The use of a immunohistochemical reaction for nestin in determining the size of brain injury after transient occlusion of the middle cerebral artery

Существенной проблемой при оценке развивающегося повреждения при экспериментальном исследовании ишемии мозга является отсутствие стандартизированных методических подходов для быстрого и точного определения зоны деструкции при использовании парафиновых срезов. Цель исследования - разработка нового подхода к оценке объема повреждения головного мозга при фокальной транзиторной ишемии в бассейне средней мозговой артерии. Методика. Проводили морфологическое исследование серийных фронтальных парафиновых срезов конечного мозга. В качестве обзорной окраски использовали окраску 0,1% водным раствором крезилового фиолетового по Нисслю. Для оценки популяции активированных глиальных клеток использовали иммуноцитохимическую реакцию на нестин, для оценки жизнеспособности нейронов использовали иммуноцитохимическую реакцию на NeuN. Иммуноцитохимическую реакцию проводили с использованием мышиных моноклональных антител к нестину. Результаты. После проведения иммуноцитохимической реакции на нестин при первичном осмотре окрашенных срезов определялся четкий линейный иммунопозитивный контур вокруг предполагаемой зоны инфаркта. Детальный анализ этого контура (демаркационной линии) показал, что он четко разделяет интактную и поврежденную ткани и состоит из астроцитов, имеющих многочисленные иммунопозитивные отростки, часть из которых участвует в образовании глиальной периваскулярной пограничной мембраны. Заключение. Яркая и контрастная реакция на нестин позволяет легко оценивать объем повреждения на серийных парафиновых срезах ишемизированного головного мозга. Гистохимическую реакцию на нестин можно считать удобным маркером зоны повреждения головного мозга при моделировании ишемического инсульта. Evaluating the condition of astrocytes is important for assessment of brain injury. Aim. To develop a new approach to assessing the size of brain injury following focal transitional ischemia in the middle cerebral artery circulation using a immunocytochemistry reaction for nestin, a protein of intermediate filaments, which is considered a marker of stem cells. Methods. Injury of the rat brain was induced by 30-min occlusion of the left middle cerebral artery followed by 48-h reperfusion. A morphological study of endbrain serial frontal paraffin sections was performed. A 0.1% aqueous solution of Nissl cresyl violet (Dr. Grubler, Germany) was used for survey staining. The population of activated glial cells was examined using an immunocytochemical reaction for nestin with mouse monoclonal antibodies to nestin; viability of neurons was evaluated using an immunocytochemical reaction for NeuN. Results. The immunocytochemical reaction revealed a clear, linear immunopositive contour around the suggested infarct zone. In two days after unilateral ischemia, the cresylic violet staining showed that the center of damage was localized in the striatum and/or basolateral area. The light microscopy study of this contour (demarcation line) showed that it divided intact and damaged tissue. Conclusion. Therefore, nestin is a convenient marker of brain injury in experimental ischemic stroke. The immunocytochemical reaction in combination with quantitative analysis of scanned images is an optimum tool for determining the size of brain injury.

Canine visceral leishmaniasis: a remarkable histopathological picture of one asymptomatic animal reported from Belo Horizonte, Minas Gerais, Brazil

A remarkable histopathological picture of one asymptomatic dog naturally infected with Leishmania infantum (syn. chagasi) has been presented. Intracellular parasites were ease found in macrophages of all exanimated organs, especially in skin. Embedded paraffin tissues of liver, spleen, axillary and popliteal lymph nodes, and skin (ear, muzzle and abdomen) were stained by hematoxylin and eosin and by immunocytochemical reaction (streptoavidin-peroxidase method) to detect parasites. All organs showed an intense parasitism associated to severe pathological changes. All lymph nodes had conspicuous histological architecture alterations. Lymphocytes were replaced by macrophages stuffed with an intense number of amastigotes forms of Leishmania. The lymphoid nodules (without germinal centers) and the mantle zones in the cortex that surround the follicles were markedly attenuated. Livers showed small intralobular granulomas composed by macrophages loaded with amastigotes. Spleens had an intense depression of the white pulp whereas the lymphocytes were replaced by parasitized macrophages. All fragments of different anatomical region of skin (ear, muzzle and abdomen) showed a diffuse chronic inflammation. The cellular exudate was composed by macrophages, plasmocytes and lymphocytes. Macrophages loaded with amastigotes were ease found in all tissue fragments, but more intense in ear and muzzle. Thus, this fact enhances the importance of asymptomatic dogs in the epidemiology of visceral leishmaniasis.

Immunocytochemistry for Drugs Containing an Aliphatic Primary Amino Group in the Molecule, Anticancer Antibiotic Daunomycin as a Model

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-male-imidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH4, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues.

Neurothekeoma of the Hand

Neurothekeomas are rare, benign connective tissue tumours probably of Schwann cell origin. We report an unusual case of a neurothekeoma involving the hand. Histological examination revealed characteristic myxoid nodules with spindle shaped cells. The immunocytochemical reaction for S-100 protein and neuron-specific enolase was positive. Complete excision proved curative as the tumour was well encapsulated.

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

Xeroxographic-assisted location of areas or structures on clinical slides for EM examination

Recent studies in our laboratories have resulted in the development of microwave-accelerated silver stains for the demonstration of basement membranes and fungi, gram-negative bacteria, type III collagen, endoneurium, perineurium, DNA, and DAB or PPD-PC cytochemical or immunocytochemical reaction products. We have also found that the very structure in a specimen that raised a question by light microscopy can be mapped with a Micro-Locator Slide and then located and examined by the SEI and BEI modes of SEM by the techniques collated in this summary.