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PubMed2003

Five-year outcome and predictability of soft tissue profiles when wire or rigid fixation is used in mandibular advancement surgery.

The purpose of this study was to follow the covariation of hard and soft tissue changes in Class II malocclusion subjects who received a bilateral sagittal split osteotomy. The subjects were randomized to receive wire or rigid fixation after the surgery. Subjects in the rigid group (n = 78) received 2-mm bicortical position screws, and those in the wire group (n = 49) received inferior border wires and 6 weeks of skeletal intermaxillary fixation with 24-gauge wires. Additionally, some subjects received genioplasty in both the rigid (n = 35) and the wire groups (n = 24). Soft and hard tissue profile changes were obtained from cephalometric films immediately before surgery and at various times up to 5 years postsurgery. Soft and hard tissue profile changes were referenced to a cranial-base X-Y coordinate system. Horizontal changes in mandibular incisor, lower lip, B-point, soft tissue B-point, pogonion, and soft tissue pogonion were calculated at each time. There was considerable skeletal relapse in the wire fixation group. Bivariate correlations and ratios between the hard and soft tissue changes were calculated for each time period. Hard to soft tissue correlations were the highest at the earlier times, although the ratios varied among the 4 groups. These results provide a solid basis for both short-term and long-term prediction.

Adult↗
PubMed1994

Alveolar septal structure in different species.

Because the retractive forces due to surface tension decrease with increasing radius of curvature, there should be a greater contribution to lung recoil attributable to the stress-bearing role of elastic elements in the lung parenchyma of species with larger alveoli. To examine alterations in lung structure that may relate to this stress-bearing role, the lungs of mice, hamsters, rats, rabbits, rhesus monkeys, baboons, and humans were preserved by vascular perfusion of fixative. The number of alveoli per lung, alveolar radius of curvature, surface area, and volume were measured by serial section reconstruction. Electron-microscopic determinations were made of the volume fraction and thickness of the epithelium, interstitium, and endothelium and of the connective tissue fibers of the alveolar septa and the portions of alveolar septa that form the alveolar ducts. The thickness of the alveolar septal interstitium increased linearly with the increase in radius of curvature of alveoli. The increase in interstitial thickness in lungs with larger alveoli was paralleled by large increases in the volume of collagen and elastin fibers present in this space. Comparable changes in the thickness of connective tissue fibers in alveolar duct walls were also found. This study demonstrates species-related changes in the structure of alveolar septa and in lung collagen and elastin fibers that are consistent with connective tissue fibers having a greater stress-bearing role in both the alveolar septa and alveolar ducts of species with larger alveoli.

Animals↗
PubMed1985

Stability of immunocytochemical reactivity of neuronal substances following delayed fixation.

The rates of deterioration of immunocytochemical staining for 4 peptide and 2 neuronal antigens were studied on human, guinea pig and rat colon and urinary bladder. It was found that if the fixation started within 10 h after surgery or death, little change was seen in the pattern and intensity of the immunostains. When fixation was further delayed, the immunostaining gradually deteriorated in quality and decreased in quantity until, when fixation was delayed for 60-72 h, most of the original immunostaining was absent. The deterioration rates of the neuronal antigens in the urinary bladder were slower than in the colon and all antigens were preserved for longer at 4 degrees C than at 20 degrees C. The deterioration curves produced from the present findings provide a standard for comparison with the immunostaining results obtained on tissues where fixation is delayed.

Animals↗
PubMed1992

Measurements of T1 and T2 over time in formalin-fixed human whole-brain specimens.

T1 and T2 were measured in 5 formalin-fixed human whole-brain specimens as a function of time. Gray matter/white matter contrast reversal was observed around the 4th day and was considered to be due to the greater decrease in T1 in gray than in white matter. A possible explanation for this is that the decomposition of the myelin phospholipid structure by formalin somewhat counteracts the general reductive effect of the fixation procedure on relaxation times.

Aged↗
PubMed1994

Problems concerning the quality of DNA measurements on Feulgen-stained imprints. A study of five fixation techniques.

Feulgen-stained rat liver imprints were investigated, and hepatocytes, lymphocytes and granulocytes were measured. Additionally, chicken erythrocytes placed on the slides were measured as an external DNA standard. The imprints were treated according to five different fixation protocols. The measured integrated optical density (IOD) was normalized according to the leukocytes and afterwards scaled according to the diploid hepatocytes. The mean IOD, coefficient of variation (CV), standard error of the mean (SEM) and IOD ratios of distinct cell groups were calculated. The CV of the IOD for hepatocytes was slightly better for air-dried preparations. It was larger for leukocytes than for hepatocytes and worst for chicken erythrocytes. The SEM of hepatocytes did not show remarkable differences between the fixation groups; in general it was near 1%. In all cases the IOD ratios of 2c, 4c and 8c hepatocytes reasonably well followed the expected ratio of 1:2:4 except for wet, formalin-fixed hepatocytes (3.8). The ratios of leukocytes to 2c hepatocytes were about 1.0 for air-dried preparations but considerably lower (0.85) for wet fixation in formalin. The IOD ratios of chicken erythrocytes to 2C hepatocytes varied from 0.33 to 0.35. The deviation of the ratios of single specimens from the estimated mean of a fixation group are described.

Animals↗
PubMed1998

Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor zeta chain and other intracellular proteins in lymphocyte subpopulations.

Expression of the T-cell receptor (TCR) zeta chain in normal individuals was studied by two-color flow cytometric analysis using digitonin-permeabilized human peripheral blood lymphocytes. Optimal detection of the TCR zeta chain involved fixation of cells in 0.25% paraformaldehyde for 2 min and permeabilization with 500 microg/ml of digitonin at 4 degrees C. Permeabilized lymphocytes and monocytes displayed decreased forward light scatter properties. Neutrophils did not survive this permeabilization/fixation/washing procedure. Permeabilization did not affect the ability of antibodies to CD3, CD4, CD8, CD16, and CD19 to detect CD antigens on lymphocytes, and the percentage of lymphocytes reacting with these antibodies was comparable between untreated and permeabilized cells. Staining of the TCR zeta chain was accomplished by incubating permeabilized cells with unconjugated antibody to the zeta chain, followed by an FITC-conjugated F(ab')2 goat anti-IgG. Following TCR zeta chain staining, cells were blocked with 25 microg/ml of mouse IgG for 20 min to saturate the goat anti-mouse antibody and then incubated with a phycoerythrin-conjugated mouse antibody to a variety of lymphocyte surface antigens. The TCR zeta chain was observed in lymphocytes displaying CD3, CD4, CD8, CD16, or TCRgammadelta markers. This permeabilization/fixation/washing procedure was also validated for detection of another intracellular T-cell protein known as the cytolytic granule-associated protein, recognized by the TIA-1 antibody. Thus the technique can be applied to detection of at least two different intracellular T-cell markers.

Antibodies, Monoclonal↗
PubMed1997

Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles.

Actin Cytoskeleton↗
PubMed2001

The EndoPearl device increases fixation strength and eliminates construct slippage of hamstring tendon grafts with interference screw fixation.

PURPOSE: The EndoPearl (Linvatec, Largo, FL), a biodegradable device to augment the femoral interference screw fixation of hamstring tendon grafts has been developed. The first objective of this study was to compare the initial fixation strength of quadrupled hamstring tendons and biodegradable interference screw fixation with and without the application of the EndoPearl device. The second objective was to determine the influence of the EndoPearl device on the fatigue behavior under incremental cyclic loading conditions in a simulation of critical fixation conditions. TYPE OF STUDY: Biomechanical study. METHODS: Fresh human hamstring tendons were harvested and grafts were fixed with biodegradable poly-L-lactide interference screws. Twenty proximal calf tibias were used to compare the initial fixation strength of the study and the control group. In the study group, the EndoPearl device was secured to the graft using two No. 5 Ethibond sutures (Ethicon, Somerville, NJ). Specimens were loaded until failure in a materials testing machine. For cyclic testing, human hamstring tendons and 20 distal porcine femurs were used. Critical graft fixation conditions were simulated by increasing tunnel diameter 2 mm over the graft diameter. Grafts were loaded progressively in increments of 100 N until failure; 100 cycles were applied per load increment. RESULTS: Graft fixation with the additional EndoPearl device had a significantly higher maximum load to failure (658.9 +/- 118.1 N v 385.9 +/- 185.6 N, P =.003) and stiffness (41.7 +/- 11 N/mm v 25.7 +/- 8.5 N/mm). Graft fixation with the EndoPearl device sustained a significant higher total number of cycles (388.5 +/- 125.6) compared with the control group (152.8 +/- 144.9, P =.002). CONCLUSIONS: We demonstrated that the augmentation of a hamstring tendon graft with the EndoPearl device increases interference screw fixation strength significantly. Under dynamic loading conditions, specimens of the study group sustained substantially higher loads and a larger number of cycles, which indicates a greater resistance to graft slippage. The application of the EndoPearl device may also allow for a secure soft-tissue graft fixation with interference screws in cases of critical fixation conditions.

Absorbable Implants↗
PubMed2006

A new method for visualization of gut mucosal cells, describing the enterochromaffin cell in the rat gastrointestinal tract.

OBJECTIVE: Enterochromaffin (EC) cells in the gastrointestinal tract have an important function as regulators of secretion, motility and sensation. The EC cell has traditionally been described as bottle-shaped, with basally located stores of serotonin. Stimuli acting on the apical membrane trigger serotonin release, which in turn activates the subepithelial sensory nerve terminals. To better describe the appearance of EC cells, we developed a new method for visualization of mucosal cells. MATERIAL AND METHODS: The stomach, small intestine and large intestine were excised from Sprague-Dawley rats and then fixed in formalin. The organs were everted and filled with pronase solution. Single cells and aggregates of formalin-fixed mucosal cells were collected by scraping the mucosa off the muscularis mucosa. EC cells were visualized by staining for immunoreactivity against serotonin. RESULTS: EC cells with luminal extensions and very long (up to 80 microM) basally located axon-like extensions, sometimes connecting to neuron-like structures, were found. Other EC cells had no or only short and blunt basal extensions. Dividing serotonin-containing EC cells were also seen. CONCLUSIONS: These findings could make an important contribution towards furthering our understanding of EC cell function in gastrointestinal physiology. This new method can also readily be used to give better visualization of the morphology of other mucosal cells.

Animals↗
PubMed1992

Golgi method without osmium tetroxide for the study of the central nervous system.

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.

Animals↗
PubMed1996

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

Blotting, Northern↗
PubMed1995

Role of glutaraldehyde in calcification of porcine heart valves: comparing cusp and wall.

Experiments were performed to better understand the relationship between glutaraldehyde and calcification of bioprosthetic heart valves, using both the cusps and the wall of porcine aortic roots. The results of the first experiment, for which 3H-labeled glutaraldehyde solutions were used, indicated that binding of glutaraldehyde in cusps and wall is concentration-dependent, that the wall contains significantly less glutaraldehyde than the cusp, and that glutaraldehyde, which penetrates in the wall at similar rates from the intima and the adventitia, is homogeneously distributed throughout the wall after 7 days of fixation, except for the intima side, where it is significantly lower. The results of the second experiment, for which cusps and 1-cm2 pieces of wall from glutaraldehyde-fixed porcine aortic roots were implanted subdermally in young rats, indicated that for both types of tissue, calcification appears to first initiate predominantly in the cell nuclei before extending to the other structures. After 8 weeks of implantation, whereas the cusps were completely calcified, calcification of the wall was limited to two longitudinal bands 150-300 microns thick, located below the adventitia and intima surfaces. The results of the third experiment indicated that cusp calcification, which decreased significantly after a 12-month storage period, was reset to high levels by reexposing the valves to glutaraldehyde at the end of the 12-month storage period. Wall calcification remained constant under all tested conditions. The results suggest that the mechanism(s) of calcification in the wall and the cusp may be different, and that calcification may be related to a particular molecular configuration resulting from exposure to glutaraldehyde.

Animals↗
PubMed2004

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells.

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 microg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

Autopsy↗
PubMed2006

A transmission electron microscopy study on effects of a modified Glutaraldehyde fixation on Acanthamoeba castellanii.

Transmission electron microscopy (TEM) can provide high resolution imaging of biological specimens. The study is to establish the effects of a modified glutaraldehyde (GA) compare to the standard GA fixation on Acanthamoeba castellanii from TEM perspectives and thus provide precise and accurate information on the ultrastructure studies of the parasite. By increasing the contrast, the ultrastructures of the parasite were more evident. The TEM images were obtained from parasites fixed with the modified GA and the standard GA and then the area of the nucleus and the gray values of the image of the nucleus of the parasites were measured. The mean areas of the nucleus were found to be significantly reduced in the standard GA fixed parasites (12210.4 nm2) compared to the modified GA fixed parasites (8676.3 nm2) (p < 0.05). The mean gray values of the image were significantly reduced from 2024 in the standard GA fixed parasites (2024) to the modified GA fixed parasites (1636) (p < 0.05). The study shows that the modified GA produced significantly better contrast on TEM images of the A. castellanii compared to the standard GA. This was because the modified GA generated more free water molecules during fixation and the uptake of modified GA by the nucleus of the parasite organizing all protein constituents in the cell into a more closely packed configuration than that of the standard GA. With such properties, the modified GA is a better fixative providing better images for ultrastructures of the parasite.

Acanthamoeba castellanii↗
PubMed1991

Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze-substitution study.

Despite a plethora of reports on the ultrastructure of secretory granule release by exocytosis, the release of coagulant activity from stimulated platelets is still being attributed to membrane vesiculation. Membrane vesiculation and the formation of myelin figures have been shown to be artifacts of glutaraldehyde GA fixation. Cells fixed by direct osmium or rapid freezing are free of such structures. Yet there is still doubt that rapid freezing interferes with vesiculation process. This study has addressed this issue by examining: (1) whether freezing and freeze-substitution affects membrane vesiculation, (2) whether paraformaldehyde-fixation also induces the phenomenon, and (3) whether the aldehyde concentration is of influence. Aldehyde fixation was carried out prior to impact freezing and freeze-substitution. In thrombin-stimulated platelets, membrane vesiculation and myelin figures were found. Glutaraldehyde induced multivesicular structures, paraformaldehyde or low aldehyde concentrations only blebs on the platelet surface. The membrane vesicles were in continuity with the cytoplasmic matrix. Unstimulated platelets did not show vesiculation or myelin figures. Control samples, without aldehyde fixation, showed instead of membrane vesiculation, granule fusion with the plasmalemma, or, instead of myelin figures, compound granules. This confirms that membrane vesiculation and the formation of myelin figures are artifacts induced by the failure of aldehydes to arrest lipid mobility within membranes undergoing rapid changes in structure. Although the presence of membrane vesiculation and myelin figures in platelets indicates that exocytotic processes were occurring at the moment of aldehyde fixation, the finding of membrane vesiculation in aldehyde-fixed platelets does not indicate a separate type of exocytosis.

Aldehydes↗
PubMed2002

Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.

Several techniques enable to inject intracellularly neurons with dyes and to use light and electron microscopy to correlate the physiological data with the morphological properties of the neuron. However, the ultrastructure of the neuron is usually obscured by the injected dye thus notably precluding the analysis of the postsynaptic specialisation and that of the other organelles. To overcome this problem, we have developed a technique based on fluorophore- and ultra small gold-conjugated streptavidins. We report, that this method facilitates the identification of intracellular organelles of the biocytin-filled neuron and of postsynaptic densities. This method is valid for the study of early postnatal neurons that are particularly refractory to this type of analysis. The procedure introduced here consists of the following steps: (1) injection of biocytin into the neuron by a patch-clamp pipette, (2) aldehyde fixation, (3) reaction with a fluorophore-conjugated streptavidin, (4) analysis with a fluorescence microscope, (5) formation of avidin-biotin complexes (ABC), (6) reaction with an ultra small gold-conjugated streptavidin, (7) silver enhancement of gold, (8) postfixation with osmium tetroxide and embedding in resin, (9) ultrathin sectioning and analysis with an electron microscope. Using this method, we show that in early postnatal hippocampal neurons, that have been injected with biocytine, it is possible to determine the morphology of the dendritic and axonal trees (including very thin details such as spines and filopodia) and to identify the localisation of the symmetric and asymmetric synapses on dendrites of the injected neuron.

Animals↗
PubMedDate not supplied

Diagnostic parameters in liquid-based cervical cytology using a coagulant suspension fixative.

OBJECTIVE: To evaluate in detail the morphology of cervical cell samples suspended in the coagulant fixative BoonFix (Finetec, Tokyo, Japan) in liquid-based Papspin slides (Thermo Shandon, Pittsburgh, Pennsylvania, U.S.A) to detect shifts in diagnostic parameters for infections and neoplasia. STUDY DESIGN: Split samples of 1,010 cases were collected. All Papspin slides were scanned with neural network technology. In 849 cases the diagnosis was "within normal limits"; in 22 cases it was preneoplasia. In 151 special cases conventional smears were compared with thin-layer slides. RESULTS: In 85% of the 151 special cases, a shift of the diagnostic parameter was observed in the Papspin slide. The parameter adhesion of inflammatory cells to epithelial cells was easier to discern in 94% of the cases, and adhesion of microorganisms varied 43-100%. Koilocytosis was more visible in 79%. Prominent nucleoli in atypical and malignant cells were enhanced in 50-100% of cases with preneoplasia. The fact that the cells on the Papspin slide were no longer present in diagnostic streaks posed a problem only in the case of follicular cervicitis. CONCLUSION: The shifts in parameters facilitated the diagnostic process. BoonFix permits the screening of liquid-based Papspin slides, which have proven to be well suited to automated neural network scanning.

Bacterial Infections↗