Kendal C. Dixon

Hydrophobic lipids in injured cells

Synopsis Introduction Dispersed and globular fat Detection of globular fat State of intracellular fat Equilibrium between dispersed and globular fat Reactivity of dispersed and globular fat Determinants of fatty globulation A. Globulation caused by interference with dispersion I. lack of phospholipid; 2. lack of oxygen; 3. lack of protein; 4. interference with dispersing power of phospholipid B. Globulation caused by initial quantitative increase of fat I. acceleration of entry or formation of fat; 2. retardation of removal of fat Fatty globulation in disease Malnutrition Cellular hypoxia Diabetes Poisoning by carbon tetrachloride Poisoning by ethanol Infection Complex hydrophobic deposits Cerebral lipofuscin CytochemicaI properties Structure Mechanism of formation References

Solubilization and cellular fatty change

Synopsis1.The micellar basis of solubilization of fat in cells has implications which must significantly affect any realistic concept of the mechanism of fatty globulation.2.Micelles have properties which are particularly relevant to the state of cellular fat. These properties are briefly considered: they include critical micellar concentration and solubilization.3.Substantial amounts of glyceryl esters occur in cells in which little or no globular fat is visible. This hidden fat, which is itself insoluble in water must, therefore, be solubilized by occlusion in some kind of micellar particle.4.Cells contain abundant potential solubilizers such as phospholipids. The solubilizing action of such amphipathic molecules may be enhanced by the formation of mixed micelles with other cellular surfactants.5.There is, however, a definite limit to the amount of insoluble oil or fat which can be solubilized in water by cellular surfactants. Once this level is surpassed, a third (oil) phase composed of visible emulsion droplets is formed in cells, where before there was only an aqueous phase and a micellar phase. Since amphipathic substances which are good solubilizers are not necessarily efficient emulsifiers, the establishment of this third phase may be followed by coalescence of microdroplets to form large plainly visible liquid globules with a minimum interfacial surface. Thus, fatty globulation can involve the spectacular appearance of large droplets of liquid lipid.6.These principles imply that fatty globulation is caused by (i) lack of suitable solubilizing agents, (ii) inhibition of their solubilizing power, or (iii) excess of fat above the amount which can be solubilized.7.Micellar equilibria which determine the state of cellular fat may profoundly be affected by changes in physical environment. Such changes occur during the processes of freezing, freeze-drying, or embedding tissues before cutting them into sections. For this reason, globules of hydrophobic lipid, seen in sections of tissues, might have little resemblance to the disposition of fat in living cells. Observations on globular lipid in rolled films of tissues, not subjected to the artefactual hazards of freezing or embedding, therefore provide necessary confirmation for the assumption that fatty globulation is a real episode in cellular life.

PSORIASIS: A DISORDER OF NECROSIS.*

NECROSIS is an essential event in tlie normal maturation of squamous epithelium. Although the manufacture of keratin from the cytoplasmic constituents of squamous cells has mainly attracted cytochemical study, it is likely that the jjrime determinants in the formation of the horny layer are nuclear rather than cytoplasmic in origin. The synchronous karyolysis in the rete mucosum is indeed the most remarkable phenomenon of epidermal develo2)ment. Little in fact is known ahout this necrosis of the epidermal nuclei. Our ignorance is in a way paradoxical, since continuous death of the epidermal eells along with loss of their nuclei is a normal and uniform process extending over our entire integument. Only, however, when disorder arises in this process of death do we deem it worthy of serious attention. Psoriasis may be regarded as a disorder of necrosis. One of the most prominent features of the disease is the retention of lamellar nuclear remnants in psoriatic scales in the place of the synehi-onous nuclear dissolution of normal epidermis. These persistent band-like incarcerated nuclei are known to retain their desoxyribonucleic acid (DNA) whieh is stainable by the Feulgen method (Braim-Falco, 1958). This communication is concerned with nuclear changes in psoriasis as well as with the effects of these changes on epidermal eytoplasm. The cytoplasm of the rete mncosum is rich in tannophilic protein stainable by the oxidized tanninazo (OTA) method for protein (Dixon, 1959). During the formation of normal keratin this tannophihc protein almost completely disappears. Indeed the loss of tannophilic protein from the cytoplasm is a striking concomitant of the karyolysis in the epidermal cells. A study was therefore made of the distribution of tannophihc protein (detectable by the OTA method) both in

Remnants of necrotic grey matter

SummaryCerebral neurones, with their intricately branching processes, have the highest surface/volume ratio of all cells. When cerebral grey matter disintegrates, the uniquely extensive surface of these ramifying cells becomes disorganised. Hydrophobic lipid remnants derived from this vast expanse of lipoprotein membranes may thus account for the highly characteristic PAS-positive granules found in phagocytes present in zones of softening produced by ischaemic necrosis of the cerebral cortex.This view was supported by further observations on these granules in phagocytes of necrotic cerebral cortex and also by the presence of granules with the same cytochemical properties in similar phagocytes which were found in necrotic cerebellar cortex. In the latter tissue, in which the neurones also possess an exceptionally large surface area owing to the extent of their axo-dendritic arborisations, neuronal disintegration was also followed by the appearance of apparently identical intraphagocytic deposits.The granular material within phagocytes of necrotic grey matter of both cerebral and cerebellar cortex was intensely coloured by Sudan black B, PAS, and paraldehyde fuchsin after pre-treatment with KMnO4. The colouration by PAS was not inhibited by previous incubation with amylase nor by previous bromination, but was suppressed after acetylation. Although faint brown in colour the granules did not contain ferric iron.The granular histiocytes found in necrotic grey matter differed from the foamy histiocytes seen in disintegrating white matter during the destruction of myelin.The granular material in the phagocytes of softened grey matter may be a hydrophobic coacervate formed by disarray of orientated lipoproteins and gangliosides during the disintegration of the extensive surfaces of axo-dendridic arborisations.Cerebral neurones, with their intricately branching processes, have the highest surface/volume ratio of all cells. When cerebral grey matter disintegrates, the uniquely extensive surface of these ramifying cells becomes disorganised. Hydrophobic lipid remnants derived from this vast expanse of lipoprotein membranes may thus account for the highly characteristic PAS-positive granules found in phagocytes present in zones of softening produced by ischaemic necrosis of the cerebral cortex.

Changes in dead and dying neurones studied by the use of rolled films of cerebellum

Introduction Substantial intracellular changes occur in neurones when they are deprived of glucose and oxygen. These changes include loss of potassium ions (DIXON 1949) entry of chlorides (VAN I-IA~EVELD and SC~AD]~), and appearance of phosphates (VAN IfAI~EVELD and POTTEI~). I t is likely, therefore, that the neurones of human brain, when examined after death, differ materially in internal composition from healthy neurones in the living state; yet, when examined in routine sections of embedded tissue, these cells usually display surprisingly little evidence of post mortem change. Conventional methods are presumably not sufficiently delicate to detect alterations in the cells, even though these changes may involve the fundamental and irreversible processes of cellular death. This communication describes the use of rolled/ilms of cerebellar vermis for the study of neurones and the post mortem changes within them. The method employed gives immediate fixation and eliminates the hazards of slow penetration of reagents into blocks of tissue. The application of this technique to cerebellar neurones post mortem revealed substantial increase of intranuclear anions in the granule cells as well as conspicuous alteration in consistency of the Purkinje cells. Intraeellular anions were demonstrated by fixing the films in alcoholic silver nitrate and subsequent reduction of the precipitated silver salts to metallic silver with 2 : 4-diaminophenol. This technique of rolling and staining the films is rapid and simple in execution and provides a convenient method for examining neurones within considerably less than half an hour after their removal from the body. The method was applied to fresh cerebellum of rabbits and to the cerebellum of rabbits kept at body temperature after death. In this way a characteristic pattern of intracellular events in the dying neurones was established. Comparable observations on human cerebellum obtained from routine autopsies showed that similar post mortem changes take place in human neurones.

Fatty globulation in the liver of fat-fed rats studied by rolled films

Synopsis(1)Globules of fat in enormous numbers appeared in the liver cells of rats fed for three days on margarine or butter as their sole food.(2)This change was studied principally in rolled films of liver incubated for 2 hr at 38°C. Films of incubated fatty liver contained numerous entire hepatocytes in which cellular structure was visible with clarity and definition. In films made from fresh liver, most of the cells were ruptured.(3)The cells in films made from incubated livers of rats fed on margarine or butter showed intense fatty globulation. This change affected most of the cells and much of their cytoplasm was occupied by globules of liquid lipid.(4)Substantial, though less severe, fatty globulation was observed in similarly prepared hepatic films made from rats fed on cows milk.(5)The cells seen in sections of frozen or wax-embedded liver of these fat-fed animals also contained fatty globules or (after wax-embedding) vacuolar spaces from which globular fat had been dissolved. But the disposition of droplets of lipid or spaces in sections of tissue, exposed to freezing and thawing or to infiltration by molten wax, may be very different from the state of hydrophobic lipid in the living cell.(6)The liquid globules seen ion rolled films, however, were not subjected to these artifactual hazards. The observations on globular fat in rolled films, therefore, confirm the assumption that globules of lipid visible in sections of fatty liver are a real entity of the living cell.(7)It is suggested that the neutral fat of normal liver cells is solubilized within micellar patticles and is thus invisible. The intense fatty globulation which occurs in liver cells of rats fed on margarine or butter probably arises because the amount of intracellular fat rapidly surpasses the quantity which can be solubilized in an invisible micellar state by the surfactants available within the cells. The excess of fat then rapidly forms an additional phase which becomes visible as conspicuous globules of liquid lipid.