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PubMed1985

Light microscopic histochemistry on plastic sections.

As compared with conventional paraffin, celloidin, and frozen sections, semithin plastic sections offer a superior quality of the light microscopic image in terms of better resolution, absence of distortion and shrinkage artifacts, and suitability for calcified tissues. Application of histochemical methods to such sections often encounters, however, serious difficulties resulting from a considerably reduced reactivity of plastic-embedded biological material. Factors involved include a poor penetration of reagents into plastic embedding media due to a steric or hydrophobic hindrance, as well as a blockade of the reactive chemical groups in the sample due to interactions with fixatives and plastics. Embedding in polar (hydrophilic) plastics, such as glycol methacrylate, permits carrying out a large number of histochemical reactions, including the demonstration of enzymatic activities, directly on sections, but is less suitable for combined light/electron microscopic studies because of an imperfect ultrastructural preservation of tissues. Embedding in nonpolar epoxy resins, particularly if combined with a double aldehyde-osmium fixation, results in a high quality ultrastructure but almost fully inhibits the histochemical reactivity of the embedded material. In order to restore this reactivity, i.e. to unmask chemical groups bound by the polymerized resin, semithin epoxy sections require the removal of the embedding matrix by alkoxides prior to the histochemical procedure. Additional steps are also often necessary: treatment of osmium-fixed sections with oxidative agents, e.g., hydrogen peroxide or periodate which reoxidize the bound osmium and remove it from tissue, and a controlled proteolytic digestion, especially useful in immunocytochemical studies, which probably cleaves the bonds between the primary aldehyde fixative, and the reactive sites. This article reviews histochemical methods which have been successfully applied to plastic-embedded material. Using polar methacrylates and/or nonpolar epoxy resins as embedding media, it has been possible to demonstrate proteins and aminoacid residues, carbohydrates, lipids, nucleic acids, biogenic amines, inorganic ions, and some enzymes, although the spectrum of methods found as suitable for plastic-embedded material is far narrower than that available for paraffin or frozen sections.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Topographical uptake of blood-borne horseradish peroxidase (HRP) in the murine testis at the light microscopic level.

Light microscopical studies on the uptake of blood-borne horseradish peroxidase (HRP) in large areas of the testis have been scarce because of the difficulty of staining HRP in testes with well-preserved morphology. However, observation of exogenous HRP in all areas of the testis enables detection of regional tissue injury induced by toxic chemicals or immunization. In the present study, the localization of blood-borne HRP in the murine testis was investigated light microscopically using plastic-embedded testes and post-embedding histochemical methods. Mice were injected intravenously with HRP, and then perfused with 2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M phosphate buffer. The fixed testes were immediately removed, dehydrated, and then embedded in plastic without cutting them into small pieces. The prepared sections treated by the diaminobenzidine method exhibited intense HRP activity with well-preserved testis morphology. It was noted that many interstitial macrophages had endocytosed HRP. In particular, HRP-endocytosing macrophages were concentrated around the tubuli recti. The testicular capsule, containing many lymphatic capillaries and vessels, was also loaded with HRP. In the subcapsular interstitium, free HRP in the lymph space accumulated, but the staining intensity was weak compared to that in testicular macrophages. No HRP infiltration into the lumen of the seminiferous tubules was observed at the light microscope level; however, HRP staining was detected in tubular walls and epithelial cells lining the rete testis and tubuli recti, indicating that these regions are permeable to HRP.

Animals↗
PubMed1999

Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping.

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.

Cold Temperature↗
PubMed2003

Histological findings in ruptured aneurysms treated with GDCs: six examples at varying times after treatment.

BACKGROUND AND PURPOSE: A novel plastic embedding approach was used to histologically evaluate inflammatory changes and scar formation over time and to better visualize the mesh attenuation within the aneurysm lumen of acutely ruptured aneurysms after treatment with GDCs. METHODS: Autopsies were performed on six patients with acute subarachnoid hemorrhage who had died between 5 and 272 days after GDC treatment. The aneurysms containing the platinum coils were embedded in plastic along with the intact parent vessels, were sliced, and were ground to a thickness of 5 to 10 micro m. In addition, 250- micro m-thick sections were prepared. Histologic examinations were performed. Three exemplary cases representing the time lapse from treatment to death are discussed in detail. RESULTS: At the three exemplary time periods (5, 13, and 272 days) after coiling, a continuing healing process could be observed. At 5 days after placement of GDCs, a blood clot consisting of erythrocytes and fibrin was found throughout the cavity. Thirteen days after the procedure, large foamy macrophages were observed near the coils in the aneurysm cavity. Two hundred seventy-two days after the intervention, scar formation within the aneurysm appeared to be completed, with vascularized connective tissue filling the cavity and embedding the coils. Large foreign body giant cells were found adjacent to the coils. A layer of long slender cells, resembling endothelium, sealed the aneurysm neck. CONCLUSION: We postulate that within days after GDC treatment, blood clotting and thrombus formation prevent rebleeding and that solid scar formation covered by a layer of long slender cells, resembling endothelium, seals the aneurysm over time.

Adult↗
PubMedDate not supplied

Temporal development of 2',3'-dideoxyinosine (ddI)-induced peripheral myelinopathy.

The anti-HIV therapeutic dideoxyinosine (ddI) has been reported to produce a painful, dose-limiting peripheral myelinopathy in HIV-infected patients after chronic administration. We have previously demonstrated ddI-induced myelinopathy in a non-HIV-infected rat model after 20 weeks of dosing, characterized by myelin splitting and intramyelin edema. The present study examined the time course needed to produce the ddI-induced neuropathy. Adult male Sprague-Dawley rats were gavaged with vehicle or 415 mg/kg ddI twice daily for up to 20 weeks. Groups of treated (n = 6-8) and control (n = 3-5) animals were killed after 5, 10, 15, and 20 weeks of dosing and the distal end of the sciatic nerve was removed. The nerve was postfixed by immersion in neutral phosphate-buffered formalin, dehydrated in graded alcohols, and embedded in plastic embedding media. One-micrometer-thick sections were cut and stained with toluidine blue and basic fuchsin. Plasma levels of ddI on the day the animals were killed were greater than 10 microgram/ml within the first hour after dosing and fell rapidly to less than 1 microgram/ml (clinical range 1-2 microgram/ml) within 3 h after dosing. The abnormalities observed in the sciatic nerve were few, if any, after 5 or 10 weeks, but very prominent after 15 weeks of dosing. Four of the six ddI-treated rats exhibited abnormal morphology as evidenced by myelin splitting and ballooned myelin sheaths. Although abnormal morphology was present at 20 weeks of dosing, the effect was not as robust as at 15 weeks. This suggests that the nerve may partially recover from the effects of ddI with time. Published by Elsevier Science Inc.

Animals↗
PubMed1996

Anatomy of the eustachian tube as demonstrated by endoluminal ultrasonography.

Using a new application of the endoluminal approach, we were able to demonstrate the sonographic anatomy of the eustachian tube in vitro and in vivo and correlate it with the plastic-embedded specimen. Five adult normocephalic cadavers, two patients, and one specimen especially prepared for embedding in plastic were examined. A specifically developed device was used to insert the ultrasound transducer employing an endo-oral approach. The investigation was performed using an intravascular ultrasound unit. In all cases the tube could be visualized in its entirety and relevant anatomic structures identified and compared with the specimen at the corresponding levels. The deeper layers, including the paratubal structures and the mucosa, could be distinguished for the first time by means of endoluminal ultrasonography. Now that endoluminal ultrasonography has revealed this anatomic information, further studies will be able to gauge the clinical efficacy of our method in cases of ventilatory, drainage, and clearance problems. The images showed no difference between the structures in vitro and in vivo, and the vivo examination was even easier than the in vitro one owing to better tissue turgor.

Adult↗
PubMed1981

Splenic alterations in hairy cell leukemia.

Spleens from 12 patients with hairy cell leukemia were studied for morphologic alterations. Paraffin-embedded, plastic-embedded, and fresh-frozen tissue sections were stained for a variety of histochemical reactions. Hairy cells were present in great numbers within the splenic sinuses and cords. Hairy cells adhered to splenic endothelial lining cells, other hairy cells, and erythrocytes. Large clumps of hairy cells filled and partially occluded splenic sinuses. Numerous dilated sinuses, abnormal sinuses, and blood-filled spaces were present in all cases. Ring fibers around abnormal sinuses were fragmented and reduced in amount. Occasionally, the number of reticular fibers appeared increased around abnormal sinuses, but, in general, it was decreased in areas around the blood-filled spaces and in splenic cords markedly infiltrated by hairy cells. The formation of abnormal sinuses and blood-filled spaces probably resulted from the obstruction of venous blood flow and from injury or death of endothelial lining cells.

Endothelium↗
PubMed1986

Preparation methods for quantitative electron probe X-ray microanalysis of rat exocrine pancreas: a review.

Pancreatic acinar cells are thought to secrete a fluid containing digestive enzymes and electrolytes and use e.g. calcium as a second messenger upon stimulation. Together with their pronounced morphological polarity, they provide a model system to study the effect of different preparation methods for quantitative biological electron probe X-ray microanalysis (EPXMA) of ultrathin sections. Several preparation methods i.e., freeze-drying and plastic-embedding, freeze-substitution (2 days) and freeze-drying of ultrathin cryosections have been applied to examine the retention of sodium, magnesium, phosphorus, sulfur, (chlorine), potassium and calcium in subcellular compartments (basal cytoplasm, apical cytoplasm, mitochondria and zymogen granules). In freeze-substituted samples the phosphorus, potassium and sulfur concentrations were 2-3 times lower in all compartments compared to freeze-dried, plastic-embedded samples. Intracellular potassium-to-sodium ratios obtained on frozen substituted and frozen-dried, plastic-embedded samples were considerably lower than for cryosections. Element gradients between adjacent organelles were large in frozen-dried cryosections, smaller in frozen-dried plastic- embedded samples and insignificant in frozen-substituted samples.

Animals↗
PubMed1994

Histological methods for assessing myelin sheaths and axons in human nerve trunks.

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.

Axons↗
PubMed1984

Glutamic acid decarboxylase-containing neurons in the dorsal column nuclei of the cat.

The retrograde transport of horseradish peroxidase (HRP) and immunocytochemistry for glutamic acid decarboxylase (GAD) have been employed to examine whether local circuit neurons (LCNs) exist in the dorsal column nuclei (DCN) and whether these neurons may be GABA-ergic. Observations focused on the dorsal part of the middle cuneate nucleus (MCd), since this region has been previously shown to contain projecting neurons whose axons terminate almost exclusively in the contralateral thalamus. After large injections of HRP in the nucleus ventralis posterolateralis and surrounding structures of the feline thalamus, the majority of neurons in MCd are labeled. These represent about 89% of the neurons in MCd as counted in 40-microns frozen sections, and about 69% as counted in plastic-embedded, 2.5-microns-thick section. Unlabeled by the same injections are some medium to large neurons at the dorsal rim of MCd, and many characteristically small (mean = +/- 250 microns2) neurons at the periphery of the cell clusters formed by thalamic-projecting neurons. These small neurons represent 10-12% of the neuronal population of MCd, as counted in 40-microns-thick frozen sections, and about 30%, as counted in plastic-embedded, 2.5-microns-thick sections. Neurons in this size range are also unlabeled after injection of retrograde tracer in the pretectal area, inferior and superior colliculi, inferior olivary complex, and/or spinal cord. These injections, however, result in the labeling of neurons along the dorsal rim of MCd and/or in other regions of the cuneate nucleus. In adult, colchicine-treated cats, the use of anti-GAD serum reveals a population of labeled neurons uniformly distributed throughout the DCN. In MCd, these are small (mean = +/- 235 microns2) neurons mainly intercalated between cell clusters, and represent about 25% of the neuronal population of this nuclear subdivision as counted in plastic-embedded, 2.5-microns-thick sections. Labeled processes densely infiltrate the cell clusters, and labeled varicosities appear to cover the soma and dendrites of unlabeled neurons. At the electron-microscopic level, most labeled profiles contain vesicles and correspond to F boutons usually involved in "axoaxonic" contacts with terminals of dorsal root afferent and presynaptic to dendrites. Other vesicle-containing, GAD-positive endings seem to correspond to the P boutons described by Ellis and Rustioni (1981) and are believed to be, at least in part, of dendritic origin. It is suggested that GAD-positive neurons are GABA-ergic LCNs and that these can mediate both pre- and postsynaptic inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1977

Localization of glomerular "deposits" in Henoch--Schönlein nephritis.

Twenty-five renal biopsies from patients with Henoch--Schönlein nephritis were examined using light microscopy, immunofluorescence, 1 micrometer plastic-embedded sections and electron microscopy. The 1 micrometer plastic-embedded sections and electron microscopy showed deposits in mesangial, subendothelial and subepithelial sites. Some of the latter were very large and similar to those which have been described as "humps" in acute proliferative glomerulonephritis. Immunofluorescence showed the mesangial deposition of IgG, IgA and C3 with extension into a peripheral position in some cases. Fibrin was frequently found associated with crescents. The case for Henoch--Schönlein disease being mediated, in part at least, by immune complex deposition, is presented.

Complement System Proteins↗
PubMed1985

Monocyte/macrophage derived cells in normal and transplanted human kidneys.

The existence of HLA-DR/Ia-like antigen (Ia)-bearing cells of the mononuclear phagocyte system, macrophages (Mac), and/or interdigitating cells (IDC), in the normal kidney is controversial. If present, such cells may be important in renal transplant rejection. We performed enzyme histochemistry using alpha-naphthyl acetate/butyrate esterases (alpha NAE, alpha NBE), 5'-nucleotidase (5'N), acid phosphatase (AcP), alkaline phosphatase (AlkP), and ATPase (ATP) as well as immunoperoxidase staining for Ia and lectin binding (Ulex europaeus I; UEA) on plastic-embedded tissue sections of normal kidneys and rejected renal allografts. Plastic embedding provides clear visualization of histologic detail and allows specific identification of immunoperoxidase-stained cells. Mac and IDC (shown to be Ia+, alpha NAE+, AcP+, ATP+ in other sites) could not be demonstrated in normal renal interstitium. IDC and Mac were not generally identified in normal mesangium, although they could not be altogether distinguished from Ia+ endothelial cells. Focal mesangial staining for alpha NAE but not alpha NBE was present. Rejected kidneys showed increased numbers of alpha NAE+ cells in glomeruli. These cells were frequently Ia negative and often appeared to be blood monocytes present in capillary lumens. Peritubular capillaries and glomerular endothelium stained strongly for UEA, 5'N, and Ia. Our results suggest that previous reports of the presence of IDC in renal tissue on the basis of staining for Ia on frozen tissue may be due to staining of compressed or obliquely sectioned vascular structures that were not adequately visualized.

Enzymes↗
PubMed1991

Dysmegakaryopoiesis predicting response to therapy in acute myeloid leukaemia. A histologic and clinical study.

With standard induction therapy between 50 to 85% of patients with Acute Myeloid Leukaemia (AML) achieve Complete Remission (CR). We investigated whether any morphological feature of bone marrow (BM) plastic embedded biopsies could predict failure of therapy. We reviewed BM plastic embedded biopsies from 54 adult patients presenting with untreated AML. The main histologic parameters analysed were cellularity, dysmegakaryopoiesis (DysM), percentage of marrow blasts and fibrosis. CR was obtained in 34 of 49 treated patients (69%). The rate of CR was significantly lower in the group of patients presenting with DysM: CR was achieved in 54% of the 28 treated patients with DysM and in 90% of the 21 treated patients without DysM (p less than 0.02). Patients with DysM had a significantly lower blood count and bone marrow blasts at presentation. Median age was not significantly different in the 2 groups. Cellularity and fibrosis were not predictive. DysM may be the hallmark of an AML subgroup with distinct clinical behaviour and lower rate of CR with conventional therapy. DysM should be carefully looked for on BM marrow biopsies and aspirate from AML patients at diagnosis.

Acute Disease↗
PubMed1993

The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.

A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.

Adsorption↗
PubMed1980

An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations.

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.

Animals↗
PubMed1980

Examination of the larynx in the histopathology laboratory.

A method is described for examination of the larynx in the histopathology laboratory. Using a slicing machine, transverse slices of the whole larynx are obtained from which representative histological samples may be prepared. This method offers the advantages of a complete gross examination of the normal and pathological structures of the larynx supplemented by histological studies using any of the methods of paraffin embedding, frozen section, plastic embedding, or electron microscopy on any part of the larynx.

Histological Techniques↗