Implementation of periodic acid-thiosemicarbazide-silver proteinate staining for ultrastructural assessment of muscle glycogen utilization during exercise

Summary Distribution of glycogen particles in semithin and ultrathin sections of biopsy samples from human muscles subjected to either short- or long-term running were investigated using PAS and Periodic Acid-ThioSemiCarbazide-Silver Proteinate (PA-TSC-SP) staining methods. Glycogen particles were predominantly found immediately under the sarcolemma or aligned along the myofibrillar Iband. After long-term exhaustive exercise type-1 fibers with a few or no glycogen particles in the core of the fibers were frequently observed. The subsarcolemmal glycogen stores of these depleted type-1 fibers were about three times as large as after exhaustive short-time exercise. Another indication of utilization of subsarcolemmal glycogen stores during anaerobic exercise was that many particles displayed a pale, rudimentary shape. This observation suggests fragmental metabolization of glycogen. Thus, depending on type of exercise and type of fiber differential and sequential glycogen utilization patterns can be observed.     

Histologic studies on the neurohypophysis of the living frog

Summary The infundibular region in the leopard frog, Rana pipiens, was studied in vivo at magnifications of up to 900 diameters. This was accomplished by the use of an operative procedure which allowed for transillumination of the region and the introduction of specially modified water immersion lenses of 50, 70 and 90 x into the field of study. This procedure is described.Details of the living histologic structure in the infundibular region were related to the capillaries of the complex vascular apparatus of that region and the region was divided into five zones on the basis of capillary patterns occurring in given areas. Living histologic structure of this region includes five different morphologic types of granular materials. Two of these materials could be identified with chrome-alum hematoxylin positive particles in fixed and stained sections. Fibers about 2, in diameter varying widely in numbers seen from animal to animal were observed in the region of the tractus hypophyseus. These fibers have not been positively identified as nerve fibers. Some of the particles and the fibers show changes in position and/or configuration with time. The location of the fibers, their appearance, and the location, appearance, and distribution of the particles are described. The implications of the observations made are discussed.Supported by a Grant from the Commonwealth Fund and United States Public Health Service Grants H-4176 and HTS-5340.Presented in part, at the VIII. Microcirculatory Conference (Dr. John Irwin, Chairman), International Anatomical Congress, New York, April, 1960.     

Virus-like particles in the brown algaStreblonema

Summary Ultrastructural examination ofStreblonema sp. revealed icosahedral virus-like particles (135–150 nm) throughout the cytoplasm of vegetative cells. The densely packed particles consist of an osmiophilic coat around a fibrillar core. Most cytoplasmic organelles are excluded from the regions where the particles are extremely abundant, but no degeneration of plastids, mitochondria or dictyosomes is evident. The virogenic stroma contains many ribosomes and fibers possibly representing DNA strands remaining from the lysed nucleus. No decrease in vigor seemed to be associated with the presence of the particles.     

Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Myofibrillar ATPase activity of intrafusal fibers in chronically de-afferented rat muscle spindles

Summary Dorsal root ganglia L₄, L₅ were removed to accomplish long-term (1 year) de-afferentation of the rat soleus muscle. Muscle spindles in the muscles deprived of sensory innervation were morphologically and histochemically abnormal. The spindle periaxial fluid space was greatly diminished with a thicker capsular investment. De-afferented intrafusal muscle fibers lacked either nuclear bags or nuclear chains at their midlengths. The intracapsular myofibrillar ATPase staining pattern of de-afferented nuclear bag fibers resembled that which the bag fibers normally display in their extracapsular regions. These abnormalities are discussed with respect to the regulatory functions of spindle sensory and motor nerves.     

Temporal progress of muscle adaptation to endurance training in hind limb muscles of young rats

Summary The soleus, rectus femoris and gastrocnemius muscles of young rats were studied after 3, 6 and 12 weeks of treadmill training. The muscle fibers were characterized histochemically by their succinate dehydrogenase (SDH) and myofibrillar ATPase activity, and morphometrically by their cross-sectional areas, which were corrected for different body weights of control and trained animals.After 12 weeks of training the mean area of fibers in the muscles studied was not significantly different from the controls, as expected. In the soleus muscle the percentage of the fast-twitch fibers was decreased as a result of their transformation into slow-twitch fibers. Trained soleus muscles were the only muscles showing pathologically altered fibers, suggesting overload. The percentages of fiber types and their areas exhibited changes specific for the muscles and muscle regions studied.From these results it is concluded that the adaptation follows the sequence proportional adaptation of morphometrical parameters, disproportional adaptation of the areas of fiber types, and disproportional adaptation of the percentages and/or the areas of the fiber types. It is shown by comparison with the literature that this sequence may be generalized to a sequence of increasing expense necessary for the adaptation to increasing stimuli, and that the most decisive factors for adaptation are work load, frequency of exercise, period of training, and the age of the subject at the initiation of the training.     

Attachment of tail fibers in bacteriophage T4 assembly: role of the phage whiskers.

The collar and whiskers of bacteriophage T4 extend outward from the top of the tail and play a role in regulating retraction of the tail fibers (Conley &; Wood, 1975). The collar and whiskers also are required for efficient tail fiber attachment during phage assembly. The structural gene for the collar/whisker protein is called wac. In vitro, infected-cell extracts that contain tail fibers activate whiskerless (wac) tail fiberless particles and ordinary (wac⁺) tail fiberless particles at equal rates if the extracts contain the wac⁺ gene product. However, extracts that contain tail fibers but no wac⁺ gene product activate wac particles about ten times more slowly. In vivo, whiskers are not essential for plaque formation, but a wac mutation causes a delay in the appearance of intracellular phage and a fivefold decrease in the burst size of infectious particles.The effect of the whiskers on tail fiber attachment is due to an interaction between the whisker and the distal half of the tail fiber, similar if not identical to the interaction that controls tail fiber retraction in complete phage. The following observations support this view: a slow rate of in vitro tail fiber attachment similar to that described above is seen with wac⁺ particles when they are pretreated with anti-whisker serum, or when the tail fibers carry a mutational alteration in gp36, a structural protein in the distal half fiber near the central kink. Lack of whiskers does not affect the slow rate of attachment of proximal half fibers to the baseplate of fiberless particles, but lack of whiskers greatly decreases the rate at which particles with attached proximal half fibers are activated by addition of distal half fibers. Since whiskers normally are attached to the phage only after head—tail union (Coombs &; Eiserling, 1977; Terzaghi et al., 1978), these findings explain why tail fibers do not attach efficiently to the baseplates of free tails.     

Fiber type-specific distribution of parvalbumin in rabbit skeletal muscle

Summary A highly sensitive sandwich ELISA for parvalbumin (PA), based on a fluorometric detection system, was developed. This assay detected PA concentrations as low as 20 pg/ml (2 pg per assay) and was used for measuring PA contents in fragments of single muscle fibers isolated from freeze-dried 100–150 m thick cross sections. The fibers were typed according to their histo-chemically assessed mATPase in parallel cross sections. Type I fibers from rabbit tibialis anterior (TA) and vastus lateralis (VL) muscles contained extremely low PA concentrations (2–5 g/g w.wt.). Type IIA fibers displayed slightly higher values with mean values of 17 and 29 g/g w.wt. (range 5–65) in TA and VL, respectively. Much higher PA concentrations were found in type IIB fibers with wide ranges from 75–1150 g/g w.wt. in TA and 440–1370 g/g w.wt. in VL. Whereas the IIB fibers of the TA displayed a continuum, two subgroups were distinguishable according to their PA contents (means of 590 and 1230 g/g w.wt.) in VL. Possibly, the population with the lower PA content which was histochemically defined as type IIB in the present study, corresponds to fiber type IID. The finding that PA is predominantly present in type IIB fibers was also confirmed by the parallel decay of PA and type IIB fibers during chronic low-frequency stimulation. The use of freeze substitution, or alternatively, of freeze-drying, made it possible to demonstrate PA immunohistochemically without artifacts and to evaluate the staining intensity by microphotometry. Performing measurements on the same fibers with the two methods, it was possible to establish a relationship between immunohistochemical staining intensity and PA concentration. This correlation can be used to assess PA contents by evaluating immunohistochemical staining intensities in comparative measurements within the same section.     

Motor innervation of the cat muscle spindle studied by the cholinesterase technique

Summary Muscle spindles were traced in serial transverse sections of cat tenuissimus muscles. Myofibrillar adenosine triphosphatase staining reaction was used to identify nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers. Typical chain fibers and long chain fibers were distinguished, the latter extending for more than 1,000 m beyond the termination of the spindle capsule. Simple rim and more elaborate plate deposits were demonstrated histochemically along the poles of the typical chain fibers in staining for cholinesterases. They were considered to correspond, respectively, to the trail and plate motor nerve terminals. Most long chain fibers and the majority of nuclear bag fibers had their motor innervation limited to plate-type endings. In addition, faint diffuse cholinesterase staining occurred along the spindle capsule and the surface of some intrafusal fibers. These histochemical observations are discussed with regard to the current concepts concerning the morphological and functional organization of the motor innervation of the cat muscle spindle.     

Metal and Silicate Particles Including Nanoparticles Are Present in Electronic Cigarette Cartomizer Fluid and Aerosol

Background

Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit. Because EC do not burn tobacco, they may be safer than conventional cigarettes. Their use is rapidly increasing worldwide with little prior testing of their aerosol.

Objectives

We tested the hypothesis that EC aerosol contains metals derived from various components in EC.

Methods

Cartomizer contents and aerosols were analyzed using light and electron microscopy, cytotoxicity testing, x-ray microanalysis, particle counting, and inductively coupled plasma optical emission spectrometry.

Results

The filament, a nickel-chromium wire, was coupled to a thicker copper wire coated with silver. The silver coating was sometimes missing. Four tin solder joints attached the wires to each other and coupled the copper/silver wire to the air tube and mouthpiece. All cartomizers had evidence of use before packaging (burn spots on the fibers and electrophoretic movement of fluid in the fibers). Fibers in two cartomizers had green deposits that contained copper. Centrifugation of the fibers produced large pellets containing tin. Tin particles and tin whiskers were identified in cartridge fluid and outer fibers. Cartomizer fluid with tin particles was cytotoxic in assays using human pulmonary fibroblasts. The aerosol contained particles >1 µm comprised of tin, silver, iron, nickel, aluminum, and silicate and nanoparticles (<100 nm) of tin, chromium and nickel. The concentrations of nine of eleven elements in EC aerosol were higher than or equal to the corresponding concentrations in conventional cigarette smoke. Many of the elements identified in EC aerosol are known to cause respiratory distress and disease.

Conclusions

The presence of metal and silicate particles in cartomizer aerosol demonstrates the need for improved quality control in EC design and manufacture and studies on how EC aerosol impacts the health of users and bystanders.     

Distribution and specific central projections of mechanoreceptors in the thorax and proximal leg joints of locusts

Summary Tactile hairs on the locust thorax can be divided into two classes by their external morphology and their central projection pattern: Short hairs, 10–100 m in length, which are assembled in distinct plates and rows, and long hairs, 100–800 m in length, which are distributed all over the body and are organized in large fields or aligned along the ridges of the appendages.The sensory fibers of the first class arborize in the lateral dorsal neuropile of thoracic ganglia and then extend further into the ipsilateral half of the corresponding ganglion in three main bundles from which fine rami of fibers end in the intermediate neuropile. In all three thoracic ganglia the projection pattern of homologous hair plates is similar.The sensory fibers of the second class exclusively terminate in special median ventral neuropiles, the ventral association center (VAC) and ventralmost ventral association center (VVAC). In addition fibers from meso- and metathoracic hairs, located close to the longitudinal midline of the animal, may terminate in the contralateral VAC and with one branch project to the next anterior ganglion through the ipsilateral connective. In contrast, fibers from prothoracic hairs were not found to leave their ganglion.With support by the DFG Neurale Mechanismen des VerhaltensSome of the studies were started at Universität Bielefeld, Fakultät für Biologie II (Abtlg. Prof. Dr. P. Görner)     

Fine structure of baroreceptor terminals in the carotid sinus of guinea pigs and mice

Summary A light and electron microscopic study was undertaken on the baroreceptor axon terminals in the carotid sinus of guinea pigs and mice, using serial semithin and thin sections.Together with their enveloping Schwann cells, numerous lanceolate axon terminals are organized into a well-defined discoid end organ, referred to as the baroreceptor unit. Baroreceptor units measure 100 to 150 m in diameter and are arranged in a hexagonal pattern. These end organs represent free branched lanceolate mechanoreceptors of complex type (Andres and von Düring, 1973) which belong to the main group of stretch receptors.In the guinea pig the lanceolate terminals enter the media and approach the innermost layers near the intima. In the mouse the terminals are seen to spread in the adventitia and along the medio-adventitial border. Only a few of them penetrate the external elastic layer. Species differences concerning the localization and extent of these visceral mechanoreceptors are discussed, as well as the modified architecture of the sinus wall in the receptor area (elastic segment).Lanceolate terminals form beaded varicosities which are equipped with finger-like or lamellar axoplasmic protrusions. These projections contain a well-differentiated receptor matrix. They are attached to collagen and elastic fibers. The varicosities include densely packed mitochondria, neurotubules, profiles of axoplasmic reticulum, clear and granular vesicles, and striking accumulations of glycogen particles, lamellated bodies and lysosomes. Four types of varicosities are discerned according to their main axoplasmic components. Various types of these varicosities occur within an individual lanceolate terminal.The adrenergic innervation of the carotid sinus was studied by fluorescence histochemistry. In guinea pigs a multilayered wide-meshed plexus of fluorescent fibers occurs in the adventitia where it is closely related to baroreceptor stem fibers. However, adrenergic axons do not enter the media. In mice fluorescent fibers are extremely rare in the adventitia of the carotid sinus.Dedicated to Prof. Dr. Drs.h.c. W. Bargmann on the occasion of his 70th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft Nr. Bo/525-1. These results were presented in part at the 17. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie, Berlin, Sept. 21.–26., 1975     

Ultrastructural duality of extrafusal fibers in a slow (tonic) skeletal muscle

Summary Ultrastructural and stereological assessment of the mature avian anterior latissimus dorsi (ALD) muscle showed that it contains two kinds of extrafusal fibers. This fine structural dichotomy of fiber types in the ALD correlated well with their previously reported histochemical duality. Distinct differences occur in sarcomere banding, myofibrillar area, sarcotubular and mitochondrial density, and in morphology of motor-nerve terminals. Both myofiber types in this muscle were interpreted as representing varieties of slow or tonic muscle fibers.Both fibers contain myofibrils that, despite differences in cross-sectional area, were large, irregular, and ribbon-shaped, typical of the Felderstruktur appearance of true slow fibers. Whereas the majority of fibers (type-1) are devoid of well-defined M-bands, the minor fiber population (type-2) exhibit prominent M-bands in the center of each sarcomere. In addition, type-1 tonic fibers contain a significantly lower mitochondrial and sarcotubular volume than the tonic fibers of type-2. While both fiber types exhibit motor-nerve terminals that are small, smooth and punctate in appearance, those on the type2 fibers often had a number of shallow postjunctional folds. Whether or not these two classes of extrafusal fiber in this muscle represent two separate and distinct types of motor units remains to be determined functionally.Supported by grants from the Medical Research Council and the Muscular Dystrophy Association of Canada. The author gratefully acknowledges the excellent technical assistance of Susan L. Shinn     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.     

Complementarity of particles and pits in freeze-fractured hepatic and cardiac gap junctions

Summary Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas.In hepatocyte gap junctions, the center-to-center distances (mean±sd) among pits, 9.57±1.49 nm, and particles, 9.70±1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510±830)/m², than for the particles, (11,230±950)/m². In the myocardium, the spacing of the regularly arrayed pits, 9.55±1.33 nm barely exceeds the value of 9.44±1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090±740)/m², appears a little higher than that of the particles (9,890±920)/m². As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the collinearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Scanning electron microscopy of the lens of the adult rat

Summary The lenses of albino rats were studied after freeze-drying with scanning electron microscopic technique.The multilamellated lens capsule is covered by adhering fibers in the equatorial region but looks otherwise almost smooth. The cuboidal, polyhedronal cells on the anterior surface of the lens are characterized by their complex interdigitations and their paucity of extracellular space. The cortical polyhedronal, mostly hexagonal lens fibers have several processes interconnecting neighbouring cells. The plasma membrane appears slightly undulated. It is covered by extracellular material. There is a well-delimited extracellular space between the lens fibers in the cortical layer. The results are discussed in relation to previous studies based upon light and electron microscopy.Supported by grants from the Swedish Medical Research Council (B70-12X-2543-02) and Magn. Bergwalls Stiftelse.     

Fluorescence microscopic distinction between elastin and collagen

Summary Distinction between elastin and collagen in arteriosclerotic lesions is difficult because immature and incompletely cross-linked collagen bind so-called elastica stains; furthermore, abnormal collagen can lack cross-striation and thus resemble elastin in electron microscopy. However, collagen and elastin differ significantly in their content of basic amino acids and hence in their affinity for heteropolyacids. This chemical difference was utilized for the development of a fluorescence microscopic method for distinction between collagen and elastin.Paraffin sections of human autopsy material were treated with a 1% aqueous solution of phosphomolybdic acid (PMA) for five minutes, rinsed in distilled water, dehydrated and mounted. Other series were treated with the PMA-molybdenum blue reaction and with various special stains.Only elastic membranes of aorta, the elastica interna and externa of sizable arteries, and true elastic fibers remained strongly fluorescent; the autofluorescence of collagen, reticulum fibers, basement membranes, pseudo-elastic fibers, and elastic membranes in small arteries was quenched. In other series PMA abolished the fluorescence of basic fluorochromes.Correlation of fluorescence and direct light microscopic observations with chemical and electron microscopic data showed that the PMA-fluorescence method permits distinction between elastin and various types of collagen.     

Scanning electron microscopy of giemsa-stained chromosomes and surface-spread chromosomes

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.