Chrome Alum Hematoxylin Research Articles

Histological, histochemical, ultrastructural and immunohistochemical identification and characterization of the neurosecretory cells of the adult rabbit's adrenal medulla.

The present investigation was conducted on the adrenal glands of 40 adult New Zealand rabbits of both sexes to characterize and identify the histological, histochemical, ultrastructural, and immunohistochemical features of the neurosecretory cells of the adrenal medulla. The obtained specimens of adrenal medulla were subjected to routine histological techniques and then stained with different histological stains, including general, non-specific, specific, and highly specific stains for neurosecretory cells, in addition to immunohistochemical reactions. The obtained results showed two types of adrenal medullary neurosecretory cells containing secretory granules (SGs) of different electron densities: adrenaline and noradrenaline (NA) secreting cells. These secretory granules showed a strong positive reaction to the Grimelius silver impregnation technique. Sections stained with Gomori's chrome alum haematoxylin stain, and the secretory granules showed a strong dark blackish-blue positive colour. The medullary cells showed typical chromaffin reactions when stained by H&E and Giemsa stains after formol dichromate 'Ortha's fluid' fixation. The noradrenaline secretory granules gave a strong positive Schmorl's test, while the adrenaline ones showed a moderate reaction. Immunohistochemically, the adrenal medullary cells were subjected to anti-chromogranin A (CHGA) antibody using the PAP technique, which gave positive reactions.

Neuroendocrine control of cocoon production in native earthworm Perionyx ceylanensis subjected to seasonal variation

Aim: The aim of the present study was to observe the role of cerebral ganglionic neurosecretory cells (NSCs) during cocoon production in native earthworm species Perionyx ceylanensis subjected to amputation and seasonal variations. Methodology: Histological studies (using Aldehyde Fuchsin and Chrome Alum Haematoxylin Phloxin stain) were carried out on brain NSCs in the two groups of earthworms (Group I and Group II) maintained in earthen culture pots (2L) with cowdung as food. Group I comprised of brain amputed earthworms was subjected to observe the role of brain NSCs in production of cocoon and Group II earthworms subjected to seasonal changes in the cerebral NSCs during cocoon production. Five replications were kept for Group I (1 individual per pot) and Group II (1 pair per pot). Results: Group I debrained earthworms started to lay cocoons from the 31st day following regeneration of cerebral ganglionic type A NSCs. In group II worms the highest neurosecretory activity was registered in the cerebral type A cells, especially during monsoon coinciding with the hike of cocoon generation. Interpretation: Appearance of type A NSCs in regenerated brain and peak of type A neurosecretory cell activity during peak reproductive period of earthworm species (as indicated by peak of cocoon production) indicates the possible role of cerebral type A NSCs in cocoon laying.

Histological, histochemical, and immunohistochemical studies of hippocampus in male New Zealand rabbits.

The present study aims to give detailed histomorphological features of the hippocampus of adult male New Zealand rabbits. Both histological and histochemical specimens were prepared to be examined microscopically by using a light microscope. The hippocampus appeared as C-shaped hippocampal proper, dentate gyrus, and subiculum. The hippocampal proper subdivided along its length according to the density and size of its major constituent pyramidal cells into four distinct regions named Cornu Ammonis (CA1, CA2, CA3, and CA4). With the histochemical preparations, each of these regions consisted of five layers, stratum alveolus, stratum oriens, stratum pyramidale, stratum radiatum, and stratum lacunosum-moleculare. The stratum pyramidale constituted the middle dark zone and contained the principal excitatory neurons and a few interneurons. Histochemically, the pyramidal neurons along all regions of the CA reacted positively to Grimelius silver impregnation, lead hematoxylin, Gomori's aldehyde fuchsin, aldehyde thionine, Gomori's chrome alum hematoxylin, and performic acid alcian blue stains. Immunohistochemically, the pyramidal neurons reacted positively to anti-NSE antibodies. The dentate gyrus was formed of three distinct layers. The subiculum was formed of proper subiculum, presubiculum, and parasubiculum.

GLUT2 Immunoreactivity in Gomori-positive Astrocytes of the Hypothalamus

A specialized subtype of astrocyte, the Gomori-positive (GP) astrocyte, is unusually abundant and prominent in the arcuate nucleus of the hypothalamus. GP astrocytes possess cytoplasmic granules derived from degenerating mitochondria. GP granules are highly stained by Gomori's chrome alum hematoxylin stain, by the Perl's reaction for iron, or by toluidine blue. The source of the oxidative stress causing mitochondrial damage in GP astrocytes is uncertain, but such damage could arise from the oxidative metabolism of glucose transported into astrocytes by high-capacity GLUT2 glucose transporters. In accord with this hypothesis, the reported anatomical distribution of astrocytes staining positively for GLUT2 glucose transporters closely matches that of GP astrocytes. To examine whether or not these two staining procedures detect the same population of astrocytes, immunocytochemistry was performed on semithin sections to detect GLUT2 protein and sections were then stained with toluidine blue to detect GP granules. It was determined that GP astrocytes are frequently immunoreactive for the GLUT2 transporter protein. These data support the possibility that GP astrocytes may have an important influence upon the reactivity of the hypothalamus to glucose and that a specialized glucose metabolism may in part underlie the development of mitochondrial abnormalities in hypothalamic GP astrocytes.

Reflection contrast microscopy within chrome-alum haematoxylin stained thick tissue-sections.

This paper introduces two innovations in reflection contrast microscopy (RCM): (1) an extended application for qualitative light microscopic investigations; and (2) a novel method for quantification in cytochemistry. (1) We found out that RCM cannot only be used for surface characterizations and in thin sections but also within thick tissue-sections. The use of the RCM technique is demonstrated on slides of the supraoptic nucleus (SON) of the rat stained with chrome-alum haematoxylin: Among all the stained structures only neurosecretory granules are found to cause reflections. The visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright field microscopy. (2) The improved differentiation allows the quantification of neurosecretion in tissue-sections by combining RCM with grey-scale image analysis.

Isolation of pseudoperoxidase-positive astrocyte granules from intact rat brain and cysteamine-treated neuroglial cultures

A subpopulation of astrocytes in periventricular regions of aging brain and in cysteamine (CSH)-treated glial cultures contain autofluorescent cytoplasmic granules that exhibit an affinity for Gomori's chrome alum hematoxylin (CAH), and non-enzymatic peroxidase activity. Although shown to be histochemically distinct from lipofuscin, the lack of pure preparations of these glial inclusions has hindered the elucidation of their precise chemical constituents. Using sucrose gradient fractionation and density centrifugation on percoll, we obtained enriched preparations of astrocyte cytoplasmic granules from intact rat brain and CSH-treated astrocyte cultures. The presence and relative purity of these inclusions were confirmed by laser scanning confocal microscopy for red autofluorescent granules, diaminobenzidine histochemistry for non-enzymatic peroxidase activity and chrome alum hematoxylin (Gomori) staining. In the enriched fractions, the smaller granules (0.5-4.0 microns) were spherical and weakly autofluorescent, whereas larger inclusions (5.0-10.0 microns) tended to be intensely autofluorescent and pleomorphic. As in situ, the purified material was argyrophilic and did not stain for lipids. Isolation of these astrocytic inclusions should permit a more thorough characterization of their biochemical contents.

Gomori‐positive astrocytes: Biological properties and implications for neurologic and neuroendocrine disorders

Granule laden astrocytes exhibiting an affinity for chrome alum hematoxylin and aldehyde fuchsin (Gomori stains) have been described in the periventricular brain of all terrestrial vertebrate species examined to date including humans. The astrocytic inclusions are rich in sulfhydryl groups, emit an orange-red autofluorescence, and stain intensely with diaminobenzidine, a marker of endogenous peroxidase activity. The distinct autofluorescence pattern and the absence of neutral lipid, acid phosphatase, and beta-glucuronidase activity exclude lipofuscin or lysosomes as components of these astrocytic granules. The emission of orange-red autofluorescence and the nonenzymatic nature of the peroxidase activity implicate the presence of porphyrins and metalloporphyrins such as heme as major constituents of these cytoplasmic gliosomes. The role of Gomori-positive astrocytes under normal and pathologic conditions is incompletely understood. In vivo, numbers of astrocytic granules increase as a function of advancing age, in response to chronic estrogen stimulation, and following X-irradiation. In vitro, these cells accumulate with increasing time in culture and following exposure to the sulfhydryl agent, cysteamine. Gomori-positive astrocytes may supply heme to neurons for the synthesis of cytochromes, catalases, and other heme enzymes. They may play a role in photostimulation of sexual cyclicity, the promotion of neuritic development, the degradation of toxic lipoperoxides, and the metabolism of various neurotransmitters. Conversely, these cells may contribute to the pathogenesis of several neurologic and neuroendocrine disorders. Examples of the latter include a) augmentation of goldthioglucose neurotoxicity, b) induction of hypothalamic anovulation and reproductive failure, c) exacerbation of porphyric encephalopathy, and d) potentiation of parkinsonism and other free radical-related neurodegenerations.

Morphogenesis of the subcommissural organ in the hamster.

Morphogenesis and cytodifferentiation of the subcommissural organ (SCO) of the hamster were studied by light, and scanning and transmission electron microscopy, from 11.0 to 15.6 days of gestation and to 60 days after birth. The cells of SCO were differentiated at 12.5 days of gestation, earlier than the adjacent ordinary ependymal cells. The SCO consisted of tall columnar epithelial cells with less developed organelles and short cilia. At 13.0 to 14.5 days of gestation, many polysomes and a small number of rER and microtubules were observed in the cytoplasm. At 15.0 days, rER, sER and microtubules were increased in number. Just before birth (15.5-15.6 days of gestation), cisternae of rER became slightly swollen and filled with flocculent substances. At 0 to 2 days after birth, the cisternae of rER were enlarged remarkably, and moderately electron-dense granules appeared in the apical cytoplasm. Irregular thread-like fibrils, apparently secretory materials, began to cover a part of the ventricular surface of the SCO, together with conspicuously stretched cilia. At 2 to 3 days after birth, secretory materials in the cytoplasm and in the ventricular surface became to be stained positively by chrome-alum hematoxylin, with the first appearance of Reissner's fiber. At 5 to 20 days, rER became more conspicuously swollen and filled with flocculent substances, as was observed in the adult.

Accumulation and identification of lipofuscin-like pigment in the neurons of Bulla gouldiana (gastropoda: opisthobranchia)

A few reports suggest that pigmented granules found in molluscan neurons accumulate with age as do lipofuscin granules in vertebrate cells; however, no reports on molluscan neurons include detailed descriptions of granule accumulation or histochemical tests to identify the pigment as lipofuscin-like. In this study light microscope observations of living ganglia from 1.7, 2.7, and 3.0 cm and larger (shell length) sized Bulla gouldiana showed an increasing accumulation of orange-red pigment in the perikaryon corresponding to increasing shell size ( i.e. age). With the electron microscope similar results were obtained, and lipofuscin-like granules were seen in the nerve cell cytoplasm of veliger larvae and in all adult sized Bulla. Staining with Sudan black B, Nile blue, chrome alum hematoxylin, PAS reagents, and exposure of the neurons to u.v. light to observe subsequent autofluorescence, yielded positive results in the areas of pigmented granule accumulation. Thus, the brillant orange-red granules that accumulate with age in the peripheral cytoplasm of adult Bulla neurons, and which are probably also present in larval stages, chemically resemble the lipofuscin granules of vertebrates. Similarities and differences between molluscan pigmented granules and vertebrate lipofuscin granules, in relation to structure and mechanisms of development and accumulation, are discussed.

On the neurosecretory system of Dendrobaena atheca Cernosvitov.

AbstractFour kinds of neurosecretory cells A, B, U and C are distinguished in the central nervous system of Dendrobaena atheca Cernosvitov. A cells, which show different morphological characteristics under different physiological states and during their cyclic changes, are the most active neurosecretory cells. They form the outer layer of the cortical cell zone in the cerebral ganglion. B cells are large and medium sized and are distributed in all parts of the central nervous system. U cells are found only in the sub‐pharyngeal ganglion while C cells are distributed in the sub‐pharyngeal as well as in the ventral nerve cord ganglion. The number and secretory activity of C cells decrease in caudal direction. Further, Gomori‐positive cells are also observed in the ganglia of the vegetative nervous system.A rudimentary neurohaemal organ, the storage zone, has been observed in the cerebral ganglion and there appears to be another neurohaemal area in the ventral nerve cord ganglion. The storage zone is formed by the terminal ends of the axons of A cells. The chrome alum haematoxylin phloxin (CHP) and aldehyde fuchsin (AF) positive substances in the form of granules are found in this area. The cerebral ganglion is richly supplied by blood capillaries. The distal end of the axons of B cells are swollen like a bulb while in some cases the axons are united to form an axonal tract. Extra‐cellular material is abundant in different parts of the nervous system. In all cell types, the perinuclear zone is the first to show activity in the secretory cycle. It appears that the nucleus may be involved in the elaboration of the neurosecretory material in the cells.

Protein granules in glial and ependymal cells in the hypophysiotropic area of the rat, stainable with aldehyde-fuchsin and chrome alum-hematoxylin.

Protein granules in glial and ependymal cells in the hypophysiotropic area of the rat, stainable with aldehyde-fuchsin and chrome alum-hematoxylin.

Some light- and electron-microscopical observations on the pars tuberalis of the pituitary gland of the rabbit.

SUMMARY The cytological appearance of the pars tuberalis both at the light- and the electron-microscopical levels is described. Two main types of cell, one, which is designated the tuberalis cell, having a rounded contour, pale cytoplasm with a few small secretory granules of about 100 nm in diameter, many polyribosomes and a relatively large ovoid nucleus; and the other, the interstitial cell, with long processes often encircling tuberalis cells, an irregular nucleus and cytoplasm with abundant microfilaments about 10 nm in diameter were seen. Beaded nerve fibres and neurosecretory material were demonstrated in the pars tuberalis with chrome alum—haematoxylin, a finding confirmed at the electron-microscopical level where nerve fibres which appeared to be making contact with tuberalis cells and containing numerous microvesicles of about 50 nm in diameter were observed.

Some light- and electron-microscopical observations on the pars intermedia of the pituitary gland of the rabbit.

SUMMARY The cytological appearance of the pars intermedia of the rabbit both at the light and the electron microscopical (E.M.) levels, is described. Two types of cell,one stainable byperiodic acid—Schiff's reagent and aldehyde—fuchsin and the other chromophilic, non-granular and intercalated between the former, were observed. In E.M. sections the first type had two kinds of granule in its cytoplasm, a few electron-dense about 250 nm in diameter, mostly confined to the Golgi area, and numerous large 'vesicles' or granules of varying density about 500–600 nm in diameter generally distributed throughout the cytoplasm. The second type of cell when viewed in the electron microscope is characterized by microvilli projecting into intercellular spaces, and long, linked processes ramifying in between the principal cells. Varicose nerve fibres and abundant neurosecretory material were demonstrated in the pars intermedia with chrome alum—haematoxylin and Alcian blue. In E.M. sections these neurosecretory granules were similar to those in the infundibular process. At the point where nerve fibres appeared to be making contact with glandular cells, synaptic vesicles of about 50 nm in diameter were also observed.

セロイド色素の本態に関する組織化学的研究

Ever since disclosure of ceroid in the uterus of vitamin E-deficient rat, the problem on its discrimination from lipofuscin has been the subject of much debate. Only when this discrimination can be satisfactorily made, the independence of ceroid can be said to be established.Heret o fore, this assay has mainly been conducted by observing either the grades of the positiveness or the negativeness of the acid fast fuchsin stain, chrome alum hematoxylin and sudan III or IV stain which seem to be defective for discriminating ceroid from lipofuscin, since, for example, the acid fast fuchsin stain not only colors ceroid, but also feebly does lipofuscin, and chrome alum hematoxylin stain, provides only an unassertive assay where, while lipofuscin is positive with this stain, ceroid negative. In contrast, the 0.02% nile blue sulpihate staining method (pH 3.0), newly developed by the author and his associates on the principle of the Lillie' s method of 1956, is an assertive method whereby ceroid, when the time of dehydration as well as mounting is shortened in all within 2-3 minutes, is stained blue, lipofuscin being scarcely stained. Mast cell granules are stained deep blue, however, can be easily discriminated from ceroid in that they indicate red metachromasia with 0.05% toluidine blue. Then it has been found that the blue coloration ef ceroid by nile blue sulphate at a pH 3.0 is resistant to a 4-hour,60° C methylation. In considering the affinity of nile blue sulphate for both the pigments, the author made a serious view of the Lillie's report that a light green coloration of lipofuscin which is effective with a dilute nile blue sulphate solution (005-0.1%) at a lower pH limit (about3.5), but not so with many basic dyes other than nile blue sulphate, is quite resistant to acetone extraction and to some extent so to alcohol.This difference between both the pigments may be elucidated according to the author's point of view that lipofuscin stains blue merely because of its fat dissolving property, but its blue coloration can not be resistant to dehydration by alcohol, while ceroid forms some kind of relatively insoluble dye complexes with nile blue sulphate, because the pigment is rich in carboxylic radicals resistant to a low pH (from 2.5 to 3.0) because of being originated from the protein possessing a low solubility. Moreover, the meaning of the highly low concentration of the dye in aqueous solution is discussed.

Observations with the light microscope on the distribution and connexions of the oxytalan fibre of the lower jaw of the mouse

Serial sections through the lower jaw of the adult laboratory mouse, stained by aldehyde-fuchsin following oxidation by potassium monopersulphate, show that the majority of oxytalan fibres run between periodontal vessels and cementum. The fibres may also be stained by chrome alum haematoxylin and alcoholic basic fuchsin, also after a stage of oxidation. These stains, which also outline cell membranes and processes, show that the oxytalan fibres are very intimately related to cell processes, and appear to be continuous with them at the resolution of the light microscope. The orientation of most of the oxytalan fibres is obliquely across the periodontal membrane, frequently perpendicular to the direction of the bundles of collagen. These fibres are also oblique to the main directions of compressional force of the tooth on the periodontal membrane. Their direction and attachments therefore suggest that they are concerned with the stability, and possible patency, of the periodontal vessels under pressure. The other major group of fibres is located towards the mesial end of the incisor periodontal membrane, where they are closely applied to the vessels and run forward to the gingiva. These fibres may also lend stability to the vascular plexus.

A system linking the third ventricle with the pars tuberalis of the rhesus monkey.

IN the course of studies of the hypothalamus of the rhesus monkey, we have investigated a restricted area of specialized ependymal cells. This area lies anterolaterally in the tuber cinereum (see Fig. 1), and is distinguished by the long processes of the ependymal cells which extend to the region of the pars tuberalis; neighbouring ependymal cells are cuboid, without processes. The elongate ependymal cells stain deeply with the Gomori chrome–alum–haematoxylin technique and may be followed with ease using the optical microscope. They are not evident after silver impregnation techniques (Bodian's silver protargol method or Bielschowsky's technique), nor are they detectable after performic acid–alcian blue staining; they can, however, be demonstrated by cresol fuchsin and aldehyde fuchsin and the periodic acid–Schiff (PAS) method.

Light-microscopic observations on the hypothalamus and pituitary gland of the rat fixed in osmium tetroxide.

SUMMARY The ventral hypothalamus and the pituitary gland of the rat were examined with the light microscope in sections fixed in an osmium tetroxide solution without further staining. The histology of the neurohypophysis could be observed with remarkable clarity. Herring bodies and axons of the supra-optico-neurohypophysial tract show as grey homogeneous structures. Pituicytes appear as round or variously shaped cells whose cytoplasm is crowded with osmiophilic granules. The granules vary in size from 0·5 to about 2·5 μ, and present either as solid or as ring-shaped black structures. They stained neither with aldehyde-thionine, nor with chrome-alum haematoxylin, nor with the PA-Schiff techniques but stained with Sudan black B. They were found in profusion in the median eminence of the hypothalamus and the infundibular stem in an apparent extracellular location surrounding fibre bundles and capillaries of the hypophysial portal plexus. Two types of cells were identifiable in the supraoptic nucleus of the hypothalamus. Smaller, angular neurones stained densely with osmium and with aldehyde-thionine and larger, more rounded neurones stained only lightly with osmium and did not stain with aldehyde-thionine. A peculiar laminated arrangement of structures in the infundibular recess in the region of the arcuate nucleus was noted. Several distinct types of cells in the adenohypophysis were identifiable on morphological grounds: thyrotrophic basophils, gonadotrophic basophils, acidophils and chromophobes. The possibility of a neuroendocrine role of the pituicytes is discussed.

Electron microscopic observation on the supramedullary nerve cell of Spheroides Niphobles.

Several morphological reports have been published, since the so-called “supramedullary nerve cell (SMC)” or “dorsale Zelle” was first discovered in the dorsal surface of the spinal cord in Cyclostomata by Owsjanikow (1854). However in the functional signification of the SMC, the opinions of authors have been incongruous, namely, Kapper et al. (1939), and Hughes (1957) presumed it to be a sensory nerve cell, while Bennett et al. (1959), Hagiwar aet al. (1959), and others denied this on the basis of their electrophysiological observations. While Ishibashi recently (1962) reported that the SMC was a sort of neurosecretory cells from the positive results of chrome-alum hematoxylin phloxine (CHP) staining preparations. Nakajima et al. (1965) are the first to study the SMC of the puffer by electron microscopy. Since they were unable to detect a correlation between SMC granules and elementary granules in the neurosecretory cells of the hypothalamus, they denied the theory of Ishibashi's, and concurrently reported the relationship between the SMC and the capillaries. We have studied the fine structure of the SMC of Spheroides Niphobles and compared our observations with those of the above-mentioned investigators.

Demonstration of an alcoholchloroform insoluble, periodic acid-Schiff reactive substance in the hypothalamus of the rat.

An alcohol-chloroform insoluble, periodic acid-Schiff reactive substance associated with the ependymal lining of the supraoptic and infundibular recesses was demonstrated in the hypothalamus of the white rat. Although the ependyma is stratified and contains cells with long processes throughout the extent of the recesses, the PAS reactive substance is present in very circumscribed areas within the recesses. It was also shown that this PAS reactive substance is not related to neurosecretory material which has an affinity for chrome-alum hematoxylin and aldehyde fuchsin. The regularity of appearance and location of the substance may be taken as evidence in favor of its being a specific material elaborated in the regions mentioned.

Histochemical Studies on the Cytoplasmic Granules in Nerve Cells

Previously we have reported on the histochemical nature of the cytoplasmic granules contained in the nerve cell.The granules are stained by the chrome-alum hematoxylin method (CAH) as are the neurosecretory granules in the neurosecretory cell in the hypothalamus. They are recognized as fine round particles and are well fixed in the Maximow-osmium fluid and are stained positively by the periodic acid-Schiff method (PAS).In order to confirm the existence of the substance, we made an attempt at autolysis and exmined the granules from the histochemical point of view.