Biomedical subjects
Publications and source records attributed to P Castano.
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A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips ('sandwich' style) and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the 'sandwich' upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e., near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.
The 3-dimensional organisation of the neural component of the human Meissner corpuscle was studied after silver impregnation and following immunofluorescence for protein gene product 9.5 (PGP 9.5) by confocal scanning laser microscopy. The morphology of the Meissner corpuscle was found to show consistent differences depending on the labelling method used. After silver impregnation by the Winkelmann technique the branches of the afferent nerve fibres of the corpuscle showed both thin regions and varicose elements, the latter probably corresponding to the portions rich in mitochondria observed by transmission electron microscopy. The bulkier elements were never more than 5-6 microns in diameter. After immunolabelling for PGP 9.5 the nerve fibre branches in the corpuscle always presented flattened and discoidal expansions with a diameter of up to 30 microns. On the basis of what is known as to the mechanism of action of silver impregnations it is considered that the black precipitate preferentially labels the parts of neurons that are rich in neurofilaments. In any case the precipitate is deposited throughout the neuronal cytoplasm except in the mitochondria and the nucleus. Accordingly, in the varicosities of the Meissner corpuscles that are rich in mitochondria, there is little space for the formation of the precipitate. The use of antiserum against PGP 9.5, which labels the larger proteinaceous component of the axoplasm, demonstrates the complete architecture of the neural component of the Meissner corpuscle, and visualises the discoidal and flattened expansions which are absent in the impregnated corpuscles. It is concluded that immunostaining provides images of the corpuscles, and of peripheral neural structures that are in general closer to reality.
Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi-Cox mercuric impregnation, however, provides specimens which are precipitate-free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He-Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.
The activities of the 5 alpha-reductase [the enzyme that converts testosterone into dihydrotestosterone (DHT)] and 3 alpha-hydroxysteroid dehydrogenase [the enzyme that converts DHT into 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol)] have been evaluated in primary cultures of neurons, oligodendrocytes, and type 1 and 2 astrocytes obtained from fetal or neonatal rat brain. All cultures were used on the fifth day. The formation of DHT and 3 alpha-diol was evaluated by incubating the different cultures with [14C]testosterone or [14C]DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type 2 astrocytes and oligodendrocytes also possess considerable 5 alpha-reductase activity, while type 1 astrocytes show a much lower enzymatic activity. A completely different localization was observed for 3 alpha-hydroxysteroid dehydrogenase. The formation of 3 alpha-diol appears to be mainly, if not exclusively, present in type 1 astrocytes. 3 alpha-Diol is formed in very low yields by neurons, type 2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase) in separate central nervous system cell populations suggests the simultaneous participation of neurons and glial cells in the 5 alpha-reductive metabolism of testosterone and possibly other hormonal steroids (e.g. progesterone, corticoids, etc.).
Glial cells are able to metabolize testosterone into DHT through the action of the enzyme 5 alpha-reductase. DHT may be further processed to 3 alpha-diol by the 3 alpha-hydroxysteroid-dehydrogenase. The aim of this study was to analyze if a modulation of two second messenger systems might be able to modify the 5 alpha-reductase and the 3 alpha-hydroxysteroid-dehydrogenase activities present in glial cells. To this purpose, the formation of DHT has been measured in rat glial cell cultures after different time of exposure to TPA, 4 alpha-Ph, an active and an inactive phorbol ester respectively, and 8-Br-cAMP. The results obtained indicate that the formation of DHT is not modified by the addition of phorbol esters. On the contrary, a statistically significant increase of 5 alpha-reductase activity, over control levels, has been observed after 6, 12, and 24 h of incubation with 8-Br-cAMP (10(-3) M). The effect of the cAMP analogue appears to be specific for the 5 alpha-reductase, since the 3 alpha-hydroxysteroid-dehydrogenase did not show any variation after exposure to the drug. In conclusion, the present data suggest that proteinkinase A (PKA) might be involved in the control of the 5 alpha-reductase in glial cells. It is postulated that nervous inputs utilizing cAMP as the second messenger might modify the activity of this enzyme in glial cells.
The formation of the 5 alpha-reduced metabolites of testosterone, 5 alpha-androstan-17 beta-ol-3-one (dihydroxytestosterone, DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) was evaluated in sciatic nerve fragments and homogenates and in the myelin purified from the sciatic nerve of adult (60-90-day-old) male rats. The effects of aging on the metabolism of testosterone in the sciatic nerve was also evaluated using 20-month-old animals. In the sciatic nerve, tissue homogenization and myelin purification are associated with a marked decrease in the 5 alpha-reductase activity; these data do not appear to be consistent with a possible association of the enzyme with myelin membranes, as it occurs in the CNS. However, the results may be subject to other interpretations which are presented in the discussion. The formation of 3 alpha-diol is higher in sciatic nerve fragments than in homogenates or in the purified myelin. This occurs when testosterone is used as the substrate. In aged animals, the same pattern is observed but at a lower level. In the incubations in which the formation of 3 alpha-diol was evaluated using DHT as the substrate in fragments, homogenates and purified myelin of the sciatic nerve of either adult or old animals, it was found that the yields of this metabolite are higher than those obtained following incubation with testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)
A case of herniation of small bowel through a defect of the transverse meso-colon with secondary herniation through the gastrocolic ligament and re-entry into the greater peritoneal cavity is reported. This form of lesser sac hernia is rare and only one hundred half cases have been reported in literature.
The ultrastructure and organization of free nerve fibers occurring in dermal papillae containing Meissner's corpuscles were studied in the fingertips of the Green Monkey (Cercopithecus aethiops L.). The course of the thin unmyelinated fibers leads in the vicinity of Meissner's corpuscles in the connective tissue of its sheaths, between the epidermis and the peripheral fibroblast layer; they never come into contact with the nervous component of the receptor. Consequently, neither a "pericorpuscular reticulum" nor an "apparatus of Timofeew" is formed. The presence of thin axons containing dense-cored vesicles is evident, but they are not autonomous fibers, as indicated by the negative results of the Falck-Hillarp test for catecholamines. The nerve fibers occasionally seem to bear a close resemblance to the "open" and "plain" endings described by other authors in papillae devoid of corpuscles. Despite failure for close mutual contact to be established between the Meissner's corpuscle and the unmyelinated fibers ramifying in the connective tissue surrounding it, the hypothesis that the two may cooperate to form a "multimodal sensory package" is not without interest.
The distribution of the 5 alpha-reductase, the enzyme which converts testosterone into its 'active' metabolite dihydrotestosterone (DHT), has been studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation and in neurons and glial cells grown in cultures. Purity of cellular preparations was examined by electron and light microscopy. Purified neurons, astrocytes and oligodendrocytes, obtained from the brain of adult male rats, are all able to form DHT from testosterone and consequently possess a 5 alpha-reductase activity. Among the 3 cell types studied, neurons appear to be more active than oligodendrocytes and astrocytes. Moreover, between the two population of glial cells, the oligodendrocytes seem to possess a slightly higher enzymatic activity than that present in the astrocytes. Neurons appeared more active in metabolizing testosterone than glial cells also in cell culture experiments. It is presently believed that the 5 alpha-reduction of testosterone to DHT provides one of the mechanisms through which the hormone becomes effective in the CNS. This is supported by the present findings, which indicate that neurons are the cell population in which the 5 alpha-reductase is more concentrated. However, the presence of a considerable 5 alpha-reductase activity in glial cells indicates that also non-neuronal cells might participate in androgen-mediated events occurring in the brain.
By removing epidermis with EDTA and a subsequent enzymatic digestion of dermis, eccrine sweat glands of rat fingertips were exposed and examined by scanning electron microscopy (SEM). Different protocols were tested to remove as much connective tissue as possible, while minimizing damage to other structures, and to expose the epithelial surface of secretory tubules in order to display vascular and nervous networks. SEM observations gave detailed information on the relationship between epithelial secretory cells and myoepithelial cells, as well as on the vascular and nervous networks which surround the glomeruli of glands.
In the central nervous system of the rat, the 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone, appears to be concentrated in the white matter and in particular to be associated with myelin. In order to verify whether a temporal correlation might exist between the formation of myelin membranes and the variations of the 5 alpha-reductase activity observed in the brain, the enzymatic activity was studied in the cerebral cortex and in the hypothalamus of male rat in the age range of 3-60 days, in myelin purified from animals of 15-60 days of life and in oligodendrocytes (i.e. in the cells responsible for the formation of the myelin) isolated from the brain of adult and very young rats (7th day of life, when the myelination process is not yet initiated). The results show that the formation of 5 alpha-androstane-17 beta-ol-3-one (DHT) in the cerebral cortex and in the hypothalamus has a peak activity in the first two weeks of life, before the beginning of the myelination process; purified myelin has an enzymatic activity always much higher than that present in the cerebral cortex and in the hypothalamus and shows a peak in the formation of DHT in the first period of myelinogenesis, on the third week of life. Finally the oligodendrocytes of young rats possess a much higher ability to convert testosterone into the 5 alpha-reduced metabolites than the oligodendrocytes of adult animals. A possible involvement of this enzyme in the myelin function may be hypothesized.
Previous results obtained in this laboratory indicate that in the rat brain the 5 alpha-reductase, the enzymatic activity involved in metabolizing testosterone into 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone), is particularly concentrated in the white matter. In the present experiments, this enzymatic activity was studied in the following white matter structures, which were microdissected using the punch technique of Palkovits: anterior commissure (CA), fornix (FX), habenulo-interpeduncular tract (HP), corpus callosum (CC), stria medullaris (SM), optic chiasm (CO), fimbria of the hippocampus (FI), cerebral peduncle (PC), pontine fibers (FP), cerebellar medulla (CMD) and corticospinal tract (TCS). Moreover brain myelin was isolated and purified by sucrose density gradient ultracentrifugation. The results obtained confirm that, in the rat brain, the enzymes involved in testosterone 5 alpha-reduction are preferentially localized in the white matter. However, clearcut differences in the metabolic activity exist between the different structures examined so far. DHT formation increases rostro-caudally, so that the highest activity has been recorded in the white matter structures punched at the level of pons (FP), medulla oblungata (TCS) and cerebellum (CMD). The high metabolic activity associated with the white matter structures appears to be linked to the presence of myelin, since the specific activity of the enzyme is particularly elevated in purified preparations of myelin sheaths.
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During an electron microscopical study of the common peroneal nerve of albino rats and pigeons, neurons were found along the course of the nerve. Their ultrastructural morphology was studied. On the basis of observed features, such as the occurrence of bare areas of the neurilemma, of thick processes of the perikarya (dendrites), and of synapses on the soma, it appears that these cells are aberrant sympathetic neurons which have migrated to a more distal position than usual.
In this relation the authors present preliminary morpho-quantitative data on the fibres of sciatic nerves of normal albino rats, compared with those of animals treated endoperitoneally with 20 and 40 mg of atrazine. This compound (2 chloro - 4 ethylamino - 6 isopropyl amino-S-triazine) is largely used in agriculture, and recently was suspected to be neurotoxic in man. In order to evaluate its action on the peripheral and central nervous systems, and other organs, a morphological, morpho-quantitative and physiological investigation was planned on groups of untreated and treated albino rats. Our data are concerned with both unmyelinated and myelinated fibres. The areas, the shapes (and their distribution spectra), the perimeter and the possible anisothropy (as a sectioning plane index) of unmyelinated axons were evaluated. These parameters were compared in the groups (treated and untreated) by the usual statistical tests. The axonal and total areas, the thickness of the myelin sheath and the number of myelin lamellae of myelinated fibres were analysed, and the related distribution histograms were carried out. A comparison in the three groups was also made in this case. All the data, particularly those resulting from comparison among the groups, are discussed in terms of the possible neurotoxic action of atrazine.
The aim of this study was to elucidate the site and detailed nature of peripheral nerve damage induced in the rat by chronic CS2 inhalation exposure in the light of the relationship between pathological and neurophysiological data. Adult male rats were exposed to 700 ppm of CS2 2 h/d, 5 d/week for 12 weeks and then followed-up for 18 weeks. The first alteration observed was a decrease in the nerve conduction velocity, discovered after only 3 weeks of exposure. Pathological lesions were first observed in the 10th week and consisted of a typical "giant axon" axonopathy. Obvious pathological lesions of the myelin sheaths were revealed much later, in the 3rd week after the end of exposure, when some nerve fibers were dying back. Recovery took place with the regeneration of new fibers which started in the 8th week after the end of exposure and was nearly complete in the 18th week. These findings demonstrate that CS2-induced polyneuropathy is an axonopathy very similar to that caused by other occupational neurotoxic agents like MnBK, n-hexane, and acrylamide. The timing of the pathological events within the nerve fibers suggests that the pathogenesis of the nerve lesions should probably be attributed to a primary energy failure of the axonal membrane induced by CS2.
Both in animals and in man the inhalation of CS2 vapor induces a chronic polyneuropathy with primary lesions in the axons of peripheral nerves. Since it was reported in several studies that the administration of gangliosides improves nerve regeneration and the functional recovery of nerves damaged by section as well as cryodegeneration, a study was undertaken to evaluate the effects of bovine-brain gangliosides administration on the experimental CS2 neuropathy in the rat. One hundred and fifty male rats were intoxicated with CS2 by a discontinuous inhalation exposure to 700 ppm for 12 weeks until a clear neuropathy developed. Thereafter the animals were subdivided at random into five groups and treated in different ways: 10 mg/kg BW gangliosides, 0.5 mg/kg gangliosides, 0.5 mg/kg vitamin B1, and 1 mg/kg vitamin B6, physiological solution, and controls without any treatment. The recovery from neuropathy was controlled for 18 weeks of treatment and assessed periodically by means of clinical, electromyographic, and morphological examination. The results of morphological studies showed more pronounced regeneration activity in the rats treated with the high dose of gangliosides than in all others, while no differences among the groups could be observed as far as clinical and neurophysiological parameters are concerned. The mechanism supporting this ganglioside-induced effect has so far not been ascertained, and further studies on this subject are in progress.