An Electron Microscope Study of Myelin Figures

In the electron microscope, thin sections of OsO₄-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO₄ with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO₄ in it.     

Fine structure of Bacillus subtilis. I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO(4), OsO(4), or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO(4) in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO(4) fixation appeared to provide much better definition of the boundaries of various structures than did OsO(4). With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO(4) fixation) as two thin lines. In cells fixed first with OsO(4) solution, and then refixed with a mixture of KMnO(4) and OsO(4) solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO(4). One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia

Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO(4) and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO(4) solution, and cardiac samples (6-mum-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO(4) accumulation were determined. Leakage of OsO(4) from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO(4) enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO(4) in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO(4) as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.     

CELLULAR INJURY IN VITRO: PHASE CONTRAST STUDIES ON INJURED CYTOPLASM

Unfixed, compressed acinar cells of rat pancreas, isolated by mechanical and enzymatic means, were examined by phase microscopy and photomicrographed using 35 mm film and electronic flash illumination. Similarly, observations were made on Walker carcinoma cells; in addition, these cells were treated with solutions containing either phosphatidase A or enzyme inhibitors. Acinar cells contained, besides nuclei, perinuclear droplets and secretion granules, various membranous and vacuolar structures. The basal cytoplasm showed parallel dark lines interpreted as endoplasmic reticulum. In some cells, fragmentation of the reticulum was followed by the direct incorporation of fragments into simple myelin figures. In other cells it appeared that phase-lucent linear structures and vacuoles were derived by dilatation of cisternae of the endoplasmic reticulum. Perinuclear fluid collections arose either by dilation of the perinuclear cisternae of the endoplasmic reticulum or by fluid dilatation of the nuclear envelope. Phosphatidase A disrupted early vacuoles of Walker carcinoma cells. From this and the direct involvement of elements of the endoplasmic reticulum in myelin figures, it was concluded that the membranes limiting the endoplasmic reticulum incorporate phosphatides in continuous layers. While many severely injured cells formed large vacuoles, others developed concentrically laminated myelin figures; it was concluded that both types of structure derived from phosphatides liberated intracellularly, the vacuoles by vesicular myelin figure formation.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

Osmium tetroxide, used in the treatment of arthritic joints, is a fast mimic of superoxide dismutase

Aqueous solutions of osmium tetroxide (OsO4) have been injected into arthritic knees for the past 45 years to chemically destroy diseased tissue, in a procedure termed "chemical synovectomy." Arthritis is an inflammatory disease. The primary inflammatory chemical species are the superoxide anion radical (O2.-) and nitric oxide (.NO), which combine to form the peroxynitrite anion (ONOO-). Here we show that OsO4 does not react with ONOO- but very efficiently catalyzes the dismutation of O2.- to O2 and H2O2. Using the pulse-radiolysis technique, the catalytic rate constant has been determined to be (1.43+/-0.04) x 10(9) M-1 s-1, independent of the pH in the 5.1-8.7 range. This value is about half that for the natural Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Per unit mass, OsO4 is about 60 times more active than Cu,Zn-SOD. The catalytically active couple is OsVIII/OsVII, OsVIII oxidizing O2.- to O2 with a bimolecular rate constant of k=(2.6+/-0.1)x10(9) M-1 s-1 and OsVII reducing it to H2O2 with a bimolecular rate constant of (1.0+/-0.1)x10(9) M-1 s-1. Although lower valent osmium species are intrinsically poor catalysts, they are activated through oxidation by O2.- to the catalytic OsVIII/OsVII redox couple. The OsVIII/OsVII catalyst is stable to biochemicals other than proteins and peptides comprising histidine, cysteine, and dithiols.     

Analysis of the mechanism of the fixation of membrane structures using osmium tetroxide. II. An electron microscopic and x-ray structural study of myelin frozen and lyophilized at low temperature]

Comparative electron microscope and X-ray studies were made on the frog sciatic nerve myelin after freeze-drying technique. The specimens were fixed with OsO4 before and after freeze-drying. In the latter case, osmium was used as a hydrophobic solution (OsO4 in CCl4), or in the high vacuum during osmium sublimation. The results obtained in this study do not fit in the accepted mechanism operating during osmium fixation of membranes. Another mechanism is proposed by the authors, and the problem of osmium localization within the space of the myelin repeated unit is discussed.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds.

Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (approximately 0.25%) considerably less concentrated with respect to OsO4 than solutions (1-2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry. These results suggest that the effectiveness of OsO4 as a fixative may, in part, be related to its nonpolarity.The infrared spectra indicate that the OsO4 molecule is tetrahedral, perfectly symmetrical and, therefore, as a whole nonpolar. As a consequence, it could be expected to readily penetrate charged surfaces of tissues, cells, and organelles. The spectral studies show that osmeth is much less symmetrical and, to that extent, polar; thus, it penetrates biomembranes less readily.     

The fine structure of developing locomotor muscles of the pelagic tunicate,Cyclosalpa affinis (Thaliacea:Salpidae)

Salps are free-swimming tunicates whose peculiar life history renders them ideal for developmental studies. The solitary salp reproduces asexually by budding a stolon containing the complete developmental sequence of the aggregate generation. The ultrastructure of developing locomotor muscle of the aggregate generation of Cyclosalpa affinis was studied. The early muscle contains essentially non-striated myofibrils. However, in transverse sections, areas indicating early I-bands and A-bands can be recognized. As development continues, the number of fibrils increases, the Z-lines appear, and the fibrils contain more recognizable striations. The fully developed muscle has the characteristic structure of striated muscle. Longitudinal sections show sarcomeres with irregular and discontinuous (perforated) Z-lines; H-zones are not apparent. No M-lines are seen. Throughout development, the ratio of thin to thick myofilaments is always 2:1, the ratio found in all vertebrate striated muscle. Other findings in C. affinis suggest that: (1) multinucleated muscle cells are formed by the fusion of mononucleated cells, (2) membranes of adjacent mononucleated cells destined to fuse form myelin figures, and (3) these myelin figures become closely associated with mitochondria.     

Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions

In sections of KMnO(4)-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside approximately 20 A dense strata of the two approximately 75 A Schwann cell membranes. The two combined outside strata make the intraperiod line bisecting each myelin lamella. The approximately 150 A mesaxon is elaborated spirally around the axon in either a right hand or left hand spiral, and its inside (cytoplasmic) approximately 20 A strata in apposition form the major dense lines of myelin. In hypotonic solutions the lamellae of adult frog sciatic myelinated fibers split apart along the outside membrane strata apposed at the intraperiod line throughout the spiral. Under similar conditions the inside (cytoplasmic) strata of the membranes, in apposition at the major dense lines, do not separate. The approximately 150 A membranous structure resulting from this is called an "internal compound membrane." The double membranes of normal and control frog sciatic unmyelinated fibers have a central gap approximately 100 to 150 A wide. After soaking in 4 to 10 times normal strength Ringer solution or 10 N sucrose-Ringer solution, this gap closes and a membranous structure approximately 150 A wide resembling developing mouse mesaxons results. This is designated by the term "external compound membrane." The latter membranes resemble internal compound membranes, but their central dense zones, each consisting of two apposed outside membrane strata, are less dense.     

Probing of DNA structure with osmium tetroxide. Effect of ligands

Fourteen OsO4 complexes with different ligands were tested as probes of DNA structure. Of these complexes, only OsO4-2,2'-bipyridine (Os-bipy), OsO4-bathophenanthrolinedisulfonic acid (Os-bpds) and OsO4-N,N,N',N'-tetramethylenediamine (Os-TMEN) site-specifically modified the ColE1 cruciform in a supercoiled plasmid pColIR215 at millimolar concentrations. Os-bipy, Os-bpds and Os-TMEN also displayed site-specific modification of the B-Z junctions in the supercoiled plasmid pRW751 containing (dC-dG)n inserts.     

Differential staining of tannin in sections of epoxy-embedded plant cells.

A staining procedure is described for the light microscopic localization of ergastic tannins in epoxy sections of plant cells embedded for study by transmission electron microscopy. Callus and cell suspensions of Pseudotsuga menziesii and Pinus taeda fixed in glutaraldehyde:acrolein and then OsO4, followed by epoxy embedding, were sectioned 0.5 mum thick, stained on a glass slide with ethanolic Sudan black B at 60 C as described by Bronner, and then mounted in Karo syrup. Tannin deposits stained brownish-orange and were easily distinguished from lipid bodies of similar size, which stained dark blue to black, and from starch grains, which were unstained. The significance of this differential polychromasia was confirmed by transmission electron microscopy. This staining procedure should prove valuable in the cytoplasmic evaluation of the plant cell ergastics (especially tannins) via light microscopy whether or not electroc microscopic examination is intended.     

ELECTRON MICROSCOPE STUDIES ON ENZYME ACTIVITY AND THE ISOLATION OF THIOHYDANTOIN-INDUCED MYELIN FIGURES IN RAT LIVER

Characteristic cytoplasmic inclusions (myelin figures), consisting of concentric multilaminar paired membranes surrounding one or more lipid bodies, were produced in rat liver parenchymal cells by incorporating high doses of an anticonvulsant agent (Bax 422Z) into the animals' diet. Enzymatic reaction product (presumably lead phosphate) was found around the central fat of these myelin figures in liver which had been fixed in glutaraldehyde, incubated in Wachstein and Meisel's medium containing adenosine triphosphate or inosine tri- or diphosphate, postosmicated, embedded in epoxy resin, and examined in the electron microscope. In an attempt to isolate myelin figures, fresh liver from medicated rats was homogenized and differentially centrifuged. Thin sections of osmium tetroxide-fixed, Epon-embedded pellets from each fraction were examined with the electron microscope. The concentric membranous whorls, which are probably derived from cisternae of the endoplasmic reticulum, broke up as the cells were disrupted and became inextricably mixed with the microsomal fraction. However, when liver previously fixed in formalin for 24 hours was homogenized, the myelin figures remained intact.     

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.     

Über die Pleomorphie von MLO in Catharanthus roseus (Vinca rosea)

About the pleomorphism of MLO in Catharanthus roseus (Vinca rosea) MLO of the flower greening (virescence)-type of Primula in the phloem of Catharanthus roseus (Vinca rosea) were fixed in two different ways. After prefixation with OsO4 they showed a diminished pleomorphic morphology, most MLO were spherical. With the fixation of KARNOVSKY (1965) and subsequent treatments with OsO4 and uranyl acetate, however, an extremely high pleomorphism was observed. Such pleomorphic MLO in single thin sections are probably the expression of a threedimensional network-like connection. The concentration of MLO in the sieve elements seems to influence the pleomorphism only insignificantly.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID : A Correlated Chemical and Morphological Study

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl₄ (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl₄) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.     

Observations on the cytoplasmic membranes of testicular cells,examined by phase contrast and electron microscopy

In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.