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Biomedical subjects

R Laschi

Publications and source records attributed to R Laschi.

At least 37 records · Page 2Linked to original sources

Scanning electron microscopy application in clinical research.

Our personal experience on the application of scanning electron microscopy in cardiology, gastroenterology and ophthalmology is reviewed. SEM has not yet significantly contributed to myocardium pathology. However, in the near future, SEM could be a reliable technique to complete the information available from other sources. As to atherosclerosis, SEM allowed us to improve our knowledge of the early stages of the disease; some pathological features, not always detected by conventional morphological examinations, can be documented. An important contribution to gastrointestinal pathology was made by SEM investigations both in the staging of some important diseases (i.e., coeliac disease, peptic ulcer, Crohn's disease, ulcerative colitis) and in the follow-up of mucosal changes during therapy. In the ophthalmological field, SEM provided three-dimensional new information to clinicians, who are familiar with the biomicroscopic images. Our experience in hematology is still limited. However, in the last few years SEM joined to immunocytochemistry allowed us to characterize cell populations in several blood diseases. Some procedures of particular interest in the management of human bioptic specimens are stressed in order to get to a complete correlative microscopy. We conclude that continuous and simultaneous correlations have to be carried out between SEM and other methods and instruments available for morphological investigation.

Animals↗

Light and electron microscopy investigation of glycoconjugates of rat hippocampus by lectin-gold technique.

The distribution of glycoconjugates in rat hippocampus was investigated by light and electron microscopy using gold-labelled lectins with different sugar specificities: ConA, WGA, PNA and LTA. A morphological correlation with cytochemical data was performed, and revealed the presence of intensely-stained (dark), unstained (light) and weakly-stained (intermediate) neurons both in Ammon's horn and in the dentate gyrus. The glycoconjugates are located in the nucleus, the perikaryal cytoplasm and the cytoskeleton of axons and dendrites: they are probably involved in the mechanisms of transport towards the synaptic membrane. The presence or absence of glycoconjugates corresponds to the particular ultrastructural aspect of the cell. This suggests the existence of different functional states of the neurons, probably correlated with the synthesis of neurotransmitter precursors or receptors.

Animals↗

Ultrastructural aspects of ageing rat hippocampus and effects of L-acetyl-carnitine treatment.

The ageing rat hippocampus undergoes ultrastructural changes, including the loss of axosomatic synapses of the granule cells. These synapses are supposed to take part in a feed-back regulation of granule cell activity, as they are inhibitory terminals of interneurons which receive afferences from the granules themselves via the giant synapses formed by the collaterals of the mossy fibres. In the present study the authors performed a quantitative analysis of axosomatic and giant synapses at the ultrastructural level in aged rats as compared with young animals. In aged rats a decrease both in axosomatic synapses and in giant synapsis vesicles was found, giving further support to the postulated feed-back mechanism. Both young and old rats were treated with L-acetyl-carnitine, a drug which favours the synthesis of acetylcholine, the main neurotransmitter deficient in old age. In aged rats the drug restored a normal number of both axosomatic synapses and giant bouton vesicles. The authors hypothesize that the drug, by a cholinergic-type mechanism, restores the excitatory afferences to the granule whose axon would thus form normal giant boutons with the interneuron, reestablishing the feed-back regulation. In young rats the drug induced a decrease only of the axosomatic synapses, suggesting that an "over-excitation" might impair the information to the local-circuit neurons, thus interrupting feed-back control.

Acetylcarnitine↗

Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM. I. A quantitative study in normal subjects.

We applied a specific cytochemical reaction to characterize the glycoconjugates produced by goblet and non-goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colloidal gold as ultrastructural marker for Transmission Electron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non-goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribution to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non-goblet epithelial cell vesicles increase.

Carbohydrate Metabolism↗

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. II. A quantitative study in dry-eye patients.

The mucus glycoconjugates produced by conjunctival goblet cells in dry-eye patients were studied by a specific cytochemical reaction in Transmission Electron Microscopy (TEM). Four lectins, proteins of vegetal origin which specifically bind glycosidic residues, (WGA, PNA, SBA and ConA) were used conjugated with colloidal gold as ultrastructural marker. We performed a quantitative analysis by counting the colloidal gold particles present on mucus granules. The results were compared with normal conditions. We found a decrease in sialic acid, N-acetyl-glucosamine, N-acetyl-galactosamine and galactose-N-acetyl-galactosamine and an increase in mannose. The different content of glycoconjugates in goblet cells may reflect in the change of physical and functional properties of mucus. We think these data may be useful in the search for a therapeutic mucomimetic drug.

Carbohydrate Metabolism↗

Characterization of mucus glycoconjugates in normal human conjunctiva by lectins in light microscopy, transmission and scanning electron microscopy.

Maintenance of tear film in normal conditions is dependent on mucus layer integrity and the presence and distribution of conjunctival epithelial cell microvilli. In the present work a new methodology has been developed to gain correlative information about microprojection assessment and mucus composition, from the same specimen, by Light Microscopy (LM), Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). We have characterized the glycosidic residues secreted by goblet cells in normal human conjunctiva, by means of four lectins (WGA, ConA, PNA and SBA), conjugated with FITC for LM and with colloidal gold for TEM and SEM. The cytochemical reactions were performed on histological sections of paraffin-embedded material and on semithin and ultrathin sections of both Epon embedded material directly processed for TEM and of blocks recovered from SEM and reprocessed for TEM. WGA, ConA, PNA and SBA receptors were found to be constituents of the mucus produced by goblet cells in human conjunctiva. The granules of the so-called "second mucus system" (SMS) cells were labelled mainly by WGA. A difference in the quality of glycoconjugates between goblet cells and SMS cells has been also demonstrated. Our results provide an improved method to evaluate alterations of tear film that occur in many conjunctival diseases.

Carbohydrates↗

Correlative scanning electron microscopy in the study of human gastric mucosa.

We studied two aspects of the human gastric mucosa: the surface morphology of mucous cells, as viewed by scanning electron microscopy (SEM); the glycosidic components of intracellular mucins, characterized by means of lectins. The latter were conjugated with fluorescein isothiocyanate and with colloidal gold-silver for the visualization of the reaction products in light microscopy (LM) and in SEM (backscattered mode) respectively. The surface morphology of mucous cells appears to be correlated to the secretory state. In gastric ulcers we found a prevalence of non-secreting cells. A decrease in glycosidic receptors for fucose-binding lectin and galactose-(1-3)-N-acetyl-galactosamine-binding lectin was also observed. This suggests the presence of an impaired mucus secretion which may play a role in the pathogenesis of gastric ulcer. Spiral bacteria, supposed to be aetiologically related to peptic ulcer and gastritis, were easily detected by SEM. Intestinal metaplasia defined "complete" in LM showed surface morphology and glycosidic components different from those of true intestinal mucosa. This implies the necessity of taking into account also these parameters when classifying this lesion. The same applies to polyps. Our data indicate that correlative SEM may contribute further information on the pathogenesis and pathology of gastric diseases.

Adult↗

Beam voltage effects in the study of embedded biological materials by secondary electron detectors.

Thin and semithin sections were extensively examined by the secondary electron (SE) detector in a conventional scanning electron microscope (SEM), and in a transmission electron microscope with a scanning attachment (STEM). Various parameters, in particular the beam voltage, were shown to affect the final SE image (SEI). As for SEM observation, a surface contrast was imaged at low primary electron (PE) voltages (0.6-2 kV), whereas a subsurface contrast predominated at higher energies (15-30 kV). In STEM, significant differences were not detected by varying the PE in the 20-100 kV range. Surface and subsurface information was simultaneously imaged even though the SEI were better resolved at the highest energy.

Carotid Arteries↗

Cell ultrastructure in disease.

The doctor of today must adopt the 'cellular way of thinking' in the evaluation of diseases. This ultrastructural outlook provides him with much indispensable information that also serves a practical purpose. A diseased cell organelle is at the basis of every clinical sign and any attempt of therapy must be aimed at that specific point of lesion. We intend, in the light of a long experience, to propose to clinicians a new way of thinking in which a precise correlation between symptoms and submicroscopic changes of the cell is considered. Many different examples amply justify this proposal. Electron microscopy can contribute by enabling identification of structural subcellular modifications suitable for the finest differential diagnosis, more and more complete understanding of pathogenic pathways of various diseases, the establishment of guidelines for precise pharmacological interventions at the molecular level.

Adult↗

S.E.M. study I: Gastric and duodenal lesions induced by non-steroidal anti-inflammatory drugs (aspirin, piroxicam) in man.

A scanning electron microscopy (SEM) study was performed on the gastric and duodenal mucosa of 14 patients treated with Aspirin or Piroxicam. Six patients with normal mucosal morphology were treated with either 1.5 g/day of Aspirin (3 patients) or 20 mg/day or Piroxicam (3 patients) for one month. In addition, 8 rheumatic patients were treated with a similar dose of Aspirin (4 patients) or Piroxicam (4 patients) for at least 4 months. SEM was used to evaluate the following parameters: mucosal structure, cellular exfoliation and anisocytosis, alteration of the microvilli (blebs). The mucosa showed various aspects of alterations ranging from minimal changes (microvillar alteration) to a completely subverted structure both in the stomach and in the duodenum. Mucosal changes, both on the edge of the macroscopic lesions and at a distance from these, were visible with SEM even when endoscopy was normal. Piroxicam appeared slightly less damaging than Aspirin, and no difference was observed between short-term and long-term treated patients, either with Aspirin or with Piroxicam.

Adult↗

S.E.M. study II: Protective effect of ranitidine against gastric and duodenal lesions induced by non-steroidal anti-inflammatory drugs.

The effect of Ranitidine on the gastric and duodenal lesions induced by Aspirin or Piroxicam was studied by scanning electron microscopy (SEM). Twelve patients were entered into the study. The first group consisted of 6 patients with normal basic morphology, of which 3 were treated with 1.5 g of Aspirin and Ranitidine 300 mg/day, and 3 with 20 mg of Piroxicam and Ranitidine 300 mg/day, for one month. The second group was composed of 6 rheumatic patients, 3 of which took 1.5 g of Aspirin and the other 3 received 20 mg of Piroxicam per day for at least four months. Ranitidine (300 mg/day) was then administered concomitantly for one month to all these 6 patients. Overall, Ranitidine appeared to have a positive effect in preventing the development of gastroduodenal lesions induced by these anti-inflammatory drugs in the first group of patients. No differences were observed in the ability of Ranitidine to promote the healing of lesions induced by chronic treatment. Ranitidine, however, appeared to be more effective in preventing lesions induced by Aspirin rather than those induced by Piroxicam.

Adult↗

Scanning electron microscopy of normal human corneal epithelium obtained by scraping-off in vivo.

The surface of normal human corneal epithelium was examined by scanning electron microscopy (SEM). The study was carried out on specimens obtained by scraping-off in vivo, and immediately fixed. Four different types of microprojections were observed on the epithelial surface: microridges, microvilli, tufted microvilli and knobs. Detailed high magnification of surface microprojections showed that they consist of subunits. Our data suggest a correlation between surface morphology and maturity of the corneal epithelial cell. An evolutive sequence is proposed, from the youngest cell, provided with microridges, to the oldest exfoliating one, with knobs.

Cornea↗

A new approach for studying semithin sections of human pathological material: intermicroscopic correlation between light microscopy and scanning electron microscopy.

In order to obtain useful and complete information on the study of pathological material, we observed by scanning electron microscopy (SEM) the same semithin sections observed by light microscopy (LM). For this purpose, the specimen must have, at the same time, chromatic and electron dense characteristics. We thus developed different specimen preparation methods, subjecting the semithin sections to specific polychromatic staining with high atomic number (Z) elements, to monochromatic staining followed by routine contrasting with uranyl acetate and lead citrate, and to specific cytochemical and immunocytochemical procedures. The specimens were examined in sequence by LM, by SEM equipped with secondary electron, backscattered electron, transmitted electron detectors and by scanning transmission electron microscopy (S(T)EM).

Actins↗

Contribution of scanning electron microscopy and associated analytical techniques to the study of atherosclerotic disease.

Human carotid atheroma has been examined by scanning electron microscopy (SEM) utilizing both secondary and backscattered electron detectors and transmission electron microscopy (TEM) both at conventional and high voltage. Different cytochemical techniques have been used to map elastic fibers, proteoglycans, calcium, non esterified cholesterol. By immunocytochemistry the distribution of factor VIII related antigen and actin has been studied. With SEM it was possible to detect aspects of carotid plaques not appreciated when using other conventional techniques. With TEM some modifications of the structural and/or functional features of connective tissue macromolecules have been observed. The occurrence of anomalous collagen has been shown. The fine investigation of endothelial cells, smooth muscle cells and the intercellular matrix components has supplied information of particular morpho-functional significance, thus allowing the development of new ideas on the complex pathogenetic mechanisms of atherosclerosis and suggesting some important anatomo-clinical correlations.

Actins↗

Scanning electron microscopy study of human cornea and conjunctiva in normal and various pathological conditions.

Relatively few morphological studies on human cornea have been performed due to the difficulty in obtaining material. The specimens may be obtained either by superficial scraping-off, limited to the epithelial layer, or from corneal buttons obtained at keratoplasty. Conjunctiva, on the other hand, may be obtained by painless biopsy. We have studied the three-dimensional ultrastructure of the normal cornea and conjunctiva and in the course of various diseases where the application of scanning electron microscopy (SEM) seemed to be useful. In the cornea, we studied Fuchs' endo-epithelial dystrophy. In the conjunctiva, we analysed the ultrastructural characteristics of adenovirus follicular conjunctivitis, hay fever conjunctivitis and keratoconjunctivitis sicca. We also considered the problem of contact lens wearing, studying by SEM both the conjunctival tissue and the discarded lenses. On the basis of our results we conclude that SEM supplies useful information which integrates that obtained by biomicroscopy, used by clinicians to interpret the lesions at microscopic level. It is, however, indispensable to carry out, in addition to SEM, an examination on sections to completely investigate the disease.

Conjunctiva↗