Cryo Sections Research Articles

Characterization of semenogelin proteins in the human retina

Semenogelin I and II are the major proteins present in semen coagulum. In the present study, semenogelin I and II were detected in human RPE lysates by proteomic analysis. We further analyzed the expression of these proteins in the retinal cells in vivo and in vitro. Western blots detected semenogelin I and II in both RPE and neural retina while the vitreous contained only SgII. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with an antibody that recognizes both forms of semenogelin proteins. Retina and RPE total lysates were evaluated for the presence of these proteins and in a human RPE cell line (D407). Both proteins were detected by western blot in human RPE and in D407 cell lysates. Immunoreactivity was detected in the ganglion cell and photoreceptor layer of the retina. Our data support the expression of semenogelin I and II in the human retina in several different compartments. Further studies towards addressing the function of these proteins in the retina are in progress.

Benefit from anti-inflammatory treatment during clinical remission of atopic asthma

Subjects with atopic asthma often experience a disappearance of symptoms around puberty. However, airway inflammation and remodeling may persist. It is unknown whether those findings warrant prolonged anti-inflammatory treatment despite the absence of symptoms. In this study, we investigated whether a short course of combined anti-inflammatory treatment would, also in this specific patient population, diminish airway inflammation and/or remodeling. A double-blind, randomized placebo-controlled trial was conducted in 28 asymptomatic subjects with a history of atopic asthma, with established bronchial hyperresponsiveness to methacholine (MCh) as non-invasive indicator of ongoing airway pathology. Intervention consisted of the salmeterol/fluticasone propionate combination (SFC) product (50/250 microg bid via the Diskus inhaler) or placebo for 3 months. The change in lung function (FEV1), bronchial response to MCh and adenosine monophosphate (AMP), the fraction of nitric oxide in exhaled air (FENO) and quality of life (QOL) scores were measured. Also, bronchial biopsies were taken and cryo sections immunostained for eosinophils (major basic protein, MBP) and mast cells (tryptase and chymase) before and after treatment. The change in reticular basement membrane (RBM) thickness, one of the parameters of airway remodeling, was also determined. SFC treatment improved hyperresponsiveness to MCh (P = 0.014) as well as AMP (P = 0.011), and reduced FENO (P < 0.001) significantly as compared with placebo. Lung function tended to improve (NS). Furthermore, SFC treatment reduced tryptase in the subepithelium of bronchial biopsy specimens (P = 0.01), and slightly reduced RBM thickness (P = 0.05). However, eosinophils in (sub)epithelium were not significantly affected; neither were chymase levels, blood eosinophils or QOL scores. We found that 3 months of treatment with fluticasone propionate and salmeterol reduced airway hyperresponsiveness, FENO and tryptase density in the airway mucosa as markers of airway inflammation. MBP density in the airway mucosa and QOL were, however, unchanged. The clinical relevance of these findings, especially with respect to the long-term outcome, has not been determined yet.

A METHOD FOR CRYOSECTIONING OF PLANT ROOTS FOR PROTON MICROPROBE ANALYSIS

By building up a cryo-preparation laboratory a method was developed which allows us to prepare cryo sections of fully differentiated plant-root material, according to the requirements of the proton microprobe. Since cryo-preparation is the only possible method delivering reliable elemental distributions, this step was a fundamental for all our further investigations in plant root physiology. As an example, PIXE-investigations of a root section of a heavy metal hyper-accumulating plant Brassica juncea L. will be presented.

Selective staining and disintegration of intestinal eosinophilic granule cells in Atlantic salmon after intraperitoneal injection of the zinc chelator dithizone

Following intraperitoneal injection of the zinc ion chelator dithizone into Atlantic salmon Salmo salar, staining and morphological changes in intestinal eosinophiic granule cells (EGC) were observed in both cryo‐preserved and chemical‐preserved tissue sections. In cryo sections rapid and selective vital staining of EGC was observed within 5 min of dithizone injection. Intensely stained red granules appeared as a result of the formation of a complex between dithizone and zinc ions. Stained EGC appeared on both sides of the stratum compactum as well as in the lamina propria. Stained EGC started to disintegrate 5 min after the injection of dithizone, and little of the zinc‐dithizone complex was observed 15 h later. The disappearance of the stain coincided with an intense degranulation and disintegration of EGC. Fifteen hours after dithizone treatment regenerating cells were observed in Masson's trichrome stained sections. Four to five days after injection, a fully regenerated continuous EGC layer was observed. Simultaneously with the disintegration of EGC an increase in plasma lysozyme activity occurred. EGC resembles mammalian Paneth cells in their possession of lysozymecontaining granules and their staining by, and response to, dithizone. EGC may represent a central component of zinc metabolism in Atlantic salmon.

Co(III)EDTA as extra-cellular marker in μPIXE-analysis of rat cardiomyocytes

In previous studies no clear difference was found between the intra- and extra-cellular compartment in nuclear microprobe elemental distribution maps of freeze-dried cryo sections of heart tissue. Probably due to artefacts during the preparation of these samples, the intra-cellular and the extra-cellular content of elements are mixed up. In this article a method, using NaCo(III)EDTA as an extra-cellular marker, was applied to deconvolute the total ion content in an extra- and intra-cellular contribution. This method was both applied to normoxic heart tissue and low-flow ischemic heart tissue. Intra-cellular ion concentrations calculated from the corrected ion contents of the normoxic tissue agrees well with literature values. Moreover a clear elevation of the intra-cellular sodium and chlorine concentration was found in low-flow ischemic tissue.

Prompt epithelial damage and restitution processes in allergen challenged guinea-pig trachea in vivo.

Little is known about the induction and the morphology of epithelial damage, and of the ensuing epithelial restitution processes in allergic airways. To examine epithelial damage and restitution in allergen challenged guinea-pig trachea. Whole-mount techniques, transmission and scanning electron microscopy, cryosectioning, and histochemical staining were used. Cell proliferation was monitored by BrdU-immunohistochemistry. Allergen challenge produced patchy, crater-like, and leucocyte-rich epithelial damage sites. At 1, 5, and 24 h damage was associated with poorly differentiated epithelial restitution cells. Already at 1 h the epithelial craters had a floor of flattened restitution cells and the damaged areas comprised < 1% of the mucosal surface area (whole-mount preparations). In contrast, cryo sections displayed large areas (approximately 20%, 1 h) of denudation. Epithelial, and subepithelial (fibroblasts, smooth muscle) proliferation was increased 5 and 24 h after challenge (P < 0.01). Within 1 h allergen challenge has induced patchy damage sites where epithelial restitution is already advanced; although easily produced by cryosectioning frank denudation was not evident in whole-mount preparations. The present findings may explain the well maintained, functional tightness of allergic airways displaying epithelial damage, shedding, and even denudation. The present data also suggest the possibility that epithelial damage-restitution may be causative to allergic airway remodelling.

Association between inflammation and epithelial damage‐restitution processes in allergic airways in vivo

Associations between allergen challenge-induced sites of epithelial damage and the distribution of leucocytes and extravasated plasma remain unexplored. To study neutrophils, eosinophils, and fibrinogen at allergen challenge-induced patchy epithelial damage-restitution sites in guinea-pig trachea. After local challenge tracheal tissue (cryo sections and whole-mounts) and lumen (selective tracheal lavage) were examined at 1, 5, and 24 h. Eosinophils, neutrophils and fibrinogen were identified by histochemistry. Neutrophils increased markedly in tracheal lavage fluids and in tissue and were strongly associated with the challenge-induced epithelial craters of damage-restitution. At 1 and 24 h eosinophils were increased in the tracheal lumen whereas the surrounding tissue displayed a reversed pattern. Gels rich in fibrinogen, neutrophils, and eosinophils were present in epithelial crater areas, protruding into the lumen. Clusters of free eosinophil granules, Cfegs, released through lysis of eosinophils, and neutrophils with long cytoplasmatic protrusions abounded in these crater areas. The present findings provide important new insights into allergic airways where sites of epithelial damage-restitution processes emerge as the major loci for eosinophil, neutrophil, and plasma protein activities, the latter likely causing leukocyte adhesion and activation in vivo. The distribution of eosinophils in this study suggests roles of these cells both in airway mucosa and in regional lymph nodes. Based on the present study we also propose that lysis of eosinophils and Cfegs generation are a major paradigm for activation of these cells in vivo.

Anatomical studies on the geometry and stability of the distal radio ulnar joint.

In fresh-frozen amputated- and cadaver arm specimens the anatomy and stability of the distal radio ulnar joint were investigated. The articulating surface of the sigmoid notch of the radius and the corresponding surface of the ulnar head facing the sigmoid notch were studied in transverse cryo sections. In each specimen the radius of the curvature of the sigmoid notch was 4-7 mm larger than that of the ulnar head and consequently pronation and supination are combined rotation-sliding movement in the distal radio ulnar joint. The radio ulnar ligament consists of a dorsal and a volar fibrous part, broadly attached to the distal rim of the sigmoid notch and converging towards the fovea of the ulnar head. The cartilaginous disc is centrally located between these fibrous strands. In neutral position the articulating surface of the sigmoid notch is optimally covering the articulating surface of the ulnar head. This contact area is gradually diminished during pronation-supination until only a marginal contact remains at the end of each movement. The distal radius is kept stable in pronation by the volar part and in supination by the dorsal part of the radio ulnar ligament.

Immunocytochemical Localization of Albumin and Factor XIII in Thin Cryo Sections of Human Blood Platelets

Albumin and factor XIII alpha 2 were localized in human blood platelets by immunofluorescence and protein A-colloidal gold immuno-electron microscopy using thin and ultrathin cryosections of mildly fixed human blood platelets. Albumin was found in alpha-granules, co-distributing with beta-thromboglobulin in double label experiments. In platelets directly fixed in plasma, without previous washing, albumin was also localized on the plasma membrane and in the surface connecting system. Factor XIII alpha 2 was localized diffusely through the entire cytoplasmic matrix.

Electron probe and electron energy loss analysis in biology

Methods, applications and limitations of quantitative electron probe analysis, X-ray mapping, electron energy loss analysis and energy filtered imaging are described, with emphais on the analysis of thin (less than 200nm) cryosections. Energy dispersion electron probe analysis can measure reliably 5 to 10mM/Kg of biologically prevalent elements in 50nm diameter areas of 100 to 150nm thick cryo sections during 100–300 sec counts. The minimal detectable mass (MDM) with a conventional thermionic electron source is approximately 10 −19g Fe (100 sec count) and can be reduced to 10 −20g through the use of a field emission gun (FEG). A spatial resolution of 8.7nm is demonstrated in two-dimensional Fourier transforms of Mo X-ray maps of stained catalase crystals. Significant biological results of quantitative electron probe analysis include the measurement of total Ca released from and Mg and K taken up by the sarcoplasmic reticulum during muscle contraction, and the demonstration that mitochondria do not contribute to the physiological regulation of cytoplasmic free Ca levels in cardiac, vascular smooth and striated muscle. Electron energy loss analysis (EELS) promises a significant improvement in sensitivity for the measurement of Ca; based on statistical errors of the measurement, 250μM/Kg Ca should be measurable with EELS in 250 sec. through the Ca L-edge loss. The use of a doubly corrected magnetic sector spectrometer as a transmission electron microscope imaging filter outside the microscope vacuum is illustrated, and the resolution of the iron core (7.5nm) and surrounding organic shell of single ferritin molecules is demonstrated in, respectively, iron M and carbon K loss images.

Immunocytochemical localization of fibrinogen and factor VIII in ultrathin cryo sections of human blood platelets

Immunocytochemical localization of fibrinogen and factor VIII in ultrathin cryo sections of human blood platelets

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-mum diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.