O. B. Bayliss

PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically‐defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of ‘normal’ white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter‐white matter distant from plaqueclose white matter‐edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base‐line of thin‐layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

Bromine-Sudan Black: a general stain for lipids including free cholesterol

SynopsisPreliminary bromination (with bromine water) increases the intensity of staining of tissue lipids with Sudan Black and certain other dyes. The mechanism appears to be due to the formation of sudanophilic bromo-derivatives of cholesterol and to the retention of certain other lipids, notably phosphatidyl choline and free fatty acids, during staining. The advantage of the bromine-Sudan Black method is that all tissue lipids are stained, except saturated fatty acids, saturated triglycerides and perhaps saturated cholesterol esters. In practice, such lipids rarely, if ever, occur alone, and normally are admixed with their stainable unsaturated counterparts.

Osmium tetroxide as a histochemical and histological reagent

SummaryFrom the evidence discussed it can be concluded that osmium tetroxide (OsO4) would be reduced to black OsO2 (or an equivalent compound) by the ethylene bonds of liquid or solid cis-unsaturated lipids or by the Δ5-double bond in cholesterol in solid state in tissues. No evidence has been obtained to suggest that OsO4 is either reduced or bound by proteins and polysaccharides in tissue-sections.

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.

Histochemistry of myelin. VIII. Proteolytic activity around multiple sclerosis plaques

SynopsisTwenty-two plaques from six cases of multiple sclerosis were examined histochemically, with particular reference to demonstrating proteolytic activity by the gelatin-silver autogram method. Proteolytic activity was increased around plaques that were characterized as active (i.e. with increased cell population and NADH dehydrogenase activity at their edges). Inactive plaques showed little or no proteolytic activity. Proteolytic activity around active lesions could not be attributed to lipid-laden microglia: oligodendroglia or the myelin sheath are considered as alternative sources. Acid phosphatase and ‘non-specific’ cholinesterase were mainly localized in lipid-laden microglia and the activity of these two enzymes was not otherwise prominent around active plaques. The oligodendroglia from unaffected white matter were found not to contain either of these enzymes. In view of previous observations that proteolytic enzymes causein vitro myelin breakdown and are also implicated in Wallerian degeneration, it is suggested that local proteolysis may be an initiating factor in the demyelinating process of multiple sclerosis.

HISTOCHEMICAL DETECTION OF TRIGLYCERIDE ESTERS WITH SPECIFIC LIPASES AND A CALCIUM-LEAD SULPHIDE TECHNIQUE

A histochemical method for triglyceride esters is described that depends on hydrolysis of triglycerides to fatty acids by pancreatic lipase, precipitation of the released fatty acid as calcium soap and formation of lead sulphide by the conventional Gomori technique. The specificity of the lipase for triglyceride was investigated by chromatography and activation-inhibition studies. The enzyme preparation used hydrolyzed triglycerides and waxes but did not split cholesterol esters or phosphoglycerides. Positive histochemical reactions were obtained with this pancreatic lipase method in medium sized and fine lipid droplets in frozen sections. Positive reactions were observed in rat brown fat; in various adipose tissues in man, rabbit and rat; in the spermatogonia of rat testis; in human epidermis and sebaceous glands; in solitary unidentified adrenocortical cells in man and rat; in human fatty liver and in fine droplets in senile human myocardium. A slight variable reaction was obtained in human atherosclerotic plaques. The reaction of brown fat was also investigated with the electron microscope. No triglyceride was demonstrated in unfixed tissue sectons when an active preparation of lipoprotein lipase was used in place of pancreatic lipase. This failure was probably due to diffusion of lipoprotein from unfixed sections. Lipoprotein lipase is inactive against serum lipoproteins fixed with formaldehyde or glutaraldehyde.

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.

HISTOCHEMISTRY OF MYELIN. XIII DIGESTION OF BASIC PROTEIN OUTSIDE ACUTE PLAQUES OF MULTIPLE SCLEROSIS

Abstract— Plaques of multiple sclerosis from a patient with a short clinical history were investigated by qualitative and quantitative histochemical methods. One of three plaques examined showed perivenous lymphocytic infiltration; this plaque was regarded as a particularly early acute lesion. In this plaque a relatively wide zone of diminished staining for basic protein extended outwards around the edge of the lesion. A narrower and irregular zone of diminished cerebroside staining was also seen around the plaque. Staining for phosphoglycerides and cholesterol was relatively normal up to the edge of the lesion; no zone of reduced staining for these lipids was seen outside the plaque. Proteolytic activity was increased throughout the lesion (pH 3.5 > 7.4). The two less acute plaques showed no obvious loss of basic protein and cerebroside outside the plaque. Addendum: Two further acute plaques of multiple sclerosis obtained recently, showed a similar loss of basic protein outside of lesion.

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.