Gallocyanin-chrome Alum Research Articles

Features of the morphofunctional state of parotid salivary glands in six-month-old rats with experimentally induced fetal macrosomia

The paper aims at studying the features of the morphofunctional state of parotid gland tissue in six-month-old rats born with induced macrosomia in its different variations. It has been known that the amount and quality of saliva are important indicators of teeth and gums health condition. The aim of the study is to examine experimentally the features of postnatal morphogenesis of parotid salivary glands in six-month-old rats with fetal macrosomia and compare the morphofunctional condition of parotid glands between two groups of 6-month-old and 3-month-old rats with induced macrosomia. Materials and methods. Four macrosomia models have been used by applying a high-calorie diet and hypokinesia to pregnant Wistar rats. After birth, the descendant rats have been weighed and divided into groups taking into account their calculated weight-length parameters. Four main groups and a comparison group have been created. The main (macrosomic) groups consist of species with the following parameters: (1) long body length and relatively decreased body weight; (2) balanced intrauterine development; (3) standard body length and intrauterine obesity; (4) standard body weight and intrauterine obesity induced by hypokinesia of a pregnant female rat. At the age of 6 months, the rats have been sacrificed. The absolute and relative weights of parotid glands have been evaluated. A histopathologic analysis revealing DNA and RNA (Gallocyanin-chrome alum technique) has been carried out after coating the parotid glands with paraffin; the PAS-reaction has been used to reveal glycoproteins. The serocytes nuclei cross section, the content of DNA in the cell nucleus, and the content of RNA in cytoplasm have been measured using computer images. Using the images obtained with confocal laser scanning microscopy, the gland acini cross section and the acini cross section perimeter of the parotid gland have been measured. The same technique has been used to measure the serocyte cross section. Caspase-3 immunehistochemical staining has been performed. Results . The macrosomic animals born with balanced intrauterine development and intrauterine obesity have shown signs of serocyte apoptosis, stroma widening and sclerosis, and the macrophage-lymphocyte infiltration of parotid gland stoma at the age of six month. Such manifestations attain their maximum in the animals whose macrosomia is induced by the hyper-calorie diet and the hypokinetic conditions in the pregnant rat females. Also, the enlargement and euchromatization of serocytes nuclei is observed in the animals in these groups, which indicate an increase in the morphofunctional activity of an individual serocyte. In the macrosomic rats born with signs of body mass deficiency, the islets of inactive serocytes are observed, the morphofunctional load of serocytes is relatively low, and in addition, there are no signs of serocytes failure or active compensatory processes. Comparison of the morphofunctional condition of the parotid gland at the age of 3 and 6 months has revealed that the age-specific enlargement of the parotid gland is less in the rats born with macrosomia and with both body mass deficiency and obesity, than that in the comparison group. The rats born with signs of body mass deficiency show an economic type of gland functioning, whereas the animals born with intrauterine obesity show signs of parotid gland atrophy with compensatory morphofunctional activation of functioning serocytes. Conclusion. The somatometric features of fetal macrosomia affect the morphofunctional features of the parotid gland of descendant animals at the age of 6 months (mature age).

Visualization of viral DNA assembly using nonfluorescent staining

ABSTRACTWe present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.

Effect of exposure to cadmium on the hippocampus in adult albino rat and the possible role of L-carnitine

Background The hippocampus is an important structure for formation of new memories. Cadmium is one of the most toxic agents that can affect hippocampal neurons. L-carnitine is an antioxidant. Aim of work To demonstrate the effects of cadmium on principal cells of hippocampus of adult rats and possible protective role of L-carnitine. Materials and methods A total of 42 adult rats aged 1 month were subdivided into four subgroups: group I was the control group, which received no treatment. Group II was administered cadmium given at a dose of 4 mg/kg/day for 1 month. Group III (sham control) was administered L-carnitine given at a dose of 200 mg/kg/day for 1 month. Group IV was administered cadmium and L-carnitine given to the rats at the same previous doses and duration. For each group, six rats were used for light microscopic study (gallocyanin chrom alum stain), and six rats were used for electron microscopy (group III studied only by light microscopy). Principal cell count and thickness were measured and statistically analyzed. Results Principal cells in group II showed degenerative changes. Morphometric data showed a significant decrease in measured parameters. In group III, the results were similar to those of the control group. In group IV, semithin sections and the ultrastructure of the principal cells showed obvious improvement of cells. Morphometric data also increased compared with those treated with cadmium. Conclusion Cadmium induces changes in the structure of the principal cells of the hippocampus in adult rats. L-carnitine plays an important in protection of the hippocampus.

Analysis of morphometric and ultrastructural characteristics of blood microcirculation in hippocampus of rats with alloxan diabetes under administration of citicoline

Aim: determine morphometric and ultrastructural features of blood microcirculation in hippocampus of rats with alloxan diabetes under experimental therapy with citicoline. Materials and methods . The research was carried out on 48 white male Wistar rats (250-300 g) randomized in 3 groups by 16 animals: I – intact (distilled water, intragastrically); II – animals with diabetes (distilled water, intragastrically); III – animals with diabetes + citicoline (500 mg/kg, intragastrically). Diabetes was reproduced by single subcutaneous injection of alloxan monohydrate (150 mg/kg). Blood glucose level was determined on the 11 th day after administration of alloxan using a glucometer. Citicoline and distilled water were introduced once a day during 20 days starting from the 11 th day after administration of alloxan. In morpho-functional study of hippocampal CA1-zone neurons the histological sections were deparaffinized and dyed with gallocyanin-chrome alum by Einarson for specific detection of RNA. Pictures were received with a microscope Axioskop (Zeiss, Germany). Using a computer-based picture analysis system VIDAS-386 (Kontron Elektronik, Germany) the density of endothelial nuclei, the nucleus area and the concentration of RNA in the nucleus were determined. The study of ultrastructural changes of blood microcirculation in hippocampus was carried out using transmission electron microscope PEM-100-01 («SELMI», Ukraine) by standard procedure and in accordance with common standards. Statistical difference between the groups were assessed by non-parametric Mann-Whitney U-test. Results . It has been established that citicoline contributed to recovery of proliferative activity of endothelium in vascular bed of hippocampus manifested with increase of endothelial nuclei density, their area and levels of nuclear RNA by 19,4% (p < 0,001), 17,2% (p < 0,05) and 25,8% (p < 0,01). Simultaneously it has been determined that hematoencephalic barrier damage in hippocampus of rats under administration of citicoline were less expressed or virtually absent. Conclusion . Thus, stimulation of revascularization processes, optimization of metabolic processes and also lack of pronounced changes in microcirculatory bed in hippocampus of rats with alloxan diabetes are the evidence of expressed endothelium-protective activity of citicoline

PDT-induced epigenetic changes in the mouse cerebral cortex: A protein microarray study

BackgroundPhotodynamic therapy (PDT) is used for cancer treatment including brain tumors. But the role of epigenetic processes in photodynamic injury of normal brain tissue is unknown. Methods5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), was used to photosensitize mouse cerebral cortex. PpIX accumulation in cortical tissue was measured spectrofluorometrically. Hematoxylin/eosin, gallocyanin–chromalum and immunohistochemical staining were used to study morphological changes in PDT-treated cerebral cortex. Proteomic antibody microarrays were used to evaluate expression of 112 proteins involved in epigenetic regulation. ResultsALA administration induced 2.5-fold increase in the PpIX accumulation in the mouse brain cortex compared to untreated mice. Histological study demonstrated PDT-induced injury of some neurons and cortical vessels. ALA-PDT induced dimethylation of histone H3, upregulation of histone deacetylases HDAC-1 and HDAC-11, and DNA methylation-dependent protein Kaiso that suppressed transcriptional activity. Upregulation of HDAC-1 and H3K9me2 was confirmed immunohistochemically. Down-regulation of transcription factor FOXC2, PABP, and hBrm/hsnf2a negatively regulated transcription. Overexpression of phosphorylated histone H2AX indicated activation of DNA repair, but down-regulation of MTA1/MTA1L1 and PML — impairment of DNA repair. Overexpression of arginine methyltransferase PRMT5 correlated with up-regulation of transcription factor E2F4 and importin α5/7. ConclusionALA-PDT injures and kills some but not all neurons and caused limited microvascular alterations in the mouse cerebral cortex. It alters expression of some proteins involved in epigenetic regulation of transcription, histone modification, DNA repair, nuclear protein import, and proliferation. General significanceThese data indicate epigenetic markers of photo-oxidative injury of normal brain tissue.

High Contrast and Homogeneous Staining of Paraffin Sections of Whole Human Brains for Three Dimensional Ultrahigh Resolution Image Analysis

The gallocyanin chromalum stain belongs to the classical DNA-RNA staining techniques in histochemistry. It has some important features for successful object orientated image analysis of whole sections of the human brain. To obtain reproducible staining results with these large sections, the method of Einar- son was adapted to image analytical requirements. We discuss staining in a warm staining solution, pH adjustment, and optimal stain composition. The embedding procedure for whole human brains is considered as well.

Systematic investigations of the contrast results of histochemical stainings of neurons and glial cells in the human brain by means of image analysis

The investigation of neurohistological specimens by image analysis has become an important tool in morphological neuroscience. The problems which arise during the processing of these images are non-trivial, especially if a pattern recognition of cells in the imaged tissue is intended. One of the major problems faced concerns the segmentation of structures of interest, whether cells or other histologic structures. The segmentation problem is often the result of an inappropriate staining procedure. For serious image analysis to be performed, the material under investigation must be optimally prepared. Spatially complex patterns, e.g. fuzzy-like neighbouring neurons, are easy to recognize for humans. But the integrative and associative performance of current artificial neuronal network schemes is too low to achieve the same recognition quality as humans do. Therefore, a general analysis of staining characteristics was performed, especially with respect to those stains which are relevant to object segmentation. Although most image analytical investigations of tissues are based on stained samples, a study of this type has not been previously conducted. Of the stains and procedures evaluated, the gallocyanin chrome alum combination staining provided the best stain contrast. Furthermore, this staining method shows sufficient constancy within different parts of the human brain. Even the fine nuclear textures are differentiable and can be used for further pattern recognition procedures.

Effects of lipid peroxidation on chromatin in rabbit and mouse spermatozoa: A cytochemical approach

To extend present knowledge on the possible interactions between lipid peroxidation and chromatin, we studied rabbit and mouse spermatozoa during spontaneous lipid peroxidation. Epididymal spermatozoa were aerobically incubated in high sodium or high potassium media to obtain spontaneous lipid peroxidation in the absence of added promoters. Quantitative cytochemical assays were performed in situ, in individual spermatozoa processed by Feulgen reaction and Gallocyanin-chrome alum (GCA) staining. Our findings show that in both species examined, the spermatozoa chromatin undergoes destabilization associated with marked alterations of the physico-chemical state of the DNA-protein complex. The decrease of Feulgen and GCA-positive material may be explained by DNA damage and implies the release from the nucleus of DNA fragments. Moreover, our data support the idea that during spontaneous lipid peroxidation, the spermatozoa chromatin undergoes destabilization sometimes correlated with increased acid lability of the DNA-protein complex. There are differences in the two species, probably correlated with differences in major protective enzymatic systems against cell damage by autoxidation and, especially, against different kinds of oxygen radicals responsible for initiation of lipid peroxidation.

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure

In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5-30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.

Somatostatin-14-like immunoreactive neurons and fibres in the human olfactory bulb.

This study describes the morphological features and the distribution pattern of neurons in the human olfactory bulb which are immunoreactive for an antiserum against the neuropeptide somatostatin-14. Immunoreactive nerve cell bodies were mainly found in the white matter surrounding the cell clusters of the anterior olfactory nucleus. Some immunoreactive neurons were also found scattered throughout the anterior olfactory nucleus and the deeper parts of the inner granule cell layer. Only a few immunoreactive neurons were localized in the glomerular layer and the outer granule cell layer. Immunoreactive fibres were found in all layers of the olfactory bulb. In addition, an impressive number of coiled and kinked immunoreactive fibres were localized within the anterior olfactory nucleus forming a dense plexus. Accumulations of twisted and coiled branches of immunoreactive fibres were rarely found either surrounding or within the olfactory glomerula. The characteristics of somatostatin-14 immunoreactive neurons as seen in the combined pigment-Nissl preparation were studied after decolourizing the chromogen and restaining the preparations with aldehydefuchsin in order to demonstrate the lipofuscin pigment and gallocyanin chrome alum for Nissl material. About 90% of the immunoreactive neurons studied in this manner turned out to be devoid of lipofuscin granules. The remaining 10% displayed different patterns of pigmentation. These findings suggest the presence of different types of somatostatin-14-like immunoreactive neurons in the olfactory bulb of the human adult.

Neuropeptide Y-like immunoreactive neurons in the human olfactory bulb

Neuropeptide Y-like (NPY) immunoreactivity was localized in the adult human olfactory bulb by the unlabeled antibody enzyme (peroxidase anti-peroxidase; PAP) technique in vibratome sections. The majority of NPY-immunoreactive somata was localized in the white matter surrounding the anterior olfactory nucleus. Immunoreactive neurons were less numerous within the anterior olfactory nucleus and within the olfactory bulb layers. NPY-immunoreactive fibres were present in the white matter, the anterior olfactory nucleus, and in the olfactory bulb layers. Fibres within the white matter were generally aligned in a straight path parallel to the long axis of the olfactory bulb and tract. Fibres within the anterior olfactory nucleus showed no clear orientation and displayed numerous branching points. Coiled plexes of NPY-immunoreactive fibres were present in the glomerular layer of the olfactory bulb. Additional characteristics of the NPY-immunoreactive neurons were studied after decolouring the chromogen and restaining the sections with aldehydefuchsin to demonstrate the presence of lipofuscin granules and also with gallocyanin chrome alum to stain the Nissl substance. This analysis showed that the neurons belong to the class of non-pigmented nerve cells.

Silver Impregnation of Alzheimer's Neurofibrillary Changes Counterstained for Basophilic Material and Lipofuscin Pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.

Somatostatin-like immunoreactivity in non-pyramidal neurons of the human isocortex.

The distribution of somatostatin-immunoreactive cell bodies and axons throughout the human isocortex and subjacent white matter was examined. Vibratome sections of cortical tissue (30-40 micrometers thick) obtained at surgery were treated to reveal the antigen by the unlabelled antibody enzyme method. Two types of somatostatin-immunoreactive axons were present: short, coiled axons and extended ones that follow a straight course in various directions. Somatostatin immunoreactive nerve cell bodies were encountered in layers II-VI and in the subjacent white matter. The majority of labelled cells were found in the white matter and layer VI, and then in layers II and III. The immunoreactive perikarya were fusiform, triangular or multipolar in shape and did not show preferential orientation of their long axis. Frequently, the fusiform neurons in layer VI and in the white matter were aligned parallel to radiate bundles of myelinated fibres. The immunoreactive neurons gave rise to a few thick dendrites. Often thin axon-like processes could also be recognized, originating either from the cell body or from a thicker dendrite. After destaining of the chromogen and counterstaining with aldehydefuchsin and gallocyanin chromealum, the formerly immunoreactive neurons displayed a light and eccentrically located nucleus. The soma contained only a sparse amount of basophilic substance and was nearly devoid of lipofuscin granules. In electron micrographs, the cisterns of the rough endoplasmic reticulum (RER) were localized near the periphery of the soma. Immunoreactivity occurred along membranes of the RER cistern, outer mitochondrial membrane, and in particles 120-150 micrometers in diameter. Rounded areas (up to a diameter of 1 micrometer) lacked immunoreactivity. Furthermore, there were a few tiny lysosomes.

The DNA-based human karyotype.

Image cytometry and computer analysis are used to determine the relative DNA content and the DNA-based centromeric index of the 24 chromosomes of the human karyotype. A two-step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q-band patterns. They then are stained for DNA using gallocyanin-chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA-based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA-based human karyotype. Analysis of the DNA-based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q- or C-band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.

Cytochemical patterns of mouse sperm chromatin during the passage along the female genital tract.

The chromatin characteristics of spermatozoa taken from different portions of the female genital tract were analyzed by quantitative cytochemical reactions specific for DNA and thiol-rich protamines, in order to investigate if chromatin undergoes any structural modifications upon leaving the male genital tract.When compared with the results obtained for vas deferens spermatozoa, all the data recorded after reactions specific for DNA (Feulgen reaction and staining with Propidium Iodide and Gallocyanin Chrome-Alum) and protamine thiol groups (DACM staining) suggest that chromatin remains highly stabilized during the passage along the female genital tract, and no sign of precocious decondensation may be detected before fertilization.

A simple method for the use of gallocyanin-chrome alum as an electron strain.

A method has been elaborated for the demonstration of DNA in the electron microscope. The method uses glutaraldehyde fixed tissue pieces from which RNA has been removed by incubation with RNase. DNA is stained by gallocyanin-chrome alum in the tissue block. Embedding and cutting is done in the usual manner. The method is based on histochemical observations at the light microscope level which show sufficient specificity and a good stoichiometry of the staining reaction.

The quantitative histochemistry of ribonucleic acid using gallocyanin.

A method for the cytophotometric estimation of ribonucleic acid in tissue sections using gallocyanin-chrome alum is described. The dye obeys Beer's law in gelatin sections. The effect of deoxyribonuclease on the staining of ribonucleic acid is also investigated. The results indicate that this method is of value in the quantitation of ribonucleic acid.

RNA and protein changes in spinal motoneurons and perineuronal glia under effects of two kinds of experimental convulsions

Clonic convulsions lasting 15 to 20 min were induced in rats either by placing the animals into hyperbaric chamber with 6 atm of oxygen pressure or by intraperitoneal injection of Metrazol (55 mg/kg). CARNOY-fixed paraffin sections of the spinal cord were stained with gallocyanin chrome alum for nucleic acids and with Amido black lOB or with Procion Brilliant Blue RS for proteins. Cytospectrophotometric determinations have shown that both kinds of the convulsions result in a marked decrease of RNA and protein content per cell in spinal motoneurons. In their glial satellite cells, only the protein content was decreased after hyperoxic convulsions. 3 h after the end of the convulsions, the decrease of macromolecule content remained in the motoneurons after the Metrazol convulsions while in the glial cells after the hyperoxic convulsions. The content of RNA in the motoneurons in the case of hyperoxic convulsions and the content of protein in the case of Metrazol convulsions increased higher than control level whereas the content of neuronal protein in the case of hyperoxic convulsions returned to this level. 18 to 24 h after the cessation of the convulsions, the content of RNA and protein was increased almost in all cases except for a decrease in glial protein content after the hyperoxic convulsions and for no changes in neuronal protein and glial RNA content after the Metrazol convulsions. The data obtained indicate similar mechanisms of the effects of various kinds of acute convulsions upon macromolecule metabolism in nerve tissue cells whereas the postconvulsive restitution can be somewhat different for each individual kind of the convulsions.

Gallocyanin-chromalum for improved scanning electron microscopy of whole nuclei without critical point drying.

Bone marrow nuclei fixed with modified Carnoy's, then stained with gallocyanin chromalum followed by air drying showed no difference in morphology when compared by means of scanning electron microscopy with similar nuclei prepared by critical point drying. Glutaraldehyde at pH 4.0 and 7.1, mercury-containing Zenker's fluid, and chromalum alone, all of which are considered to be nuclear protein cross-linking fixatives, failed to preserve the nuclear morphology as well as gallocyanin-chromalum or critical point prepared bone marro nuclei.