Joan A. Hardy

The value of alcohol for fixation of skin.

Stained sections of skin fixed in 70% alcohol were compared with others from pieces fixed in 4% formaldehyde-saline. The sections of alcohol-fixed material were much more susceptible to the action of deoxyribonuclease and lipase than those from formalin-fixed, as demonstrated by a standardized hematoxylin staining method and by fluorescence microscopy. After formalin, cytoplasmic basophilia was increased, presumably because formalin fixation caused ribonucleic acid to diffuse from nuclei to cytoplasm. Both types of fixation damaged collagen, as seen in fluorescence induced by 5-anvmo-2-chloro-7-methoxyacridine, but alcohol caused less distortion than formalin. Probably fluorochroming of fresh tissue is the only satisfactory method for studying collagen in pathological conditions.

Ultrastructure of the contrasting types of keratinization seen in the tail epidermis of the laboratory mouseMus musculus

SummaryThe mouse tail epidermis undergoes contrasting forms of keratinization. Around the hair follicle there is a granular layer containing keratohyalin granules, and nuclei are absent from the horny layer. In the scale regions keratohyalin is not formed and nuclear remnants are retained in the horny cells as in parakeratosis generally. These findings from light microscopy were confirmed by transmission electron microscopy. The complete breakdown of organelles in the follicular regions contrasted with the retention of effete nuclei in the scales. Some of these nuclear remnants were pyknotic as in abnormal human parakeratosis, but most were further degraded with loss of nuclear membranes. In the boundary zone between the follicular and scale regions the epidermal cells had a few small keratohyalin granules and also showed incomplete degradation of nuclear remnants in the horny cells. The change from living epidermal cells to dead keratinized cells was abrupt in both the follicular and scale regions. In both sites the plasma membranes of the horny cells were thickened and there was a cytoplasmic meshwork of microfibrils in the cells.ZusammenfassungDie Verhornung der Epidermis im Mäuseschwanz verläuft in unterschiedlicher Weise. In der Umgebung der Haarfollikeln ist eine granuläre Schicht, die Keratohyalin-Körnchen enthält; die Zellkerne fehlen in der verhornten Schicht. In der Schuppenregion wird kein Keratohyalin gebildet, und Kernreste bleiben in den verhornten Zellen, wie allgemein in der Parakeratose. Die Befunde des Lichtmikroskops konnten durch Transmission-Elektronmikroskopie bestätigt werden. Der vollständige Zusammenbruch der Organelle in der Region der Follikeln stand in Gegensatz zu dem Weiterbestehen der alten Zellkerne in den Schuppen. Manche dieser Kernreste waren pyknotisch wie in anormaler Parakeratose des Menschen, aber die meisten waren mehr degeneriert und hatten die Kernmembranen verloren. Die Epidermiszellen in der Grenzzone zwischen der Follikel und Schuppenregion enthielten ein paar Keratohyalin-Körnchen, und der Verfall der Nuklearreste in den verhornten Zellen war unvollkommen. Der Übergang zwischen lebenden Epidermiszellen und toten verhornten Zellen war scharf abgegrenzt sowohl in der Follikel als in der Schuppenregion. Die Plasmamembrane der Hornzellen waren in beiden Lagen verdickt, und die Zellen enthielten ein cytoplasmisches Netzwerk von Mikrofibrillen.

Some ultrastructural observations on keratohyalin granules of guinea pig epidermis

SummaryKeratohyalin in the cavy back and plantar epidermis was examined by transmission electron microscopy. These cytoplasmic aggregates are made up of numerous fine particles uniformly stained by osmium, probably the protein component. Phospholipid in the interstices is removable in lipid solvents before osmium post-fixation. After glutaraldehyde fixation without osmium, tissue stained in uranyl acetate showed keratohyalin aggregates composed of mixtures of darkly stained and weakly stained groups of fine particles which indicate heterogeneity in composition. More weakly stained aggregates of keratohyalin composed of these particles were found in the interiors of keratinized cells in the lower part of the stratum corneum not previously demonstrated at this level. Oxidation in peracetic acid followed by staining in uranyl acetate showed dark areas in keratohyalin aggregates, which it is thought contain cystine. Keratohyalin aggregates are not surrounded by a membrane and they are not organelles. Their heterogeneous composition suggests different derivations and destinies of component substances, some of which may be synthetic and others autolytic products. It is suggested that keratohyalin is a cytoplasmic coacervate of protein, phospholipid, and bound calcium precipitated under unstable physiochemical conditions associated with keratinization involving extensive cytolysis.ZusammenfassungDas Keratohyalin der Meerschweinchenrücken- und Sohlenepidermis wurde transmissionselektronenmikroskopisch untersucht. Die cytoplasmatischen Komplexe bestehen aus zahlreichen feinen Partikeln, die, wahrscheinlich Proteinanteile, sich mit Osmium homogen färben lassen. In den Spalträumen gelagerte Phospholipide können mit fettlösenden Substanzen entfernt werden. Glutaraldehydfixierung ohne Osmium und anschließende Färbung mit Uranylacetat zeigte, daß Keratohyalin aus einer Mischung von dunkel gefärbten und schwach gefärbten Gruppen feiner Partikel bestehen, die auf die Heterogenität dieser Komplexe hinweisen. Schwächer gefärbte Keratohyalinkomplexe wurden in den verhornten Zellen der unteren Stratum corneum-Abschnitte erstmals nachgewiesen. Essigsäureoxydation mit nachfolgender Uranylacetatfärbung zeigte innerhalb der Keratohyalinkomplexe dunkle Areale, die vermutlich Cystin enthalten.Keratohyalinkomplexe sind nicht membrangebunden und stellen keine Organellen dar. Ihre heterogene Zusammensetzung läßt unterschiedliche Herkunft der Einzelsubstanzen vermuten, von denen einige neu synthetisiert werden, andere als Folge von Autolyse entstehen. Es wird angenommen, daß Karatohyalin ein Komplex von Proteinphospholipid und gebundenem Calcium darstellt, das unter unstabilen physikochemischen Bedingungen im Verlaufe der Keratinisation und Cytolyse präcipitiert wird.

Enzyme changes in lichen planus

SummaryUntreated cases of lichen planus have been studied by histochemical techniques. The acid phosphatase reaction in the transitional zone has been quantitatively estimated and compared with the adjacent relatively normal epidermis. It was found that despite a thickened and accentuated granular layer as seen by routine histological methods there was a marked reduction in the intensity of the acid phosphatase reaction.The glucose-6-phosphate dehydrogenase reaction was marked in the upper layers of the epidermis in active lesions of lichen planus. This is similar to psoriasis, but different from normal human epidermis. The suggestion by other authors that lichen planus is an inborn error of metabolism is discussed.The dendritic cells of the epidermis as studied by the ATPase reaction are virtually absent in regions of active lichen planus and the possible significance of this is mentioned.The horny layer gives a dense reaction for phospholipids in lichen planus and this is similar to psoriatic keratin. The significance of this finding is considered.ZusammenfassungUnbehandelte Fälle von Lichen planus wurden mit histochemischen Methoden untersucht. Die Saure-Phosphatase-Reaktion in der Übergangzone wurde größenmäßig geschätzt und mit der danebenliegenden relativ normalen Epidermis verglichen. Der Befund ergab trotz einer verdichteten und hervorgehobenen Granulosumschicht, wie es sich bei routinemäßigen histologischen Methoden zeigt, eine deutliche Reduktion in der Intensität der Saure-Phosphatase-Reaktion.Die Glukose-6-Phosphat-Dehydrogenase-Reaktion war in den oberen Schichten der Epidermis bei aktiven Läsionen von Lichen planus auffallend vermehrt. Dieses Verhalten ist der Psoriasis ähnlich, aber unterscheidet sich von der normalen menschlichen Epidermis. Der Vorschlag anderer Forscher, wonach der Lichen planus ein angeborener Fehler des Metabolismus sei, wird diskutiert.Die mit der ATPasen-Reaktion untersuchten dendritischen Zellen der Epidermis fehlen im wesentlichen in Zonen von aktivem Lichen planus. Die mögliche Bedeutung dieser Tatsache wird erwähnt.Die verhornte Schicht ergibt beim Lichen planus eine dunkle Reaktion für Phospholipide, was dem psoriatischen Keratin vergleichbar ist. Die Bedeutung dieses Fundes wird erörtert.

The action of solvents on the ultrastructural appearance of keratohyalin and the horny layer in guinea‐pig back epidermis

Various solvents were used to remove osmium‐reactive phospholipids and free fats from guinea‐pig back epidermis before post‐fixation in osmium. After extraction, the osmium stained material which remained was probably due to proteins with reactive amino acids. After brief fixation in glutaraldehyde, followed by immersion of the tissue in pyridine, a phospholipid solvent, followed by osmium post‐fixation, the unkeratinized epidermal cells were practically unstained. Removal of phospholipid from the keratohyalin granules revealed them as aggregates of smaller particles, probably the protein component. The thickened plasma membranes of the horny cells were darkly stained in osmium. Removal of material from the interiors of these cells left empty spaces between the meshwork of filaments, indicating absence of matrix keratin. The contrast between the darkly stained keratinized cell membranes and filaments, and unstained unkeratinized cells was marked after lipid extraction.

Ultrastructural demonstration of cystine in guinea pig back stratum corneum.

SummaryA method for the ultrastructural demonstration of cystine in keratinized epidermal cells of guinea pig back hairy skin is described. This involves selective oxidation by peracetic acid and formation of an electron dense reaction product with uranyl acetate.Unkeratinized cells were unstained except for nuclei and keratohyalin. The thickened plasma membranes of transitional and keratinized cells were darkly stained. In transitional keratinized cells darkly stained membranes occurred initially in relation to desmosomes and then spread to intervening membrane areas. Dense material, probably matrix keratin containing cystine was found in the interiors of basal horny cells but not in the intermediate or superficial cells of the stratum corneum. It is suggested that the unstable matrix keratin is probably broken down by enzymatic hydrolysis above the basal zone of the stratum corneum.ZusammenfassungDer ultrastrukturelle Nachweis von Cystin in keratinisierten Epidermiszellen der pigmentierten Meerschweinchenhaut wird mit einer neuen Methode dargestellt. Das Verfahren beruht auf selektiver Oxydation und der Bildung eines elektronendichten Reaktionsproduktes durch Uranyl-Acetat.Nicht-keratinisierte Zellen blieben abgesehen von den Kernen und Keratohyalin ungefärbt. Die verdickte Plasmamembran der Übergangszellen wie auch der verhornten Zellen sind stark gefärbt. In Übergangszellen zeigten sich stark gefärbte Zellmembranen zuerst in der Näbe von Desmosomen, um dann auch an den übrigen Membrangebieten nachweisbar zu werden. Im Cytoplasma der unteren Hornzellen konnte ein dichtes Material, wahrscheinlich cystinhaltig Matrixkeratin, gefunden werden, das in den mittleren und oberen Zellagen des Stratum corneum fehlte. Es wird vermutet, daß das nichtstabile Matrixkeratin dicht oberhalb der Hornschichtbasis durch enzymatische Hydrolyse abgebaut wird.

Some ultrastructural observations on human palmar stratum corneum

Ultrastructure of human palmar stratum corneum was examined for cell junctions and cytoplasmic material. Interdigitated microvilli are shorter and less uniform in man. Most of these processes are either conical or truncated in shape, as distinct from the relatively smooth cell surfaces except for sigma-shaped junctions in hairy sites. It is suggested that this provides strength against shearing force in the palm and sole. There is less cytoplasmic breakdown in palmar horny cells than in hairy cells.

Plantar epidermis of the guinea pig and characteristics of the stratum corneum

The guinea pig plantar epidermis was examined by light-microscopical histochemical methods and by transmission electron microscopy. Autolysis of cell structure was much less complete in guinea pig plantar horny layer than in the back, and stainable cytoplasm was retained in keratinized cells but organelles were lost except for some degraded ultrastructural remnants. By light microscopy the whole thickness of the horny layer showed bound phospholipid and bound cysteine, and there was a weak cystine reaction at the peripheries of the keratinized cells. In ultrastructure the keratohyalin contained slightly larger subparticles than in the back skin. The horny layer was not divisible into basal, intermediate and superficial regions as in hairy skin. The stratum lucidum of light microscopy was not defined in electron micrographs. Osmium-stained cytoplasmic material was retained in horny cells about to be desquamated, in contrast to the empty appearance of these cells in hairy skin. Epidermal cells in plantar skin have ultrastructural cytoplasmic processes which are longer than they are broad. In the horny layer these interdigitate with those of neighbouring cells and are held together by lateral demonsomal junctions. Probably this gives mechanical strength against shearing forces experienced by the plantar horny layer.

Acid nucleases and other hydrolases in human skin with special reference to elastic tissue

SynopsisThe presence of ribonuclease and deoxyribonuclease is reported in human elastic tissue. Differences between the effects of various agents on these enzymes in epidermal cells and in elastic tissue are described. Other acid hydrolases in elastic tissue have also been investigated.It is thought that the presence of these acid nucleases and other hydrolases in elastic tissue may be related to its removal and thus provide the means whereby a dynamic system of breakdown of old fibres and the formation of new fibres is continually occurring in dermal tissues.