R. S. Morgan

THE DEPOSITION AND DISPOSAL OF (4‐14C) CHOLESTEROL IN THE BRAIN OF GROWING CHICKENS

MUCH has been learned in recent years through the use of electron microscopy and X-ray diffraction studies about the laminated structure of the myelin sheath (FERNANDEZ-MORAN and FINEAN, 1957). Such studies have disclosed its distinctively organized character, and the likelihood that the various types of protein and lipid molecules recoverable from it by chemical extraction are mutually linked in a highly orientated manner. Little biochemical work has yet been done, however, to determine whether this apparent stability of internal structure in the sheath has as its counterpart any tardiness in the metabolic ‘turnover rate’ of any or all of its known constituent materials. Of the various lipids typical of myelin (JOHNSON, MCNABB and ROSSITER, 1948), cholesterol possesses several notable advantages for such ‘turnover’ studies. In addition to being quantitatively one of the principal members of this group of lipids, it is well characterized chemically, readily extractable and capable of accurate estimation. It can furthermore be obtained labelled with radioactive carbon, so that the presence and duration of persistence of this identifiable form of the compound can be ascertained with considerable accuracy. In the following study, radioactive cholesterol in trace amounts of high specific activity has been introduced into newly-hatched chickens at a time when myelination is proceeding with great rapidity. By extracting their brains for lipids at various intervals afterwards, it has been possible to determine the extent to which this cholesterol had become incorporated in the central nervous system and to gain some indication of its ‘turnover rate’ from the rate at which it subsequently disappears.

No regression of atheroma over one year in rabbits previously fed a cholesterol-enriched diet

Abstract Rabbits were fed a cholesterol-enriched diet for 12 weeks and then injected intravenously with a pulse-label of tritium cholesterol. Animals were killed at intervals from 1 day to 1 year after stopping the experimental diet. The inner aorta, outer aorta, plasma and liver were estimated for content and specific activity of cholesterol in both free and esterified forms. The aorta showed slow uptake of cholesterol, reaching plasma specific activity at about 3 weeks; thereafter it maintained its specific activity and cholesterol content at about the same level for 1 year. In the later part of the regression-period the amount of free cholesterol rose at the expense of esterified. By contrast, the plasma and liver showed a relatively rapid fall in cholesterol content and specific activity; the content falling to normal at about 1 month after stopping the diet. Histological and histochemical studies confirmed that the atheroma lipids had not been resorbed, and also revealed that atheromatous lesions were progressively converted into fibrous atherosclerosis over the 1-year regression-period.

Regression of atheroma in the rabbit

Ten studies in the literature concerning regression of rabbit atheroma were re-examined. In studies where cholesterol content was referred to weight, a degree of regression was noted in 3/4 studies. Such regression might at least partly have resulted from the dilution effect of the atheroma contents when results were expressed on a weight basis. By contrast, when results were referred to length or protein, partial regression was seen in only 1/4 studies. Mild atheroma induced by short-term cholesterol feeding did seem to regress in 2/2 studies.

Permeability in atherosclerosis

A new microscopic fluorescence method for trypan blue at 570 nm has been used to follow the entry of albumin into the atheromatous rabbit aorta. Permeability into the inner aortic wall increases before the onset of gross lesions and seems just to precede intraendothelial deposition of lipid. Thereafter, permeability of the inner wall progressively increases until streaks or small plaques develop. These raised lesions stain and fluoresce variably, some intensely so while others are almost unreactive. This variability might reflect the difference between progressive and quiescent lesions. However, a zone of increased permeability surrounds many raised lesions, suggesting that the edge is a major site of growth and progression.

The differential entry of [125I]albumin into mildly and severely atheromatous rabbit aortas☆

Abstract The penetration of [ 125 I]albumin into the aorta was investigated in normal rabbits and in those fed on a cholesterol-enriched diet for 4–14 weeks. Radioactivity levels in multiple layers from the inside to the outside of the aorta were determined by well-crystal counting. In normal vessels and where atheromatous plaques were thinner than 150 μ (ascending thoracic aorta) and 110 μ (descending thoracic aorta), radioactivity gradients sloped from outside-inwards. When atheromatous plaques were thicker than the above-mentioned dimension, radioactivity gradients reversed and tended to slope inside-outwards. From these observations we concluded that albumin normally enters the rabbit aorta mainly from the outside but, in the presence of severe atheroma, albumin (together with other plasma proteins and lipoproteins) leaks directly through the damaged intima from the lumen.

Differential resorption rates of subcutaneous implants of [3H]cholesterol, various [3H]cholesterol esters and [3H]cholesterol-[1-14C]linolenate

Summary Free [ 3 H] cholesterol, various [ 3 H] cholesterol esters and [ 3 H]cholesterol-[ 14 C]linolenate were implanted subcutaneously in rats. Three weeks after implantation [ 3 H] cholesterol linolenate and arachidonate were resorbed to a significantly greater extent than free [ 3 H]cholesterol, [ 3 H] cholesterol linolenate and the more saturated labelled sterol esters. [ 3 H]Cholesterol was more rapidly mobilized than the saturated and monounsaturated [ 3 H] cholesterol esters. The resorption of esterified cholesterol did not appear to depend on rapid hydrolysis of the fatty acid moiety and slow removal of the sterol nucleus, for 3 H and 14 C labels disappeared at nearly equal rates from implants of [ 3 H]cholesterol-[l- 14 C]linolenate. Addition of antioxidants to the polyunsaturated ester implants did not significantly accelerate resorption, even though it did reduce chromolipid formation. We infer that esterification of cholesterol with linolenic or arachidonic acids would accelerate its removal from the normal or atherosclerotic arterial wall.

A graticule for measuring atherosclerosis

Abstract The preparation is described of a simple graticule for measuring atherosclerosis. Transparent squared-paper is directly contact-printed on to Linefilm and then printed on to photographic glass plate. No camera is required. The size of the graticule squares can be accurately adjusted with a photographic enlarger.

The results of direct injections of botulinum toxin into the central nervous system of rabbits

A close similarity is now known to exist between the actions of the toxins of Clostridium tetani and Cl. botulinum on the neuromuscular mechanism of the rabbits iris (de Lavergne, Helluy, Faivre & Henry, 1948; Ambache, Morgan & Payling Wright, 1948a, b; Ambache, 1949). After their injection into the anterior chamber of the eye, both toxins effect a prolonged paralysis of the sphincter pupillae muscle, which becomes irresponsive either to the light reflex or to direct stimulation of the 3rd cranial nerve. That neither toxin acts directly on the muscle fibres themselves is shown by the readiness with which such paralysed irises contract under the stimulus of small doses of acetylcholine or carbachol. The site of action of both toxins seems to be at the post-ganglionic nerve terminals under the control of the 3rd cranial nerve, for after intoxication with tetanus toxin these fibres lose most of their ability to produce or release acetylcholine (Ambache et al. 1948 b). The specificity of the intoxication is well shown by the finding that neither toxin affects materially, if at all, the adrenergic mechanism of the dilator pupillae, which remains normally responsive to electrical stimulation of the sympathetic supply. Since, in addition to its paralytic action on the cholinergic mechanism of the iris, tetanus toxin leads to exaggerated activity of central nervous structures when injected into the spinal cord or medulla (Meyer & Ransom, 1903; Firor, Lamont & Shumacker, 1940; Wright, 1951, 1953) it seemed important to determine whether botulinum toxin had any similar action. Because tetanus exerts its lethal action most potently when injected into the medulla oblongata, the botulinum toxin used in most of the following experiments was inoculated at the same site.

The resorption rate of atheroma lipids in situ and implanted subcutaneously

Resorption rates have been investigated in the rabbit for atheroma-lipids and cholesterol injected subcutaneously, atheroma lipids in situ in the mildly atheromatous aorta and lipid in the degenerating peripheral nerve. Atheroma lipids injected subcutaneously resorb relatively rapidly, subcutaneous cholesterol and lipid in Wallerian degeneration are intermediate in this respect, whereas only the group with an initially low blood cholesterol (small than approximately equal to 100 mg/100 ml) showed even a suggestion of lipid resorption from their mild or trivial atheromatous lesions. Ti is not clear whether or not atheroma resorbed at all in the groups with initial blood cholesteroles above 100 mg//00 ml. The relative inertia of the arthromatous lesion is mainly attributed to the relative absence of reticuloendothelial phagocytes in it.

Exchange of plasma radioactive cholesterol with atheroma lipids in situ and implanted subcutaneously

Physicochemical exchange of radioactive cholesterol was investigated in mildly hypercholesterolaemic rabbits. Such exchange was examined in erythrocytes and atheroma lipids in situ in the aortic wall and in such lipids when implanted subcutaneously. The implanted atheroma lipids gained radioactivity from intravenously injected [3H]cholesterol, even though cholesterol was being resorbed from the implants. Autoradiography showed exchange between plasma cholesterol and both intracellular and dispersed extracellular lipid in these implants. Cholesterol crystals in both implants and atheromatous lesions labelled only sluggishly. The results are consistent with the view that physicochemical exchange is a major factor in cholesterol movement in vivo.