Healing of prosthetic arterial grafts.

Numerous synthetic biomaterials have been developed as vascular substitutes. In vitro, ex vivo and in vivo studies have demonstrated that in animals, selected materials, i.e., Dacron and ePTFE (expanded polytetrafluoroethylene) grafts, are successfully incorporated in both the large and the small caliber host arteries through a process which is generally referred to as graft healing. Morphologically, this process consists of a series of complex events including fibrin deposition and degradation, monocyte-macrophage recruitment and flow-oriented cell-layer generation, this last event being the complete endothelialization of the arterial substitute. In contrast to experimental animals, the flow surface of synthetic vascular grafts remains unhealed in humans, particularly in the small caliber conduits. Healing in man consists of graft incorporation by the perigraft fibrous tissue response with a surface covered by more or less compacted, cross-linked fibrin. It is therefore obvious that: i) marked differences in graft healing exist between animals and man; and ii) the usual mechanisms of graft endothelialization are partially ineffective in man. In order to guarantee the patency of synthetic vascular grafts for human small artery bypass, new strategies and approaches have recently been attempted. In particular, the endothelial cell seeding approach has been successfully accomplished in animals and is being experimented in human clinical studies. The problems and results of this biological approach are outlined in this paper.

Ultrastructural aspects of aging rat hippocampus after long-term administration of acetyl-L-carnitine.

In aged rats a decrease in axosomatic synapses of granule cells as well as a decrease in the number of synaptic vesicles of giant synapses was found. These phenomena were supposed to be correlated on the basis of a feed-back circuit existing at the level of the dentate gyrus. In fact the axosomatic synapses of the granules are inhibitory gamma-aminobutyric acid-ergic terminals of interneurons. Interneurons receive excitatory afferences from granules via the giant synapses of the mossy fibre collaterals. This results in a feed-back regulation of granule cell activity. The long-term administration of acetyl-L-carnitine to aged rats restores a synaptic pattern comparable to that of young rats. This effect on synaptic plasticity is transient.

Electron microscopy of lipid deposits in human atherosclerosis.

The filipin probe associated with tannic acid stain was used to study intra- and extracellular lipids in surgically removed human atherosclerotic lesions (n = 20). In particular, intimal thickenings, fatty streaks and fibrolipidic plaques have been investigated by using mainly transmission and scanning electron microscopy. In the intimal thickenings, the lipid deposits were mainly localized in the subendothelial space as homogeneously sized particles (40-140 nm) and more heterogeneous uni-multilamellar vesicles (35-700 nm). Intermediate lipid forms were also observed. In the fatty streaks, the lipid deposits were intracellular and mainly observed in cells with a monocyte/macrophagic phenotype. Lipid inclusions, lipid lysosomal bodies and intracellular cholesterol crystals very similar to those observed in experimentally induced atherosclerosis were documented. In the fibrolipidic plaque the lipid deposits were found both in the intracellular and in the extracellular compartments. Lipids accumulated within arterial macrophages and smooth muscle cells, usually as lipid droplets. Clusters of lipoprotein-like particles (50 nm in diameter) as well as larger uni-multilamellar lipids (700 nm) with an occasional compound appearance were particularly observed bound to elastic tissue and collagen fibers. These morphological observations outline the complexity of lipid metabolism in the various histological aspects of human atherosclerosis.

An ultrastructural and immunocytochemical analysis of human endothelial cell adhesion on coated vascular grafts.

Human adult endothelial cells were enzymatically harvested from adipose tissue. Cell viability was established by Trypan blue exclusion and transmission and scanning electron microscopy. Endothelial cells were identified by immunocytochemical investigation at light microscopy, transmission electron microscopy, and scanning electron microscopy. Isolated cells were positive for actin and vimentin, negative for desmin. Factor VIII RA was mainly expressed at cell surface and occasionally disclosed in the cytoplasm. Reactivity for UEA I and J15 was weak or undetectable. Human endothelial cells were seeded and left to adhere for one hour onto different nonvascular substrates (glass, poly-l-lysine, formvar-carbon, fibronectin, Teflon). Scanning electron microscopy defined surface features, suggesting tenacious cell adhesion on the substrate. Different vascular substrates were tested (preclotted Dacron, albumin Dacron, Hemashield Dacron, Gelseal Dacron, ePTFE, fibronectin-ePTFE). Commercially available coated grafts showed qualitative and quantitative differences in cell adhesion. In particular, Gelseal Dacron provided the best quantitative results, even though a wide variability was observed. In contrast, fibronectin-coated ePTFE gave more reliable results and high spreading efficiency. In the short term, coated grafts do not seem to offer greater advantages than fibronectin-coated ePTFE. However, specific incubation times for each coated graft should be selected and the long-term approach (graft culture) should also be attempted.

Endothelial injury in human atherosclerosis.

A light and electron microscopic investigation (scanning and transmission electron microscopy) was performed on 51 human atherosclerotic carotid lesions. The purpose of this study was to establish whether features of endothelial injury such as those described in animals occur in man and whether these features can be related to specific stages of human atherosclerosis. Irrespective of their histological appearance the atherosclerotic lesions were covered with endothelium which showed non-specific changes in cell shape and size. However, all complicated lesions appeared denuded. Moreover, a peculiar interaction of endothelium with monocytes and lymphocytes as well as blood components (e.g., fibrin and lipoproteins) was observed in intimal thickenings, fatty streaks and uncomplicated plaques. The surface exposure of macrophage-derived foam cells was seen on florid fatty lesions. Large areas of the arterial surface lacking any endothelial coverage were characteristic of complicated plaques. They appeared to be a consequence of the arterial wall degeneration with an associated failure in endothelial repair.

Mucus alteration and eye dryness. A possible relationship.

From a very early phase we studied 15 patients suffering from a dry-eye condition ant associated to systemic diseases. Conjunctival biopsies were studied in Transmission (TEM) and Scanning (SEM) electron microscopy. Moreover, the lectin-gold cytochemistry at ultrastructural level was applied to investigate the distribution of some glycosidic receptors produced by both the goblet cells and the vesicles belonging to the Second Mucus System (SMS). No evidence of epithelial stratification and only a decrease in the goblet cell population was observed. The SMS vesicles and the superficial cell microvilli did not appear greatly reduced in number. A difference in the mucus composition in terms of content of glycosidic residues was detected in dry-eye patients compared to the normal subjects. The role of the mucus produced by both the goblet cells and the SMS vesicles in debated. A possible correlation between the alteration of the mucus content and the failure of the tear film stability is proposed. On the basis of these data, a new therapeutic approach for eye dryness is suggested.

The lectin-gold technique: an overview of applications to pathological problems.

Lectins are proteins, mainly of vegetal origin, which recognize glycosidic residues with high specificity; for this property they have been used for many studies of molecular biology. The colloidal gold represents at present the most popular electron dense marker employed in immunocytochemistry, since it offers intrinsic and unique characteristics which are superior to those displayed by the other markers. The cytochemical method which utilizes the gold-labelled lectins takes advantages from both the two systems, in order to optimize the localization of the glycoconjugates. The present paper reviews both the technical aspects of the preparation of the lectin-gold complex and its application to some selected pathological problems. In particular, the papers concerning the eye and ear tissues, the urinary, reproductive, nervous and digestive systems and the blood cells are quoted.

Distribution of diffusible ions in the aging rat lens. An X-ray microanalytical investigation.

The elemental content of the anterior and posterior cortex and of the nucleus of the rat eye lens, and the changes occurring as a function of age, were investigated by energy-dispersive X-ray microanalysis of cryostat sections. In contrast to most other cells investigated by this technique, the main element detectable in the lens was sulfur, not phosphorus. This is likely to be due to the lack of cell organelles and of nucleic acids in the lens cells. Up to 19 months of age, the concentrations of Na, Cl and K are much lower in the nucleus than in the cortex, whereas the concentration of S is highest in the cortex. At 25 months the differences in elemental content between nucleus and cortex, while still present, are less pronounced. The age-related changes in ion content are rather complex and appear to be different for different parts of the lens. In general, the ion content of the lens increases with age. In the nucleus, the concentration of K increases gradually with age, whereas the Na concentration only appears to increase after 19 months. The Na/K ratio in the lens nucleus is lower in the oldest groups. The Ca concentration increases with age in the nucleus and the posterior cortex, but not in the anterior cortex.

Scanning electron microscopy in the study of Campylobacter pylori associated gastritis.

The close association between Campylobacter pylori (CP), gastritis and peptic ulcer is now well established. Moreover increasing evidence has been collected of a major etiological role of CP in type B chronic gastritis. For this reason, searching for CP is essential in all patients with upper gastrointestinal symptoms. Scanning electron microscopy (SEM) is a most reliable technique for studying the distribution of microorganisms and their relationship to the gastric mucosal surface. The aim of this paper is to compare SEM to other routine methods of detection for CP, such as Giemsa staining on histological sections and Urease Microtiter Test (MT) on fresh tissue and to investigate the surface morphology of gastric mucosa colonized by CP and to correlate it with the histopathological picture. Thirty-seven biopsies taken from the gastric body and the antrum of 22 patients were used for each type of determination. The different parameters were graded semiquantitatively. Histology showed a normal mucosa in 4 cases, chronic superficial gastritis in 12 and chronic atrophic gastritis in 21 cases. SEM was more sensitive than histology and Urease MT in detecting Campylobacter pylori. This is due to the patchy distribution of this bacterium on gastric mucosa. For this reason SEM should always be performed when routine tests are negative. The presence of CP correlated significantly (p less than 0.001, Spearman Rank Correlation Test) with the neutrophilic infiltrate, thus with the "activity" of the gastritis. The CP associated gastritis has no distinctive surface features other than the presence of the bacterium. SEM morphology of surface gastric mucous cells suggests that CP does not damage the lining epithelium directly. Neutrophils and inflammatory mediators could be involved in the production of the mucosal lesions.

Dry eye before and after therapy with hydroxypropyl methylcellulose. Ultrastructural and cytochemical study in 20 patients.

We have utilized hydroxypropyl methylcellulose in the therapy of 20 patients suffering from dry eye in the initial stage. The effects of the substance have been evaluated both from a clinical and a morphological standpoint. In particular, transmission and scanning electron microscopy were applied to investigate the ultrastructural features of the conjunctival epithelium before and after treatment. In addition, lectin cytochemistry was carried out to analyse semiquantitatively the presence of glycoconjugates in the conjunctival mucus produced by goblet and non-goblet epithelial cells. Hydroxypropyl methylcellulose did not improve the tear film stability as demonstrated by the clinical tests. The normal surface of the conjunctival epithelium did not appear recovered as well. Only a minimal increase in the density of the second mucus system vesicles was observed. From a cytochemical point of view, the content of glycosidic residues appeared altered in dry-eye patients both before and after treatment. This alteration can lead to changes in the physical and functional properties of the mucus, impairing its role as a surfactant. We conclude that the common treatment by means of lubrificant-like substances palliates the dryness condition. The search for a drug which reintegrates the correct composition of the conjunctival mucus should be seriously considered as a valid alternative.

Ultrastructural and immunohistochemical study on the effect of topical cyclosporin A in the rabbit eye.

The effect of topically administered cyclosporin A (CyA) was evaluated in this experimental study by means of transmission and scanning electron microscopy, and immunohistochemistry. CyA dissolved in sterile olive oil was injected in the retrobulbar area and in the subconjunctival space, or administered as eyedrops in both non-grafted and corneal xenografted rabbit eyes. The vehicle rather than the drug was thought to be responsible for the inflammatory reaction observed in the conjunctiva of the injected eyes as well as for the surface epithelial defects developing in the cornea of the topically administered eyes. CyA was found to be effective in prolonging the survival of the xenografts after 1 month follow-up. The immunohistochemistry indirectly demonstrated the CyA effectiveness in impairing the T-cell function, and this is, to our knowledge, the first documented report on this subject. We conclude that another type of vehicle for the drug has to be introduced and that the administration of CyA as eyedrops is the best route to provide fast diffusion and to avoid adverse side effects.

Ependymoma of the foramen of Monro: ultrastructural characterization.

The characteristics of clear cells of an ependymoma of the foramen of Monro have been studied by electron microscopy to precisely define its organellar composition and to establish the tumor histogenesis. Our data confirm that the once-thought oligodendroglial is, in fact, an ependymal tumor. Both the scarce number of organelles, owing to the low degree of differentiation, and the abundance of hyaloplasmic lipid vacuoles can account for the clear appearance of these tumor cells.

Elastofibroma: an in vivo model of abnormal neoelastogenesis.

Thirteen cases of elastofibroma have been studied by conventional light and electron microscopy, as well as by histochemistry and immunohistochemistry. By light microscopy elastinophilic material appeared as huge fibers crossing collagen bundles. Immunohistochemistry demonstrated a strong positivity for elastin in numerous and circumscribed areas of the extracellular matrix. By electron microscopy, collagen consisted of 40-50-nm wide fibrils, and elastin was made of large aggregates of moderately electron-dense material surrounding a very thin, apparently normal, elastin core. At high magnification these aggregates consisted of short tubules, often in regular arrays, surrounded by microfibrils and microfilaments. These data, associated with selective digestions on thin sections with elastase, purified collagenase, hyaluronidase, and chondroitinase ABC, revealed that elastic fibers in elastofibroma seem to be made of true elastin surrounded by an enormous amount of hydrophilic material, in which some elastin, chondroitin sulfates, and collagenase type-VII sensitive material are aggregated forming a rather ordered array of short tubules.

Immunocytochemical analysis of contact lens surface deposits in transmission electron microscopy.

We studied 8 soft contact lenses from asymptomatic wearers by means of an immunocytochemical method, in transmission electron microscopy. In particular, the presence of IgA, IgG, IgE, Clq complement fraction within the surface deposits was analyzed. All the lenses were found positive for the immunoglobulins and the Clq, being the tarsal side more heavily coated than the corneal one. IgA was the predominant Ig, followed by IgG, IgE, and Clq in this descending order. New, never worn lenses were found completely negative for any of the proteins under investigation. We conclude that the Igs come from the tear fluid and speculate about the Clq as a possible sign of involvement of the host immuno-defense mechanism against the prosthesis.

Ultrastructural and immunohistochemical contribution to the histogenesis of human cardiac myxoma.

The ultrastructural features of 8 human cardiac myxomas were analyzed and correlated with immunohistochemical data, with the aim to clarify the characteristics of the cell lines involved in the tumor genesis. Immunohistochemical studies were performed to detect the presence and the distribution of intracytoplasmic filaments (vimentin, desmin, actin, myosin) as well as myoglobin and factor VIII-related antigen, albumin, and lysozyme. Eighty percent of myxoma cells were simultaneously positive for vimentin, desmin, and actin, whereas 30% of them stained with antifactor VIII and antivimentin antibodies. The submicroscopic analysis revealed two main cell populations: (1) one composed of stellate-shaped cells with scanty organelles and sparse hyaloplasmic filaments scattered throughout the myxoid stroma and forming a loose network with their projections; (2) another one included cells with more cytoplasmic organelles, intermediate filaments, and myofilaments arranged either singly or in both solid and hollow cord-like structures. Our results support the hypothesis that cardiac myxoma may originate from a reserve multipotent mesenchymal cell able to differentiate more or less completely along two major evolutional lines: myoid and endothelial. The tumor tissue thus seems to be involved in vessel formation, suggesting a growth pattern akin to that manifested in other forms of endocardial pathological reactivity in which reserve mesenchymal cells are engaged.

Scanning electron microscopy, X-ray microanalysis and immunohistochemistry on worn soft contact lenses.

The deposits accumulated on the surfaces of soft contact lenses are a cause of problems for the wearer of these lenses, as the deposits are never completely removed by the available washing solutions. Therefore it appears of interest to investigate the composition of these deposits. In this paper we review the major findings in the literature and, in addition, present our personal experience. We have studied new, continuously and daily worn soft contact lenses by scanning electron microscopy (SEM), X-ray microanalysis and immunohistochemistry. We have carefully evaluated preparative methods, and we can conclude that SEM and X-ray microanalysis are best carried out on unfixed, air-dried lenses. The deposits present consist mainly of mucus, especially on the tarsal side of the lenses. Chloride and potassium, coming from the tear fluid, as well as sulfur, derived from proteins, were found. Calcium was very rarely detected. IgG, IgA, IgE and C3c complement fractions were found only on the outer surfaces and not within the lens. We believe that the best characterization of the deposits is achieved by means of correlative techniques on the same lens. In fact, this approach integrates morphology and composition.

Detection of mucus glycoconjugates in human cervical epithelium by lectin-colloidal gold technique in transmission electron microscopy. I. A quantitative study during the menstrual cycle.

We investigated the presence and distribution of five glycosidic residues at the level of the endocervical columnar epithelial cells during the various phases of the menstrual cycle in healthy subjects. We utilized the lectins Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Soybean Agglutinin (SBA), Concanavalin A (ConA) conjugated with colloidal gold particles as ultrastructural markers in Transmission Electron Microscopy. This technique proved to be particularly suitable for detecting glycosidic residues at the cellular level, where the mucus is produced. All the lectin-binding sites (except ConA binding sites which were not detected) appeared to be produced in significantly different amount during the cycle, supporting the hypothesis that the cyclic-related changes of the cervical mucus viscoelasticity is due to glycoconjugate cyclic variations.

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. III. A quantitative study in asymptomatic contact lens wearers.

We characterized the mucus glycoconjugates produced by goblet and non-goblet epithelial cells in asymptomatic contact lens (CL) wearers. We employed four lectins (proteins of vegetal origin which specifically recognize glycosidic residues: WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultrastructural marker, at Transmission Electron Microscopy. A computerized quantitative analysis was carried out in order to compare the results from the CL wearers to those from the control patients. Goblet cells produce different amount of glycosidic residues, in particular, a significant decrease in the distribution of sialic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose-N-acetylgalactosamine and mannose was observed. The content of glycosidic residues in the mucus vesicles of the non-goblet epithelial cells appeared unchanged as to the normal situation. We speculate that the CL could possibly contribute to the failure of the tear film stability by altering the production of mucus.