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PubMed1996

Helicobacter pylori and the surface mucous gel layer of the human stomach.

BACKGROUND: The colonization of Helicobacter pylori in the surface mucous gel layer (SMGL) was investigated. MATERIALS AND METHODS: Surgically removed stomachs were obtained from patients and included gastric ulcer (4 cases), duodenal ulcer (2), and gastric cancer (24). Five of these cases were examined at 8, 19, 28, 143, and 171 days after the end of eradication therapy. For the preservation of the SMGL, these specimens were fixed in cold Carnoy's solution, cleared in xylene, and embedded in paraffin. Serial sections were obtained and were stained by dual staining with the galactose oxidase-cold thionin Schiff reaction followed by paradoxical Concanavalin A staining and immunostaining for H. pylori. RESULTS: H. pylori characteristically attached to surface mucous cells and colonized in the SMGL H. pylori in the SMGL was more abundant than that attached to the surface mucous cells. The degree of H. pylori infection both on the surface of surface mucous cells and in the SMGL correlated well with the severity of gastritis. In the SMGL, this organism obviously preferred to colonize in the layer of surface mucous cell-type mucins, and the multilaminated structure of the SMGL deteriorated markedly. Eradication of H. pylori restored the structure of the SMGL, and the inflammatory reaction decreased gradually. CONCLUSION: The SMGL is an indispensable site of H. pylori colonization, and this organism damaged the gastric mucosa partially by causing deterioration of the SMGL. Removal of the organism from the SMGL should be considered for eradication of this organism.

Acetic Acid↗
PubMed1991

Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantages of microwave fixation.

Glutathione peroxidase (GSH-PO), a highly soluble, selenium-dependent enzyme metabolizing lipid peroxides, is allegedly distributed in both the cytosol and mitochondria. With the pre-embedding method of immunoelectron microscopy for GSH-PO employing conventional immersion-fixation, the nuclei of rat hepatocytes stain positively, whereas mitochondria are negative. Such observations are inconsistent with the results of biochemical and immunoblot analyses using isolated subcellular fractions. In the present study, we employed the combination of microwave irradiation and fixation in 4% paraformaldehyde (PFA), with or without 0.1% glutaraldehyde (GA), to enhance the accuracy of ultrastructural localization of GSH-PO in rat liver. A small block of liver was irradiated by microwave for 10 sec in cold cacodylate-buffered 4% PFA containing 0.1% GA. After further immersion of the tissue in 4% PFA at 4 degrees C for 1-6 hr, the standard procedure for pre-embedding immunoelectron microscopy was employed. We observed partial inhibition of artifactual diffusion of cytosolic GSH-PO into the nuclei and consistent GSH-PO localization in mitochondria. Dual localization of this enzyme in the cytosol and mitochondria of normal rat hepatocytes was thus confirmed.

Animals↗
PubMed1983

The use of specific antibodies to demonstrate the glycocalyx and spatial relationships of a K99-, F41- enterotoxigenic strain of Escherichia coli colonizing the ileum of colostrum-deprived calves.

Electron microscopy was used to study the interaction between the glycocalyx of enterotoxigenic Escherichia coli strain 210 (09:K30+;K99-;F41-:H-) and the glycocalyx of epithelial cells in then ileum of experimentally infected newborn colostrum-deprived calves. Fixation of tissues in anti-K30 antibody and ruthenium red was used to stabilize the bacterial glycocalyx so that the spatial relationship between the bacteria and the intestinal epithelial cells could be characterized. When strain 210 was grown in vitro and reacted with anti-K30 antibody prior to staining with ruthenium red, the extensive glycocalyx could be clearly visualized surrounding the bacterial cells. By negative staining, an unidentified pilus was also seen. Sections of ileum from infected calves, which were not fixed in antibody nor stained with ruthenium red, revealed attached bacteria which were surrounded by an electron-translucent zone and no visible bacterial glycocalyx. When ruthenium red staining was used, the bacterial glycocalyx partially collapsed during the dehydration steps of fixation, but could be seen as either a fibrous capsule or an electron-dense accretion on the bacterial cell surface. When ileal tissue was reacted for one hour in anti-K30 antibody before staining with ruthenium red, the bacterial glycocalyx was seen as a discrete electron-dense structure up to 1.0microm thick which was in intimate contact with the glycocalyx of the epithelial cells. The importance of the bacterial exopolysaccharide to microcolony formation on the villi could be clearly visualized.

Animals↗
PubMed2006

Using a freeze substitution fixation technique and histological crimp analysis for characterizing regions of strain in ligaments loaded in situ.

Type I collagen fibrils in tendons and ligaments assume a sinusoidal wave shape, or crimp, which straightens only with tensile load. The load response of crimp has been studied primarily in isolated subunits and not in complex, intact structures. The purpose of our study was to determine if freeze substitution fixation of an entire ligament could preserve changes in crimp morphology induced by functionally relevant loading conditions. We hypothesized that, in ligaments prepared by freeze-substitution fixation under load, crimp would progressively extinguish with increasing loads, and nonuniform strain following partial section could be detected from crimp morphology. Tensile loads ranging from 0 to 220 N were applied to patellar ligaments of 16 fresh rabbit stifle joints using simulated isometric quadriceps pull through the patella. The loaded joints were flash frozen with isopentane cooled in liquid nitrogen, then fixed using freeze substitution. Another six ligaments were loaded to 150 N following incision of the anterior third and evaluated under polarized light microscopy for crimp distribution. Ligaments with no or low loads could be identified by the presence of crimp on mid-sagittal sections. Strain distribution was inhomogeneous, in that the ligament displayed a consistent pattern of collagen fiber recruitment among three morphologically distinct bands seen on coronal sections. At very low loads (about 18 N), the fibers in a central band were uncrimped; anterior and deep bands uncrimped at higher loads. The crimp in the entire specimen was extinguished at about 67 N, which correlates closely with the previously reported toe-region of the stress-strain curve of the rabbit patellar ligament. When the anterior third was transected, fibers within that segment retained a crimp in ligaments prepared under loads that ordinarily would ablate all crimp. These findings suggest that freeze fixation could be used to map the functional microstructure of ligaments or tendons.

Animals↗
PubMed1998

Improvement of the measurements on skinned muscle fibres by fixation of the fibre ends with glutaraldehyde.

Experiments with activated skinned muscle fibre segments are limited by the structural and mechanical instability of the preparations. The present study shows that fixation of the muscle fibre ends with glutaraldehyde significantly improves the reliability of such experiments. We tested the effects of a specific glutaraldehyde fixation technique on the structural stability and the mechanical properties of skinned rat and rabbit skeletal muscle fibres in an approach where the fibre segments are attached to the apparatus by gluing. Preparations with fixed and unfixed ends were compared. During the first few minutes of maximal activation, fibres with fixed and unfixed ends exhibited similar mechanical properties to one another, suggesting that our fixation procedure selectively impregnates the fibre ends without contaminating the remaining active fibre part. During prolonged maximal activations (3-60 min), preparations with fixed ends exhibited a better stability, both in the sarcomere length signal (detected by laser diffraction) and in the unloaded shortening velocity. Thus, our technique of muscle fibre end fixation caused a substantial improvement in the mechanical measurements on skinned muscle preparations.

Adhesives↗
PubMed1995

Bone changes after experimental osteotomies fixed with absorbable self-reinforced poly-L-lactide screws or metallic screws studied by plain radiographs, quantitative computed tomography and magnetic resonance imaging.

The healing of the distal femoral osteotomy fixed with self-reinforced poly-L-lactide (SR-PLLA) or metallic screws in 16 rabbits was evaluated with plain radiographs, quantitative computed tomography and magnetic resonance imaging (MRI). At 36 weeks in the metallic fixation there was significantly more external callus than in the SR-PLLA fixation. On the metallic fixation side the cortical bone mineral density was significantly lower than on the non-operated side at 6 weeks as well as at 36 weeks. This decrease was not found in the SR-PLLA fixation. On the MRI on T1-weighted images a dark zone (signal void), and on the T2-weighted images a bright zone (increased signal intensity), was seen surrounding the screws, indicating oedema. At 36 weeks these oedematous zones were significantly smaller in the SR-PLLA fixation group than in the metallic one. The results give indirect evidence towards a more rapid and better osteotomy healing with the more physiologically elastic SR-PLLA screws than with the metallic screws. Fixation with SR-PLLA screws may prevent stress-protection atrophy and weakening of the fixed bone usually caused by the rigid metallic fixation.

Absorption↗
PubMedDate not supplied

Fixation of testes and eyes using a modified Davidson's fluid: comparison with Bouin's fluid and conventional Davidson's fluid.

Most recent revisions of regulatory guidelines for testing effects of chemicals on reproduction recommend Bouin's fluid (BF) or a "comparable fixative" instead of formalin to preserve the morphologic detail of testes for histopathological evaluation. However, picric acid in BF is a health and safety hazard, as well as a laboratory waste disposal problem. Furthermore, use of BF is labor intensive, requiring multiple alcohol rinses to remove picric acid for optimum preservation and immunohistochemical (IHC) detection of testicular antigens that may potentially be used to identify and quantify cells and functional proteins with critical roles in spermatogenesis. Recently a modified Davidson's fluid (mDF) has been reported as an altemative to BF to fix testes for routine histopathological examination. This study compared the overall histomorphologic clarity and the immuno- and histochemical staining of testicular specimens fixed in BF and mDF. Additionally, because conventional Davidson's fixative (DF) is used routinely for optimum fixation of eyes, preservation of ocular histomorphology by DF and mDF was compared. mDF resulted in noticeably less shrinkage of the seminiferous tubules and superior overall morphologic detail compared to BF. Unlike DF, the mDF also supported excellent staining of acrosomes with periodic acid-Schiff (PAS) reagent when staging of spermatogenesis was required. IHC detection of androgen receptor and PCNA (to directly and indirectly identify Sertoli cells) as well as protein gene product 9.5 (to label spermatogonia) was Superior in mDF compared to BF-fixed specimens. For histopathological examination of the eye, apposition and preservation of rods and cones, and nuclear layers of the retina were slightly inferior with mDF compared to DF. This paper has demonstrated that mDF provides comparable, and in many respects superior preservation of the testes to that of BF, both for IHC staining and for detailed histopathological examination. It also provides an acceptable fixative for eyes, although the quality of cellular preservation is inferior to that of DF.

Animals↗
PubMed2006

Rapid fixation of brains: a viable alternative?

AIM: Recent events surrounding postmortem practices have necessitated several changes in the way necropsies are conducted both within the medicolegal and hospital settings. These changes have resulted in a striking reduction in the frequency of brain retention for neuropathological examination. Aberdeen Royal Infirmary, responding quickly to rising concerns among bereaved relatives regarding organ retention, instituted a change in practice regarding the examination of brains at necropsy as early as 2001. The new protocol involved subjecting the brain to rapid fixation using combined immersion and perfusion techniques. This allows return of the organ to the body without unreasonable delay to the funeral. In this article, we present the results of a retrospective audit comparing the efficacy of the new method against the conventional fixation protocol. METHODS: An equal number of postmortem brains were selected randomly from the year 1998 (following the usual fixation protocol), and from the year 2001 (following the new method). A three tier scoring system was devised to assess section and staining quality, and a single observer scored all the cases. RESULTS: No statistical difference was noted in the quality of sections prepared by rapid fixation those by conventional fixation. Furthermore, the rapid method was more frequently adequate for diagnosis (considering scores of 2 and 3 together) than the conventional method. CONCLUSION: Rapid fixation offers a viable alternative for detailed examination of brains at necropsy, without necessarily prolonging the retention of the organ.

Autopsy↗
PubMed1996

Bovine gamma/delta T-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes.

Bovine gamma/delta T cells have been shown previously to proliferate when cocultured with gamma-irradiated bovine monocytes in the 'autologous mixed leucocyte reaction' (AMLR). It was suggested that the response may be to culture-derived or culture-induced antigenic epitopes. Data presented here indicate that the gamma/delta T-cell stimulatory activity is attributable to a self-derived cell-surface molecule of mononuclear phagocytes that is constitutively expressed in vivo. The ability to induce an AMLR did not require in vitro culture or stress associated with in vitro isolation of cells or increased temperature since it could be induced by monocytes fixed by paraformaldehyde during blood collection from normal animals. Furthermore, stimulation by monocytes did not depend upon secreted molecules since fixed monocytes that had been incubated overnight at 37 degrees to allow secretion of preformed molecules, or subjected to hypotonic shock in H2O for 10 min before addition to the cultures, induced an AMLR as did plasma membranes prepared from ex vivo monocytes. In contrast, enzymatic treatment of monocytes to digest surface molecules followed by fixation destroyed their ability to stimulate an AMLR. The ability of monocytes to stimulate proliferation of gamma/delta T cells was distinguishable from their ability to stimulate alpha/beta T cells, since the former was destroyed by glutaraldehyde fixation whereas stimulation of alpha/beta T cells by major histocompatibility complex (MHC)-presented antigenic epitopes is not. Moreover, induction of proliferation of bovine gamma/delta T cells was not MHC-restricted. Finally, bovine alveolar macrophages, sheep monocytes and transformed bovine monocytes stimulated proliferation of bovine gamma/delta T cells whereas none of the following did so: human monocytes, murine macrophages, bovine myeloid cells other than mononuclear phagocytes, other nucleated cells found in bovine blood including activated MHC class II-bearing B cells, and a variety of species of bacteria. Thus, the stimulatory epitope is unique to and conserved among mononuclear phagocytes of ruminants. Demonstration of stimulation of bovine gamma/delta T cells by self-derived molecules is consistent with reports for murine gamma/delta T cells.

Animals↗
PubMed2005

Evaluation of bone plug and soft tissue anterior cruciate ligament graft fixation over time using transverse femoral fixation in a sheep model.

PURPOSE: To evaluate the mechanical role of bone-anterior cruciate ligament (ACL) graft junction in comparison with primary fixation of the graft. TYPE OF STUDY: Ex vivo controlled biomechanical study. METHODS: An ACL reconstruction was performed on 2 groups of 40 sheep each. The ACL graft was patellar tendon in group 1, and free tendon in group 2. Load-to-failure tests were performed at 1, 2, 3, and 6 months. Each group was divided into 2 subgroups: In subgroup A the femoral fixation devices were removed before testing and in subgroup B they were left in place. RESULTS: At 1 month, structural properties of subgroup 2A were significantly lower than other subgroups. Until the third month, structural properties of each subgroup were significantly lower than normal ACL. Grafts always failed at midsubstance, except for subgroup 2A at 1 month, which pulled out of the femoral tunnel. CONCLUSIONS: Bone plug incorporation was stronger than graft strength at 1 month whereas soft tissue tendon incorporation was not stronger than graft until 2 months. At 3 months, both groups failed at approximately 30% of the native ACL strength, and at 6 months, both groups were nearly equal in terms of load to failure and stiffness. CLINICAL RELEVANCE: High fixation strength and stiffness of ACL graft does not allow earlier return to sports activities.

Animals↗
PubMed1993

Preservation of cardiac myocytes subjected to different preconditions: a comparative morphometric study of beating, fibrillating, and cardioplegically arrested canine hearts.

This study compares the ultrastructure of beating canine hearts with that of hearts subjected to different clinically common forms of cardiac arrest. The contraction state per test field was ascertained according to a specially developed classification. The volume density of myofibrils and the surface to volume ratio of mitochondria were used as parameters for cellular and mitochondrial swelling. Contraction bands were not found in any of the differently pretreated hearts. Following immersion fixation, contractions as well as over- and hypercontractions in beating, fibrillating, and St. Thomas-arrested hearts are significantly more pronounced than in HTK-arrested hearts. Cellular and mitochondrial volumes were similar in beating and fibrillating hearts. St. Thomas-perfusion significantly decreased cellular and mitochondrial volume compared to beating hearts, but these values were in the same range as in fibrillating hearts. Only HTK-solution actually led to a strong reduction of these compartments. Compared to immersion, perfusion fixation after coronary perfusion with cardioplegic solutions led to comparable cellular volumes, but significantly elevated the percentage of relaxed sarcomeres and significantly reduced mitochondrial swelling. The best structural preservation of myocytes was found after HTK-perfusion and perfusion fixation. Such ultrastructural quantitative and morphometrical parameters are powerful tools since results confirm that the degree of myocardial preservation depends on the method of cardiac arrest. This forms the basis for the choice of preconditions for subsequent ischemia. Furthermore, significant alterations of myocardial ultrastructure depend on a combination of the functional state of the heart, the method of cardioplegia, and the technique of fixation.

Animals↗
PubMed1994

Brain fixation for acetylcholine measurements.

Brain fixation using a commercially available microwave oven (power output: 750 W) has been investigated as a means for enzyme inactivation preventing post-mortem changes in brain acetylcholine (ACh) and choline (Ch) levels. Rats and mice were decapitated, and the severed heads immediately irradiated for 5.5 and 3 s, respectively, resulting in a complete inactivation of brain acetylcholine esterase (AChE) and choline acetyltransferase (ChAT). The ACh and Ch contents measured in various brain regions of rat and mouse were: (in rat) striatum 60.5 and 32.4 nmol/g, hippocampus 20.4 and 30.9 nmol/g, cortex 24.2 and 19.6 nmol/g; (in mouse) striatum 70 and 47.2 nmol/g, hippocampus 22. 1 and 30.2 nmol/g, cortex 22.9 and 27.9 nmol/g. These values were found in accordance with those reported in the literature by irradiating whole animals in instruments of higher power capabilities. Thus, the procedure described in the present work may be a simple and valuable means of brain fixation for neurochemical studies of brain ACh in small animal species.

Acetylcholine↗
PubMed2001

An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis.

OBJECTIVE: To improve existing preimplantation genetic diagnosis fixation techniques. DESIGN: Prospective randomized in vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The intensity and clarity of fluorescence in situ hybridization (FISH) signals and the percentage of successfully fixed blastomeres. RESULT(S): The described fixation technique resulted in 100% fixation and 100% adequate FISH signals. Two conventional techniques resulted in 94% and 87% fixation, and in adequate FISH signals in 81% and 87%, respectively. CONCLUSION(S): This newly developed fixation technique simplifies the process of fixation of blastomeres for preimplantation diagnosis while essentially eliminating the possibility of losing a cell during fixation. It will hopefully allow more IVF programs to offer their patients preimplantation genetic diagnosis using the FISH technique.

Blastomeres↗
PubMed1996

Microwave fixation method for cytochemistry. For conventional electron microscopy, enzymo-immunocytochemistry, autoradiography elemental distribution studies and staining methods.

More than a decade ago, we introduced a microprocessed household microwave oven for microwave-stimulated fixation. In this paper we present an overview of our experience. We have introduced the use of tannic acid (TA, 0.1%), which is very effective for fixing soluble peptides or proteins, except in the case of enzymo-cytochemistry. In the latter case, a combination of TA with aldehyde in the microwave-fixation step brings perfect fixation, but inactivation of the enzymes and their enzymatic activities.

Alkaline Phosphatase↗
PubMed2000

Fixation of the electrode array with bone paté in cochlear implant surgery.

The aims of this study are to report on our experiences using a bone paté (a mixture of bone dust and fibrin glue) fixation technique of the Combi 40/Combi 40+ electrode array during cochlear implant surgery. A retrospective study involving 57 patients implanted with Combi 40/Combi 40+ cochlear implants over 31 months fixated with bone paté was performed, utilizing our electrical fitting data as well as re-operation and X-ray findings. We found no signs of electrode retraction in any of the 57 patients. Electrical fitting data showed no reduction in the number of active channels between the first and last times patients were evaluated (a period of 4-31 months). Furthermore, a comparison of X-rays performed within the first week following cochlear implant surgery and again 7-27 months later showed no signs of electrode retraction in 10 patients. Three patients who required re-operations after implantation allowed us the opportunity to examine the effectiveness of the bone paté fixation and revealed new bone growth with the electrode fixed in place. Histological examinations of the newly built bone showed laminar and compact bone structures. The use of bone paté was thus found to be a suitable method of electrode fixation in cochlear implant surgery. Compared with other fixation methods, it is inexpensive and uses no foreign materials.

Adult↗
PubMed1991

Automatic fixation facility for plant seedlings in the TEXUS Sounding Rocket Programme.

Automatic chemical fixation of plant seedlings within a 6 min period of reduced gravity (10(-4)g) was performed on three ballistic rocket flights provided by the German Sounding Rocket Programme TEXUS (Technologische Experimente unter Schwerelosigkeit = Technological Experiments in Microgravity). The described TEXUS experiment module consists of a standard experiment housing with batteries, cooling and heating systems, timer, and a data recording unit. Typically, 60 min before launch an experiment plug-in unit containing chambers with the plant material, the fixation system, and the temperature sensors is installed into the module which is already integrated in the payload section of the sounding rocket (late access). During the ballistic flight plant chambers are rapidly filled at pre-selected instants to preserve the cell structure of gravity sensing cells. After landing the plant material is processed for transmission electron microscopy. Up to now three experiments were successfully performed with cress roots (Lepidium sativum L.). Detailed improvements resulted in an automatic fixation facility which in principle can be used in unmanned missions.

Acceleration↗
PubMed1997

High and low risk prostate carcinoma determined by histologic grade and proliferative activity.

BACKGROUND: Histologic grade of differentiation is a strong prognostic factor for prostate carcinoma. However, most tumors fall in the intermediate group. Nuclear and nucleolar morphometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) were performed to improve prognosis, especially for patients with intermediate histologic grade tumors. METHODS: Core needle biopsies from 65 patients with primary prostate carcinoma at diagnosis were studied. Patients received only hormone therapy. Formalin fixed and paraffin embedded sections were stained with the method of Ploton. The mean AgNOR count was calculated in 100 tumor cells for each case. Nuclear and nucleolar areas from 100 cells were measured with an automated image analyzer. One-way analysis of variance and uni- and multivariate survival analyses were used for statistical evaluation. RESULTS: In the whole series, World Health Organization (WHO) tumor grade, nuclear and nucleolar areas, and AgNOR counts were correlated with survival time. By multivariate analysis, only AgNOR counts retained independent prognostic significance. In WHO Grade 2 carcinoma, the 5-year survival rate for patients with AgNOR/cell < or = 7.84 was 77%, but was only 12% for those with higher counts (P < 0.0001). These survival rates were similar to those obtained when patients with WHO Grade 1 carcinoma and Grade 2 carcinoma plus low AgNOR counts were compared with patients with Grade 3 carcinoma and Grade 2 carcinoma plus high AgNOR counts. In Gleason intermediate Grade 6 and 7 carcinomas, the 5-year survival rate for patients with AgNOR/cell < or = 7.84 was 71%, but was only 7% for those having higher counts (P = 0.0001). CONCLUSIONS: Nuclear and nucleolar areas, as well as AgNOR counts, supplement histologic grading in the prognostic assessment of prostate carcinoma in patients receiving only hormone therapy. AgNOR count also is a prognostic factor for patients with intermediate grade tumors. The combination of histologic grade and proliferative activity allows the stratification of patients into low and high risk groups.

Aged↗