Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens.

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.

In vitro-in vivo evaluation of chitosan-PLGA nanoparticles for potentiated gastric retention and anti-ulcer activity of diosmin.

BACKGROUND: Diosmin showed poor water solubility and low bioavailability. Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles were successfully used to improve the drugs solubility and bioavailability. Coating of PLGA nanoparticles with chitosan can ameliorate their gastric retention and cellular uptake. METHODOLOGY: PLGA nanoparticles of diosmin were prepared using different drug and polymer amounts. Nanoparticles were selected based on entrapment efficiency% (EE%) and particle size measurements to be coated with chitosan. The selected nanoparticles either uncoated or coated were evaluated regarding morphology, ζ-potential, solid-state characterization, in vitro release, storage stability, and mucoadhesion. The anti-ulcer activity (AA) against ethanol-induced ulcer in rats was assessed through macroscopical evaluation, histopathological examination, immunohistochemical localization of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transmission electron microscopic examination of gastric tissues compared to free diosmin (100 mg/kg) and positive control. RESULTS: Based on EE% and particle size measurements, the selected nanoparticles, either uncoated or coated with 0.1% w/v chitosan, were based on 1:15 drug-PLGA weight ratio and 20 mg diosmin employing methylene chloride as an organic phase. Examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed nanoscopic spherical particles. Drug encapsulation within the selected nanoparticles was suggested by Fourier transform-infrared, differential scanning calorimetry (DSC) and X-ray diffractometry results. Chitosan-coated nanoparticles were more stable against size enlargement probably due to the higher ζ-potential. Only coated nanoparticles showed gastric retention as revealed by SEM examination of stomach and duodenum. The superior AA of coated nanoparticles was confirmed by significant reduction in average mucosal damage, the majority of histopathological changes and NF-κB expression in gastric tissue when compared to positive control, diosmin and uncoated nanoparticles as well as insignificant difference relative to normal control. Coated nanoparticles preserved the normal ultrastructure of the gastric mucosa as revealed by TEM examination. CONCLUSION: The optimized chitosan-coated PLGA nanoparticles can be represented as a potential oral drug delivery system of diosmin.

A tissue-engineered stomach as a replacement of the native stomach.

BACKGROUND: Despite recent advances in reconstruction techniques, total gastrectomy is still accompanied by various complications. As an alternative treatment, we propose a tissue-engineered stomach that replaces the mechanical and metabolic functions of a normal stomach. The objective of this study was to demonstrate the function of a tissue-engineered stomach as a replacement of the native stomach. METHODS: Tissue-engineered stomachs were formed in recipient rats from stomach epithelium organoid units isolated from neonatal donor rats. After 12 weeks, the animals underwent a second operation for replacement of the native stomachs. RESULTS: Tissue-engineered stomachs were successfully used as a substitute of the native stomach in a rat model. An upper gastrointestinal tract study revealed no evidence of bowel stenosis or obstruction at both anastomosis sites. Histologically, the tissue-engineered stomachs had well-developed vascularized tissue with a neomucosa continuously lining the lumen and stratified smooth muscle layers. Immunohistochemical staining for alpha-actin smooth muscle showed that the smooth muscle layers were arranged in a regular fashion. Scanning electron microscopy showed that the surface topography of the tissue-engineered stomachs resembled that of native stomachs. CONCLUSIONS: It has been demonstrated that a tissue-engineered stomach can replace a native stomach in a rat model. Replacement of the native stomach by a tissue-engineered stomach had beneficial effects on the formation of neomucosa and smooth muscle layers in the tissue-engineered stomach.

A new technique for staining sections and recovering celloidin film in high resolution autoradiography.

Ultrathin sections are stained immediately after cutting by placing them in contact with staining solution and then placed on a slide covered by a celloidin film. This method largely avoids precipitates of heavy metals. The recovering of celloidin film is improved using a stainless steel basket. This technique is far more reliable than that involving use of a filter paper.

Biaxial strength of multilaminated extracellular matrix scaffolds.

Xenogeneic extracellular matrix (ECM) can be harvested and configured to function as a bioscaffold for tissue and organ reconstruction. The mechanical properties of the ECM vary depending upon the tissue from which it is harvested. Likewise, the manufacturing steps required to develop ECMs into medical grade devices will affect the surface morphology and the mechanical properties of the bioscaffold; important properties for constructive tissue remodeling. The present study compared the ball-burst strength of five different ECM scaffolds before and after treatment with peracetic acid (PAA): porcine small intestinal submucosa (SIS), porcine urinary bladder submucosa (UBS), porcine urinary bladder matrix (UBM), a composite of UBS + UBM, and canine stomach submucosa (SS). This study also compared the mechanical properties of 2- and 4-layer ECM scaffolds. Results showed 2-layer SS devices had the highest ball-burst value of all 2-layer ECM devices. Moreover, all 4-layer ECM devices had similar ball-burst strength except for 4-layer UBM devices which was the weakest. PAA-treatment decreased the ball-burst strength of SS and increased the ball-burst strength of UBS 2-layer devices. This study showed the material properties of the ECM scaffolds could be engineered to mimic those of native soft tissues (i.e. vascular, musculotendinous, etc) by varying the number of layers and modifying the disinfection/sterilization treatments used for manufacturing.

In vitro assessment of the mucoadhesion of cholestyramine to porcine and human gastric mucosa.

Previous in vivo studies have suggested that the extended gastric residence and uniform intragastric distribution of cholestyramine may be due to mucoadherent properties. This series of in vitro investigations explored the possibility of the anion exchange resin exhibiting bioadhesive behaviour, and investigated the characteristics, such as particle size and surface charge, that may affect it. Tensile strength measurements were carried out to determine the mucoadhesion of cholestyramine and other test materials (resin particulates, polymers and hydrogels) with varying adhesive properties, to isolated porcine and human gastric mucosa. Optimal instrumental parameters for the system were determined initially and used; all procedures were carried out at room temperature (22 degrees C). The particle size of cholestyramine did not affect mucoadhesion to either porcine or human gastric mucosa (P=0.673, porcine; P=0.969, human), whilst anionic exchangers were found to provide better mucoadhesion than cationic exchangers (P=0.0002, porcine; P=0.0009, human). In some instances, it was found that the detachment forces recorded were lower with human gastric mucosa than with porcine gastric mucosa, although this was not consistently statistically significant. A rank order of mucoadhesion was constructed from a comparison of cholestyramine with eight other test materials. Cholestyramine produced the second highest degree of mucoadhesion, with Carbopol producing the greatest adhesion. Dextran and polyethylene glycol did not display good mucoadhesion under these conditions. From the findings presented here, we have found that cholestyramine demonstrates good mucoadhesion to both porcine and human gastric mucosa when compared to other known bioadhesives. It is suggested that particle size does not contribute to this mucoadherent behaviour but the surface charge of the resin has a significant part to play.

[Interaction of indigenous parietal microorganisms with cells of the digestive tract mucosa]. / Vzaimodeistvie indigennykh pristenochnykh mikroorganizmov s kletkami slizistoi obolochki pishchevaritel'nogo trakta.

By means of light, electron microscopy and stereomorphometry the interaction of microorganisms (MO) and cells of the mouth, stomach and gut mucous membrane was studied in different pathologic conditions on clinic and experimental material. No penetration was noted of MO into cells of the keratinized squamous epithelium, they were present in intercellular spaces. In the stomach, MO as a rule interact with mucocytes altering their surface. Spirillum-like MO sometimes penetrate into the parietal cells. In the small bowel MO may penetrate the cells causing lysis of apical membranes and photocytosis changing the microvilli structure. MO may enter the goblet cells during the secretion. They may settle in the cell cytoplasm without causing its alteration. In crypts, near Paneth cells, MO were subjected to lysis. The peculiarities of MO interaction with cells depend on their structural-functional status.

The use of nano-quercetin to arrest mitochondrial damage and MMP-9 upregulation during prevention of gastric inflammation induced by ethanol in rat.

Gastric ulcer is a multifaceted process that involves reactive oxygen species (ROS) generation, extracellular matrix degradation and mitochondrial damage. Mitochondria play a crucial role for homeostasis of ROS and cell survival. In our study, we investigated the efficacy and mechanism of polymeric nanocapsuled quercetin (NQC) over the free quercetin (QC) molecule in prevention of ethanol-induced gastric ulcer in rat. NQC possessed significantly higher efficacy (~20 fold) than free QC while preventing gastric ulcers. Our data show that prior administration of NQC and/or QC significantly blocked synthesis and secretion of matrix metalloproteinase (MMP)-9 as well as infiltration of inflammatory cells and oxidative damage in rat gastric tissues. As compared to free QC, NQC protected much better the mitochondrial integrity and size along with mitochondrial functions by controlling succinate dehydrogenase and NADH oxidase in rat gastric tissues. In addition, both free QC and NQC down regulated PARP-1 as well as apoptosis during protection against ethanol-induced gastric ulcer. Herein, the effect of NQC was greater than QC on expression of enzymes like cyclooxygenase and nitric oxidase synthase (NOS)-2. We conclude that NQC with greater bioavailability offers significantly higher potency in downregulating MMP-9 and NOS-2 as well as oxidative stress in blocking ethanol-induced gastric ulcer.

Morphological features of the stomach of Malayan pangolin, Manis javanica.

The morphology of the stomach of Malayan pangolin, Manis javanica was studied at macroscopic, light microscopic, and scanning electron microscopic levels. The stomach of M. javanica was C-shaped with short lesser curvature. At the oesophageal junction, the inner smooth muscle was thickened in the greater curvature side. The entire stomach was lined by a thick cornified stratified squamous epithelium, except at the duct orifices of glands and in the pyloric gland region. The wall of the fundus was thin and devoid of glands. The gastric glands consisted of mucous, oxyntic, and pyloric glands. The mucous glands were observed in the lesser curvature (Mg-L), in the greater curvature (Mg-G), and in the pyloric canal (Mg-C) respectively. The oxyntic glands were organized into gland mass, making an oval mound elevated to the gastric lumen, in the middle of the greater curvature. The oxyntic gland mass has a single common duct with opening directed to the pyloric side. This duct was surrounded by mucus gland (Mg-G). The pyloric glands were located caudal to the pylorus. There was no sphincter at the pyloric-duodenal junction. Large mucosal protuberance, the torus pyloricus was observed in the side of the lesser curvature of the pyloric canal. In the lumen of pyloric canal region, numerous spines and small pebbles were observed. The muscle layers in the wall of this region were considerably thickened. The present results on the stomach of M. javanica are thought to be closely related to the toothless and eating habits of this animal species.

An investigation into the role of mucus thickness on mucoadhesion in the gastrointestinal tract of pig.

Mucoadhesion in the gastrointestinal tract is a complex phenomenon and both formulation and physiological features need to be well understood and considered. Mucus thickness has been inferred to play a role in this process; however no definitive influence has been established. This study aimed to investigate the influence of mucus thickness on the mucoadhesion process, using a large animal (pig) as a model to closely resemble the human physiological features. The mucus thickness of different regions of the gastrointestinal tract of pig was fully measured by means of a histochemical method (hematoxilin/eosin) employing cryostat sections. Mucoadhesion was evaluated ex vivo on porcine mucosa by tensiometry using a polyacrylic acid polymer (Carbopol 974P NF) as a mucoadhesive model material, both in a dry and swollen state. Mucus was thickest in the stomach (body 67.9+/-54.7 microm) and mucus thickness increased from proximal to distal segments in both the small intestine (duodenum 25.9+/-11.8 microm, ileum 31.0+/-15.7 microm) and large intestine (caecum 19.4+/-8.7 microm, ascending colon 31.9+/-17.2 microm, descending colon 35.1+/-16.0 microm and rectum 40.8+/-12.5 microm). Swollen polymer exhibited lower mucoadhesion than the dry form in all sections analysed. Mucus thickness plays a role on the mucoadhesion, as thicker mucus provides deeper polymer chain diffusion and entanglements; however, other factors are also involved in this complex process.

A histological, histochemical and ultrastructural study of the digestive tract of Dentex dentex (Pisces, Sparidae).

Dentex dentex has a short esophagus, a large caecal type stomach, three to six pyloric caeca and a short intestine. Light and electron microscope studies reveal that the esophageal mucosa displays primary and secondary folds, a stratified squamous epithelium with fingerprint-like microridges alternating with a few zones formed by a single layer of columnar cells with apical microvilli. Only primary folds are present in the stomach, which is rich in simple tubular glands, these being absent in the pyloric valve. Two cell types occur in the gastric glands, one with a well developed apical intracytoplasmic membrane system consisting of a vesicular network of smooth membranes, and the other with a supranuclear tubulovesicular system. Pyloric caeca and anterior and posterior intestine mucosae display the same pattern of folding, with primary and secondary folds, without following a definite pattern in their orientation. In the rectum, the folds are oriented longitudinally. Small dense particles containing chylomicrons appear in groups in the intercellular spaces of the caecal and anterior intestinal epithelia. Eosinophilic granular cells (mast cells) appear along the digestive tract mainly within the stratum compactum. Histochemical studies reveal no differences in the composition of goblet cell mucus along the digestive tract. No histochemical differences were detected between enterocytes of the intestine, pyloric caeca and rectum. Neutral mucosubstances dominate in the stomach epithelium and in the goblet cells of the esophagus, pyloric caeca and anterior intestine. Results of the present study are discussed in relation to descriptions of the digestive tract in other sparids.

Ultrastructural distribution of lectin-binding sites on gastric superficial mucus-secreting epithelial cells. The role of Golgi apparatus in the initial glycosylation.

Normal human gastric epithelial cells were examined by electron microscopy using each of five biotinylated lectins [Ulex europaeus agglutinin I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA)] as a probe. We employed 35 gastric surgical specimens removed from complicated peptic disease. The lectin-binding sites were revealed with streptavidin-colloidal gold complex. All specimens were embedded in Spurr and LR White resins. In superficial foveolar epithelial cells, the lectins used were generally positive in all cell types (mainly UEA-1 and PNA) on the Golgi region and mucus cytoplasmic vacuoles, with many variations among cells in the same case. On the other hand, extracellular mucus was negative for WGA. Labelling with PNA revealed a biphasic pattern (peripheral positivity) on mucous droplets in surface and foveolar cells. The cis side of the Golgi apparatus was labelled with SBA and PNA and rough endoplasmic reticulum with SBA (only five cases). Lectin-binding variability could be related to heterogeneous composition of gastric mucus. Our results with SBA suggest initiation of O-glycosylation at the Golgi apparatus; however a role of the rough endoplasmic reticulum cannot be excluded (N-glycosylation). We propose the following sequence of sugar addition to the carbohydrate side-chains of gastric glycoproteins: (1) GaNAc (Golgi apparatus cis-side), (2) GlcNAc (Golgi apparatus intermediate face), (3) GalNac or Gal, alpha-L-fucose (Golgi apparatus trans-side).

Calcium transport and calcium activated ATPase activity in microsomal vesicles of rat gastric mucosa.

1. Microsomal and plasma membrane vesicles, isolated from rat gastric mucosa, were found to exhibit Ca(2+)-dependent ATPase activities of 14.1 +/- 1.4 and 7.8 +/- 1.1 mumol/mg/hr, respectively. The optimum conditions for the microsomal Ca(2+)-ATPase was pH 6-7, and required Mg2+, while divalent cation such as Cu2+, Zn2+, Fe2+, Ba2+ and Cd2+ had no significant effect. 2. As in the case of Ca2+, Mg(2+)-ATPase, the Ca2+ uptake activity of the microsomal membrane required Mg2+. Both processes were stimulated by submicro molar concentrations of Ca2+ and the apparent Km for Ca2+, Mg2+ ATPase and Ca2+ uptake activities were 0.06 microM and 0.02 microM, respectively. 3. Divalent cations Ba2+ and Fe2+, inhibited both microsomal activities, while Zn2+ and Cd2+ showed no effect on them. However, the monovalent cation K+ did not stimulate Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities. 4. The Ca2+ pumping ATPase of rat gastric mucosal microsome cross-reacted with a monoclonal antibody (mAb-5F10) against the human erythrocyte Ca2+ pump. The apparent molecular weight of mucosal Ca2+ pump was 98 kDa. 5. Close relationship between the kinetic parameters of Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities, and the cross reaction of 98 kDa protein of mucosal microsome with erythrocyte Ca2+ pump antibody, strongly suggest the expression of Ca2+ pump in rat gastric mucosa.

Post-embedding immuno-electron microscopy with rapid freezing and freeze-substitution techniques for the localization of manganese superoxide dismutase.

Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen-antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.

Comparison of the effects of NaF and CaF2 on rat gastric mucosa. A light-, scanning- and transmission electron microscopic study.

Rat stomachs were studied after intragastrically administered, repeated doses of 0.1 N HCl, 100 mmol/l NaF, 50 mmol/l CaF2 in 0.1 N HCl, respectively. NaF produced extensive desquamation and cell injury, while CaF2 caused some desquamation and a slight decrease in secretory activity as revealed by light microscopic, SEM and TEM examinations.

[Ultrastructural identification of argyrophil and argentaffin cells of the gastric mucosa in the rat]. / Ul'trastrukturnaia identifikatsiia argirofil'nykh i argentaffinykh kletok slizistoi obolochki zheludka u krys.

Ultrastructure of endocrine cells impregnated in the rat gastric mucosa by Grimelius method (identification of argyrophilia) and by Masson--Hamperl method (identification of argentaffinity) and influence of various fixatives on the structure and properties of the secretory granules in these cells have been studied. Fixation of the material in paraformaldehyde or glutaraldehyde varies in its effect on the granule structure of EC-, D1- and ECL-cells, while its influence on the granule structure of G-, D- and AL-cells is identical. The granules of EC-, ECL- and G-cells are argyrophil, and only those of EC-cells are argentaffin. Weak argyrophilia, which is evidently not appearant at the light-optical level, is specific for granules of D1- and AL-cells. Fixation in paraformaldehyde and especially the subsequent treatment in osmium tetroxide results in increasing argyrophilia of the endocrine cells, as compared to fixation in glutaraldehyde. Varieties in the effect of the fixatives do not prevent ultrastructural and histochemical identification of the endocrine cell types.

Recovery of rat gastric mucosa following single fluoride dosing.

Rats' stomachs were intubated with 0.1 N HCl or 100 mM NaF in 0.1 N HCl and excised after 1, 12, 24, 48 h or 7 days for light microscopy. The NaF solution caused erosive injury to the oxyntic glandular mucosa 1 h after application. Progressive stages of recovery were seen at 12, 24 and 48 h after fluoride dosing. Complete recovery was seen 7 days after exposure to fluoride. The kinetics of the recovery process from fluoride injury appear to be similar to those which have been reported for gastric injury produced by other substances.

Immunocytochemical demonstration of the secretory dynamics of zymogenic contents in rat gastric gland processed by high-pressure freezing/freeze substitution, with special references to phospholipase A(2) and phospholipase Cgamma1.

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.

Lysophosphatidylcholine and taurodeoxycholate increase stomach permeability to different-sized molecules.

The influence of lysophosphatidylcholine (LPC), taurocholate (TC), and taurodeoxycholate (TDC) on gastric mucosal permeability was studied in a rat experimental model, with different-sized polyethylene glycols (PEGs) in the 722-1206-dalton range as permeability markers. Gastric mucosal morphology was also studied by transmission electron microscopy. We found that 2.5 mM and 5 mM LPC and TDC, but not TC, caused an increase in the passage of PEGs across the gastric mucosa. LPC altered the permeability significantly more than did TDC. Morphologically damaged intercellular microvillous structures could be seen after LPC treatment, whereas no obvious changes could be seen after TC or TDC treatment. These findings indicate that LPC and TDC may damage the gastric mucosa and enable permeation of molecules in the 722-1206-dalton range. Molecules within this range could potentially be toxic or antigenic, and this therefore represents an aspect of interest in the pathology of enterogastric reflux. Furthermore, the results indicate that dihydroxy secondary bile acids (TDC) have a more pronounced effect on gastric mucosal permeability than trihydroxy primary bile acids (TC).

Surface changes in rat gastric mucosa induced by sodium fluoride: a scanning electron microscopic study.

This paper describes the ultrastructural, topographical changes seen in rat gastric mucosa following application of 0.1 N HCl or 1, 10 or 50 mM NaF in 0.1 N HCl to the stomach. No morphological differences were noticed between the 0.1 N HCl (control) and 1 mM NaF in 0.1 N HCl specimens. Ten milliomolar NaF in 0.1 N HCl produced some desquamation of surface mucous epithelial cells while 50 mM NaF produced extensive damage to cells surrounding the gastric gland openings (foveolae) as well as interfoveolar cell loss.