Mallory's Phosphotungstic Acid Hematoxylin Research Articles

A histochemical study of ultraviolet B irradiation and Origanum hypericifolium oil applied to the skin of mice

Ultraviolet (UV) rays cause skin damage. Chronic exposure to UV irradiation causes decreased collagen synthesis, degenerative changes in collagen bundles, accumulation of elastotic material and increased epidermal thickness. Origanum hypericifolium, an endemic Turkish plant, belongs to Lamiaceae family. The main constituents of its oil are monoterpenes including cymene, carvacrol, thymol and γ-terpinene. The effects of undiluted O. hypericifolium oil on UVB irradiated skin of mice were investigated histochemically. Four groups of female BALB/c mice, whose dorsal hair was shaved, were allocated as follows: non-UVB irradiated (Group 1), UVB-irradiated (Group 2), O. hypericifolium oil treated (Group 3), and O. hypericifolium oil treated and UVB irradiated (Group 4). Sections of dorsal skin samples were stained with Mallory's phosphotungstic acid hematoxylin for collagen fibers and Taenzer-Unna orcein for elastic fibers. Sections also were stained with hematoxylin and eosin to measure epidermal thickness. We observed intense staining of collagen and homogeneous, scattered thin elastic fibers in Group 1; scattered and weakly stained collagen and curled, amorphous, accumulate elastic fibers in Group 2; and intense staining of collagen in Groups 3 and 4. Accumulation of elastic fibers in the dermis was unremarkable in Groups 3 and 4. In Groups 3 and 4, O. hypericifolium oil treatment thickened the epidermis. Epidermal thickness was greatest in Group 4. We suggest that O. hypericifolium oil may block UVB induced alterations of collagen and elastic fibers, and increase epidermal thickness.

Spice up the hypertension diet - curcumin and piperine prevent remodeling of aorta in experimental L-NAME induced hypertension

BackgroundIncrease of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension.MethodsWistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated.ResultsThe increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself.ConclusionsAdministration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.

Improved Methods for Staining Fibrin

Two methods used to demonstrate fibrin are the Fraser-Lendrum and Mallory's phosphotungstic acid-hematoxylin. One problem with the Fraser-Lendrum is that it requires the use of saturated alcoholic picric acid. To prepare this solution, a considerable amount of picric acid is required, which is an explosive. Federal and state regulations prohibit the storage of this chemical in the laboratory unless it is stored under water. We found that saturated aqueous picric acid could be substituted for the saturated alcoholic solution. Other changes were made to simplify the method. Mallory's method for fibrin requires treatment of tissue sections with potassium permanganate and oxalic prior to staining them with phosphotungstic acid-hematoxylin. We found that better staining results are obtained if the tissues are also treated with Zenker's fixative. However, Zenker's fixative contains mercuric chloride, a hazardous chemical that many laboratories have discontinued using. We found that zinc acetate can be substituted for mercuric chloride in preparing Zenker's fixative with excellent staining results when used in the Fraser-Lendrum and Mallory methods. (The J Histotechnol 26:127, 2003)Submitted April 10, 2003; Accepted April 22, 2003

Characterization of Lesions Caused by a South American Virulent Isolate ('Quillota') of the Hog Cholera Virus

In this study, macroscopic and histopathological lesions produced by a virulent South American isolate ('Quillota') of hog cholera virus were studied. The virus was inoculated in doses of 10(5)TCID50 in each of 35 pigs of 20 kg live weight. The animals were slaughtered from 4 to 18 days post-inoculation. The presence of virus antigens in lymphatic tissue was confirmed by both direct immunofluorescence and Avidin-Biotin-Peroxidase techniques in formalin-embedded tissue samples. Histological sections were stained with haematoxylin-eosin and Mallory's phosphotungstic acid haematoxylin methods. The 'Quillota' isolate used in this study caused a disease characterized by vascular lesions (splenic infarcts, haemorrhages in the lymph nodes and the urinary system and disseminated microthrombosis), and necrosis of lymphocytes, particularly in the B-areas of the lymphoid organs, lesions that are characteristic of the acute form of the disease. Other lesions observed were a non-purulent meningoencephalitis, the necrosis of the epithelial cells of tonsils, the presence of fibrin nets in the red pulp and a marked thickening of the alveolar septa.

Evaluation of agonal artifacts in the myocardium using a combination of histological stains and immunohistochemistry.

The problem of discrimination between agonal artifacts and intravital ischemic myocardial lesions was studied with four histochemical stains [hematoxylin-eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), modified luxol fast blue, and Lie's hematoxylin basic fuchsin picric acid (HBFP)] and with immunohistochemistry using two antibodies (antimyoglobin and anti-C5b-9). Seventy-five forensic autopsy cases were divided into six groups designed to represent successively shorter periods of agonal myocardial ischemia: (a) sudden deaths with coronary artery disease, macroscopically visible myocardial infarction, and/or fresh coronary thrombus; (b) unexplained sudden deaths without coronary artery disease; (c) accidental CO poisoning; (d) suicidal CO poisoning in cars; (e) suicidal hangings; and (f) instant traumatic deaths, i.e., total brainstem laceration or rupture of the thoracic aorta. From each heart, five pieces were removed from standardized locations, and six parallel sections were stained with each method (i.e., 30 sections from each heart). Hematoxylin-eosin and anti-C5b-9 were only positive in the first three groups, thus indicating specificity for intravital necrotic changes. The other staining methods were "positive" in one or more cases in all six groups, thus implicating a high degree of sensitivity for artifactual, agonal ischemic changes. The latter methods cannot be used alone in the diagnosis of myocardial infarction. By staining parallel sections with different stains and antibodies, it seems possible to estimate the relative length of the agonal period in cardiac and noncardiac deaths.

Role of Na(+)-H+ exchange on reperfusion related myocardial injury and arrhythmias in an open-chest swine model.

The inhibition of Na(+)-H+ exchange (NHE) with amiloride analogues in vitro has been shown to prevent reperfusion arrhythmias and additional cell necrosis. Inhibition of intracellular Ca2+ overload via NHE inhibition has been suggested as a mechanism of these protective effects. The aim of this study was to examine whether treatment with amiloride analogues reduces the incidence of reperfusion arrhythmias and limits infarct size in vivo. Open-chest swine were exposed to a 30-minute left anterior descending artery (LAD) occlusion and 180 minutes of reperfusion during atrial pacing at 150 ppm. Intravenous 5-(N,N-dimethyl)-amiloride (AML, 5 micrograms/kg per min) was administered in the treatment group (n = 7) and intravenous saline in the control group (n = 7), starting 10 minutes before coronary occlusion. The infusion was continued during ischemia and reperfusion. The area at risk was defined by monastral blue dye and infarct size by triphenyltetrazolium chloride staining. Limb leads ECG and monophasic action potentials (MAPs) from the epicardium in the ischemic area were recorded. There was no significant difference in the size of the area at risk and hemodynamic parameters between the groups. However, the infarcted area was 0.4% +/- 1.0% of the area at risk in the treatment group, whereas it was 62% +/- 29% in the control group (P < 0.05). Pathological examination (Hematoxylin-eosin and Mallory's phosphotungstic acid-hematoxylin staining) revealed that all of the infarcted area consisted of contraction band necrosis. MAP duration in both groups was significantly shortened during ischemia. After reperfusion, MAP duration in the treatment group recovered earlier than that of control group. However, there was no significant difference in the incidence of ventricular tachyarrhythmia between the groups. Inhibition of NHE with AML prevented reperfusion related cell necrosis in the in vivo swine model, but did not reduce the incidence of ventricular tachyarrhythmia.

Intracytoplasmic eosinophilic hyaline globules in cartilaginous neoplasms: A surgical, pathological, ultrastructural, and electron probe x-ray microanalytic study

Hyaline globules (HGs), spherical intracytoplasmic eosinophilic droplets, have been associated with a variety of conditions, including hepatocellular carcinoma, lung adenocarcinoma, and Kaposi's sarcoma, but they have not been described in cartilaginous tumors. In specimens of 60 cartilaginous neoplasms we found that 22 of 33 chondrosarcomas (67%), eight of 16 enchondromas (50%), and three of seven soft tissue chondromas (43%) exhibited HGs. HGs were seen more commonly in low grade chondrosarcoma (70%) and were relatively rare in high grade chondrosarcoma (25%). No HGs were identified in three osteochondromas, one synovial chondromatosis, or 15 normal cartilaginous tissues taken from various sites. Cartilage associated HGs ranged in size from 2 to 20 μm, were diastase resistant and periodic acid-Schiff (PAS) stain positive, demonstrated autofluorescence, and variably stained with Mallory's phosphotungstic acid-hematoxylin stain (PTAH). A panel of immunostains did not show any specific staining reactions with HGs. Ultrastructurally the HGs were spherical, non-membrane-bound bodies having complex architectural features associated with profiles of rough endoplasmic reticulum. Electron probe x-ray microanalytic (EPXMA) study showed significant peaks of sulphur and calcium. We conclude that HGs represent secretory products of probable glycoprotein nature, may accumulate in a variety of cartilaginous neoplasms, and may be seen more frequently in low grade chondrosarcomas.

Screening of myocardial contraction bands: A comparison between two histological staining methods

Two histological stains for demonstration of contraction bands (cb): Mallory's phosphotungstic acid hematoxylin (PTAH) and a modification of luxol fast blue (LFB) were compared. Microscopical sections from hearts from 60 autopsies were screened. The regional distribution of the cb lesions was also studied. It was found that PTAH showed cb more consistently than LFB. None of the stains were suitable for screening in the lowest magnification. In cases of cardiac death the cb were often extensive and scattered throughout the myocardium. In non-cardiac deaths with cb these were always discrete and seen only in one location which might be a means of differentiating lesions of pathogenetic importance from agonal ones.

Neoplastic Paneth cells in the experimental murine carcinoma of the small intestine

The purpose of this study is to elucidate the participation of Paneth cells in experimentally induced adenocarcinoma of the intestine. The rats were fed with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in drinking water ad libitum at a concentration of 100 micrograms/ml for 28 weeks. They were sacrificed 12 weeks after the last MNNG administration. A number of tumor cells containing large eosinophilic granules in their supranuclear cytoplasm (Paneth cells) were observed in about 20% of the experimentally induced adenocarcinoma of the small intestine. The granules were stained positively with Lendrum, periodic acid-Schiff, Masson's trichrome, and Mallory's phosphotungstic acid hematoxylin. Ultrastructurally, the granules were round, osmiophilic, and relatively even in size. We compared the morphologic features of the Paneth cell-containing small intestinal adenocarcinomas (Group I) with those without Paneth cells (Group II). Group I was distinguished from Group II by its better differentiation, larger tumor size and lower incidence of calcification. Although Paneth cells are extremely rare in human gastrointestinal carcinomas, twenty percent of MNNG-induced intestinal carcinomas harbor Paneth cells. The neoplastic Paneth cells in experimental carcinomas may differentiate from uncommitted cells in the deeper portion of the crypt.

Eosinophilic intracytoplasmic globules in pulmonary adenocarcinomas: A histochemical, immunohistochemical, and ultrastructural study of six cases

Intracytoplasmic globules have been described in a variety of neoplastic and nonneoplastic conditions, but remain poorly defined. In a review of 100 consecutive cases of lung carcinomas, six cases of mucin-positive adenocarcinoma demonstrated eosinophilic intracytoplasmic globules that ranged in size from < 1 to 20 μ in diameter. The globules were often located adjacent to areas of tumor necrosis, and occurred either singly or multiply within individual tumor cells. Globules were similar in morphologic appearance to Russell bodies in plasma cells or the eosinophilic globules in hepatocytes of patients with alpha-1-antitrypsin deficiency, but were morphologically distinct from intracytoplasmic mucin vacuoles. The globules were brightly positive with PAS stain with diastase, were brick red with Masson's trichrome stain, and showed variably positive staining with Mallory's phosphotungstic acid-hematoxylin and Ziehl-Nielson stains. Immunoperoxidase staining showed slight sataining of some globules with albumin, IgG, IgA, and alpha-1-antitrypsin. Ultrastructurally the globules had a homogeneous density and were often associated with profiles of rough endoplasmic reticulum. We suggest that these globules represent secretory glyco-protein accumulated in the cytoplasm of tumor cells in areas of tumor cell injury.

Importance of immunoenzyme histochemical reaction in diagnosis of disseminated intravascular coagulation in human and animal material.

Renal tissues from 208 human necropsies were observed histologically for disseminated intravascular coagulation (DIC). The tissues were stained with hematoxylin-eosin, Mallory's phosphotungstic acid hematoxylin (PTAH) and cationic ferric hydroxide colloid stabilized with cacodylate (Fe-Cac), and tested by immunoenzyme histochemical (IEH) reaction for fibrin-related materials (FRMs). The use of the IEH method increased FRM recognition, and FRMs were detected in a total of 80 cases (38.5%). In 26 cases diagnosed clinically as DIC, FRMs were shown in 23 of the cases (88.5%). Thus, 57 patients with FRMs were clinically asymptomatic. In rats with DIC induced by endotoxin injection, glomerulus FRM was effluxed into the tubulus through the Bowman's capsule and was excreted into urine. The electric charge was reduced on the endothelial surface of the glomerular capillaries in both human and rat DIC. Under the scanning electron microscopy, the endothelial surface appeared coarse in the glomerular capillary and fibrin degradation was present. Our conclusions are: (a) PTAH is non-specific for FRMs, (b) IEH aids the pathohistological diagnosis of DIC, especially in asymptomatic forms including the compensated DIC state, (c) FRMs in tubuli suggest DIC, and (d) DIC is possibly initiated by a reduction in the capillary electric surface charge.

DISSEMINATED INTRAVASGULAR COAGULATION (DIC)

Renal tissues of 208 autopsied cases were examined. Malignant neoplasm with hematological malignancies often accompanied DIC. Tissue sections were stained with hematoxylin and eosin and Mallory's phosphotungstic acid hematoxylin (PTAH), and were applied for immunoperoxidase method (IP), using antisera against human fibrinogen, FDP-D and FDP-E. Histologically in 80 cases (38%) fibrin or fibrinogen related materials (FRMs) were observed in the glomerular capillary or the intratubular area or in both. FRMs were PTAH or IP positive or both in 23 of the 26 cases (88%) clinically diagnosed as DIC. In the remaining three cases anticoagulants probably interfered with FRMs observation. This study showed the PTAH stain was nonspecific and insensitive to FRMs, and that IP was necessary for a pathological diagnosis of DIC. The presence of FRMs in the renal tubuli is an important finding in confirming DIC. DIC may be present histologically in the absence of clinical DIC symptoms.

Immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) in nasal gliomas

Using the Sternberger method (Immunoluk Histoset KIT) GFAP (glial fibrillary acidic protein) was demonstrated immunohistochemically in 4 nasal gliomas. In these histologically complex tumour-like lesions mesenchymal, epithelial, and neuroglial tissues as well as small groups of scattered glial elements could be differentiated specifically by the highly sensitive GFAP immunoperoxidase technique. GFAP was present in astrocytes and astrocyte-like differentiations. The reactivity of cell processes was essentially lower. The GFAP immunostain does not always correlate with Mallory's phosphotungstic acid hematoxylin (PTAH) stain and Gallyas' silver impregnation method for astrocytes. Additionally the immunohistochemical investigation of semithin sections prepared by the so-called pop off technique after Bretschneider et al. (1981) allows the correct localization of GFAP in astrocytes and their modulations. Furthermore, in this study, the intimate connection of epithelium and glial cells as well as astrocytes containing hemosiderin granules could be demonstrated. The latter findings suggest a possible phagocytotic activity of astrocytes. Our results show that the demonstration of GFAP by the Sternberger method is a valuable aid in establishing astrocytic glial differentiations and modulations in complex tumour-like lesions such as nasal gliomas.

Astroblastoma of pure type

A rare case of astroblastoma of the pure type occurring in the left occipital lobe of a 54-year-old female is reported. The predominant histologic feature was that of perivascular pseudorosettes. The tumor tissues were not stained by the Mallory's phosphotungstic acid-hematoxylin, but the perikarya of some tumor cells were positively stained for the glial fibrillary acidic protein.

Fibrin linkage: a precursor for new attachment.

Previous studies indicated that connective tissue attachment to the root surface appeared to be dependent upon a chronologic healing sequence related to fibrin and collagen interactions. It was the purpose of the present study to try and substantiate this hypothesis by using histologic techniques designed to differentiate between fibrin and collagen during healing at the root surface interface. In four squirrel monkeys, 24 normal teeth were extracted and reimplanted after either (i) surgically denuding the coronal root surface of connective tissue fibers and cementum by root planing or, (ii) surgical denudation followed by topical application of citric acid (pH = 1; 3 minutes). Three specimens were available for histological analysis 1, 3, 7 and 21 days after reimplantation. Mallory's phosphotungstic acid hematoxylin staining technique was used to differentiate between fibrin and collagen. Epithelium migrated rapidly along the denuded, non-acid-treated, root surfaces, had reached the alveolar crest at 3 days, and was within the ligament space to the level of root denudation at 21 days. Epithelium did not migrate apically along denuded root surfaces treated with citric acid. At 1 and 3 days, inflammatory cells were enmeshed in a fibrin network which appeared to be attached to the root surface by arcade-like structures. At 7 and 21 days, the region had repopulated with connective tissue cells, and collagen fibers had replaced the fibrin. It was concluded that collagen fiber attachment to the root surface was preceded by fibrin linkage, and that the linkage process occurred as an initial event in the wound healing response.

Staining of isolated rabbit neurons and neuroglial clumps.

Isolated rabbit neurons stained just as intensely as neuroglial clumps did with Mallory's phosphotungstic acid haematoxylin, Weil and Davenport's, Marsland, Glees and Erikson's and with Gallyas's silver stain.

Oral leiomyomas

Oral leiomyomas are considered to be rare neoplasms, but they may be encountered more frequently than generally believed. Three types of leiomyomas are commonly described: solid leiomyomas, angiomyomas, and epithelioid leiomyomas. Three cases of solid leiomyoma are presented, all of which occurred in the anterior mandibular mucobuccal fold. Leiomyomas can be easily confused with other spindle-cell tumors. The necessity of using special stains, especially Mallory's phosphotungstic acid hematoxylin, is discussed.

A rapid method to demonstrate muscle cross-striations with thionin.

Frequently used methods for the demonstration of cross-striations of muscle fibers are Mallory's phosphotungstic acid hematoxylin (PTAH) (Mallory 1939 and Heidenhain's iron hematoxylin (McManus and Mowry 1960). Both require long staining periods; in addition, Mallory's method specifies fixation or mordanting in Zenker's fluid. A modified Mallory's PTAH procedure has been developed to accelerate the staining of formalin fixed tissue (Bohacek 1966). Preparation of solutions and actual staining time are still lengthy. The method of Menzies (1961) using bromphenol blue, though no mordanting is required, specifies 4-24 hours staining. None of these techniques are practical when time is a major factor. This report describes a staining procedure which makes the differentiation of muscle cross-striations quick and reliable.

Abnormal protein synthesis in facioscapulohumeral muscular dystrophy.

Recently we have observed that muscle polyribosomes obtained from patients with Duchenne muscular dystrophy (DMD) synthesize abnormally large amounts of collagen when combined with soluble enzymes derived from the same patient's muscle.' Increased protein synthesis in the carrier state of DMD has also been observed and has proved useful in the detection of hetero~~~otes.~ In this study, we tested four biochemical parameters in skeletal muscle from patients with fa cioscapulohumeral (FSH) muscular dystrophy: (1) total ribosome concentration, (2) ribosome distribution, (3) in vitro amino acid incorporation by fractionated ribosomes, and (4) in vitro collagen synthesis. Because three of these parameters are abnormal in patients and two are abnormal in carriers of DMD,'~~ we wanted to know if similar distwbances in ribosomal protein synthesis are found in FSH muscular dystrophy. Material and methods Eight patients with FSH muscular dystrophy and five normal controls matched for sex and age were examined. Muscle samples of patients and controls were obtained from the left vastus lateralis. Technical details about the muscle biopsy, the procedure for the preparation of the muscle extracts, and the evaluation of ribosomal protein synthesis were presented in previous papers. ' 74 The specimens for light microscopy were fixed in Susa's fixative, and longitudinal and transverse sections were stained with hematoxylin and eosin, Mallory's trichrome, phosphotungstic acid-hematoxylin, and periodic acid-Schiff reagent. Determination of noncollagen protein in the muscle homogenate was done by the method of Lowry and associates5 using bovine serum albumin as standard. A statistical analysis of the data was done by estimation of Fisher's t coefficient. The method of ~u~hes,~ as modified by Bray and ~errendelli,' was used for the determination of serum creatine phosphokinase (CPK) levels. Results

Application of standard micro-anatomical staining methods to epoxy resin-embedded sections.

Staining of semi-thin sections of osmium-fixed epoxy resin (Epon or Araldite)-embedded tissues with haematoxylin and acid dyes as counterstains (eosin or phloxin) was successfully achieved after intense oxidation using hydrogen peroxide as the oxygen donor. Results are closely similar to those obtained with paraffin sections (haematoxylin and eosin): nuclei stain blue, cytoplasm and extracellular structures (eg, collagen, elastica, basement membranes) stain with varying shades of red. Preliminary studies show that similarly good results, comparable with standard paraffinembedded histological materials, can be obtained, using H(2)O(2) pretreatment, with Weigert's iron haematoxylin, Mallory's phosphotungstic acid haematoxylin, Alcian blue, PAS reaction, Sudan III, Sudan black, oil red, Gomori's aldehyde fuchsin, and Baker's acid haematein.