sho~,n that blood-borne lipid particles may leave the lumen of these vessels by two pathways, intercellular and intracellular. (a) An intercellular pathway: Some capillaries, venous capillaries and venules contain intramural , extracellular deposits of lipid which is presumably hematogenous. In some animals these deposits are quite numerous; available evidence suggests that they are a consequence of intercellular gaps, too small or too transient to be observed except in rare instances. Plasma apparently escapes through these gaps and filters across the basement membrane, while lipid particles are retained, usually in sufficient number to fill the small defect; some lipid particles are then taken up by endothelial cells and pericytes, while a few escape and are incorporated into free phagocytes. These focal defects, though few in number, may explain the apparent incapacity of blood vessels of newborn rats to leak any further after a local injection of histamine. Discontinuities in the endothelium were found also in the renal glomerulus, sometimes accompanied by extensive interstitial accumulations of lipid particles. Similar intercellular gaps are known to exist in other types of immature endothelia. (b) An intracellular pathway: This is best demonstrated in the capillaries, venous capillaries and venules which supply the developing subcutaneous adipose tissue. Here the lipid particles adhere in large numbers to the endothelial surface; the morphologic evidence suggests that they are also taken up into the endothelium through phagocytosis by "flaps," or into pockets or crevices. The lipid is apparently metabolized in the vascular wall; some is found in the multivesicular bodies. There was no evidence of active transport by vesicles or vacuoles. Neither pathway was demonstrable in the adult. The passage of materials across the endothelial barrier has been studied, at the level of the electron microscope, mainly with the aid of tracer particles injected intravenously. With the exception of ferritin, these tracers are colloidal suspensions of substances foreign to the body. The chylornicrons, which are normally carried by the blood stream, could be considered physiologic tracer particles; they are recognizable by electron microscopy, and when sufficiently numerous they can be used for "labeling" leaking vessels almost as effectively as foreign materials (1). The present study was undertaken for two purposes. In the first place, the physiologic lipemia of the suckling rat offered an opportunity to study the behavior of the endothelium towards blood-borne lipid particles. Furthermore, it appeared useful to study the capillaries of the newborn, since relatively little is known about immature endothelia. A detailed survey of the literature on the fine 163 on O cber 9, 2017 jcb.rress. org D ow nladed fom Key to Abbreviations for Figures B, basement membrane E, endothelial cell F, endothelial flap L, lumen M, striated muscle fibre MB, multivesieular body N, nucleus 0, osmiophilic material P, pericyte R, red blood cell S, extravascular space FIOURES 1 to 3 Comparison of pericapillary basement membranes in a newborn rat (Figs. 1 and 2) and in a young adult rat (860 gin) (Fig. 3). Fig. 1 (skin) illustrates the thin, somewhat irregular basement membrane typical of many capillaries in the newborn. In Fig. ~ the basement membrane is more poorly developed; the arrow points to an area in which it is virtually absent. Fig. 3 shows that the adult basement membrane is thicker, denser, and more even in width. (Figs. ~ and 3, striated musc]e. Uranyl acetate, lead citrate.) X 33,000. structure of the endothelium, both mature and immature, is available elsewhere (2). M A T E R I A L S A N D M E T H O D S Pregnant female rats of the Sprague-Dawley and Wistar strains were procured. Preliminary experiments indicated that competition among sucklings in large litters could lead to very different levels of milk consumption and thus of lipemia. Therefore, shortly after delivery, the size of each litter was reduced to 4 to 6 sucklings. Despite these precautions, the degree of lipemia remained quite variable, a factor which made this study very time-consuming. In an attempt to circumvent this difficulty, intravenous injections of homologous chyle were administered; the resulting lipemia was short-lived and did not compare in intensity with the spontaneous lipemia (when present). The sucklings were sacrificed when they were 1 to 2 days old and weighed 6 to 8 gin. Tissues examined from 19 normal sucklings included skin, subcutaneous adipose tissue and striated muscle from the lower abdominal quadrants, heart, lung, kidney, liver, and adrenal gland. Six additional animals were examined 3 to 70 minutes after intravenous injection of homologous chyle. Thirteen adult male Holtzman rats weighing 250 to 300 gm were made lipemic by 2 cc of melted butter 164 Tn~ JOURNAL OF CELL BIOLOGY • VOLUME ~7, 1965 on O cber 9, 2017 jcb.rress. org D ow nladed fom FIG~ES 4 and 5 Cellular processes (arrows) arising from the outer surface of the endothelium in small vessels of the newborn rat. Where the surface of these processes is cut at right angles, the lack of basement membrane is obvious. Note chylomicrons in the lumen (L). Fig. 4: skin. Fig. 5: muscle. Lead hydroxide, X 16000. or corn oil fed by stomach tube after a fasting period of 24 hours. The tissues sampled from these rats (and 4 normal controls) were the same as for the newborn animals. All the tissues mentioned above were also examined by light microscopy, on frozen sections of formolfixed samples stained with oll red O and Fettrot, as well as on whole mounts of tissue cleared in glycerin and stained with Sudan Black and Fettrot. Some of the thick Epon sections were also stained for fat (3). Tissues for electron microscopy were removed from the living animals under ether anesthesia, fixed for 2~/'~ to 31~ hours in Palade's fixative (4) with 2 per cent OsO4, dehydrated in ascending concentrations of acetone, and embedded in Epon. Some blocks were stained with KMnO4 following dehydration (5). Sections were cut on an LKB ultratome with diamond knives, stained with Karnovsky's method A (6), or with lead citrate (7) preceded by saturated uranyl acetate, and examined with an RCA 3D or a Philips 200 electron microscope. Thicker sections from the same blocks, stained with 0.1 or 1 per cent toluidine blue, were used for orientation. To obtain the greatest number of small blood vessels it was found practical to embed the skin with the epithelium towards the cutting surface; this was then trimmed away to the depth of the tips of t i le papillae. Perpendicular sections through the whole thickness of the skin were also studied. Chyle was obtained by cannulation of the main lacteal of an adult rat made lipemic as described above. Aliquots (0.005 or 0.02 cc) were immediately injected intravenously, into newborn animals which were sacrificed after 3, 20, 30, and 70 minutes.