The Role of Scanning Electron Microscopy in Ophthalmic Science

The eyeglobe is one of the classical domains of Scanning Electron Microscopy (SEM) in biology as it exposes several inner and outer surfaces. Both corneal and conjunctival epithelia towards the tear film as well as the corneal endothelial cells facing the anterior chamber may be accurately evaluated. The architecture of the angle and particularly the morphology of the Schlemm\u27s canal inner wall are clarified by SEM more than by TEM serial reconstruction. The surfaces of the iris and ciliary body, the zonula and the choroidal vessel arrangement are described in great detail. Three distinct types of membrane anchoring devices are demonstrated among the lens fibers. SEM impressively describes the retina, but it has not yet added any new information as to previous observations in a more conventional way. SEM plays a fundamental role in teaching ocular anatomy and physiology as it makes more comprehensive the interrelationships among different structures. In addition, it represents a proper structural approach for the clinician who is familiar with the three-dimensional observations obtained by means of biomicroscopy, ophthalmoscopy and fluorescein angiography. Therefore, SEM application should be further spread and possibly joined to immunocytochemistry, in order to obtain a more dynamic and functional analysis of the eye

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\u27s 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 lost 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

One month use of Systane® improves ocular surface parameters in subjects with moderate symptoms of ocular dryness

Piera Versura, Vincenzo Profazio, Emilio C CamposDepartment of Surgery and Anesthesiology, Section of Ophthalmology, Alma Mater Studiorum University at Bologna, Bologna, ItalyThe data in this paper were first presented at the 9th International Ocular Inflammation Society (IOIS) Annual Meeting, September 17–20, 2007, Paris, France, and the European Association for Vision and Eye Research (EVER) Congress, October 3–6, 2007, Portoroz, SloveniaObjective: To evaluate the efficacy of Systane® Lubricating Eye Drops in improving the symptoms of moderate ocular dryness.Methods: Fifty subjects with moderate symptoms of ocular dryness were enrolled in this open label study. The mean age of subjects was 57.6 ± 15.4 years. To be eligible, subjects’ tear film break-up time (TFBUT) had to be <10 seconds, and subjects had to have at least one ocular discomfort symptom in addition to dryness. Saline was used for a wash-out period of 3–5 days. Subjects were re-examined, and those continuing to meet the inclusion criteria were dispensed Systane® and re-examined again after 28 days. At each visit, slitlamp examination was conducted, and ocular discomfort symptoms and TFBUT were evaluated. Subjects rated their overall satisfaction at baseline and on the last visit.Results: No significant changes in TFBUT or ocular discomfort symptoms were observed after saline use, compared with screening visit. After 28 days of Systane® use there was statistically significant improvement of TFBUT (p = 0.0001) compared with baseline. Subjects experienced significant symptomatic relief for all 6 ocular discomfort symptoms at the endpoint visit.Conclusion: Systane® effectively relieved the symptoms associated with moderate ocular dryness, with measurable improvement in objective TFBUT, subjective symptoms, and overall satisfaction.Keywords: Systane, lubricant eye drops, TFBUT, ocular dryness, ocular symptom

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

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 \u3c 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

Neurotrophic keratitis: Current challenges and future prospects

Neurotrophic keratitis (NK) is a degenerative corneal disease caused by damage of trigeminal corneal innervation, which leads to spontaneous epithelial breakdown and corneal ulceration. The impairment of corneal sensory innervation causes the reduction of both protective reflexes and trophic neuromodulators that are essential for the vitality, metabolism, and wound healing of ocular surface tissues. A wide range of ocular and systemic conditions, including herpetic keratitis, ocular chemical burns, corneal surgery, diabetes, multiple sclerosis, and neurosurgical procedures, can cause NK by damaging trigeminal innervation. Diagnosis of NK requires careful investigation of any ocular and systemic condition associated with the disease, complete ocular surface examination, and quantitative measurement of corneal sensitivity. The clinical stages of NK range from corneal epithelial alterations (stage 1) to persistent epithelial defect (stage 2) and ulcer (stage 3), which may progress to corneal perforation. Management of NK is based on clinical severity, and the aim of the therapy is to halt the progression of corneal damage and promote epithelial healing. Although several medical and surgical treatments have been proposed, no therapies are currently available to restore corneal sensitivity, and thus, NK remains difficult and challenging to treat. The purpose of this review is to summarize available evidence on the pathogenesis, diagnosis, and treatment of NK. Novel medical and surgical therapies including the topical administration of nerve growth factor and corneal neurotization are also described

Comparison of Trehalose/Hyaluronic Acid (HA) vs. 0.001% Hydrocortisone/HA Eyedrops on Signs and Inflammatory Markers in a Desiccating Model of Dry Eye Disease (DED)

Background: Dry eye disease (DED) is a multifactorial disease where ocular surface inflammation and damage play key etiological roles. Purpose: To compare a combination of 3% trehalose (T) and 0.15% hyaluronic acid (HA) (Thealoz duo(R), T/HA) with a tear substitute containing 0.001% hydrocortisone (I) and 0.2% HA (Idroflog(R), I/HA), with respect to changes on signs and inflammatory markers in a mouse DED model. Methods: Thirty 12-week-old C57BL/6 mice were exposed in a controlled-environment chamber as a desiccating stress model of DED for 35 days. At day 14 (T1), administration of 5 mu L T or I in the right eye (RE) or NaCl 0.9% in the left eye (LE) started, twice a day. Animals were sacrificed after 7 (T2), 14 (T3), 21 (T4, endpoint) days from the beginning of treatment. Corneal fluorescein staining ratio (Image J), histological and histochemical assessment of ocular surface tissues (goblet cell GC density and characterization -PAS, Alcian blue pH 2.5, pH 1.0, and MUC4 expression-in the superior and inferior conjunctiva), and levels of inflammatory markers HLA-DR, IL-1 beta and TNF-alpha in cornea and conjunctiva were measured. Results: No animal fully recovered from DED signs at the endpoint. Difference between arms was observed at T3 and T4, with T treated eyes showing a higher corneal damage reduction, PAS-positive GC recovery, lower inflammatory marker expression as compared to the I treated ones. Conclusions: Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA. Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA

Efficacy of two-month treatment with Xiloial® eyedrops for discomfort from disposable soft contact lenses

none4noneVersura P.; Profazio V.; Balducci N.; Campos E.C.Versura P.; Profazio V.; Balducci N.; Campos E.C

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 1) mucus layer integrity and 2) 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

Ocular surface analysis in patients affected with rheumatic diseases

Objective. The international criteria for primary Sjogren's Syndrome (SS I) diagnosis (Vitali et al. 2002) include the Schirmer test I and vital dye staining as tests for ocular surface involvement, but diagnosis can be reached also when the item for ocular signs is not satisfied. The purpose of our study was to evaluate the ocular surface in patients with Sjogren's Syndrome, non- Sjogren's autoimmune diseases and Sicca Syndrome, to understand whether the SS I diagnosis can be targeted also on other tests related to the ocular surface status. Methods. Clinical and cytological data were collected from 122 patients: 40 patients had diagnosis of Primary Sjogren's Syndrome, 51 a non Sjogren's autoimmune disease and 31 had symptoms of dry eye. A validated questionnaire on symptoms was filled by each patient; clinical tests included: Schirmer test I, Jones test, Ferning test, Break Up Time, corneal aesthesiometry, tear clearance test, vital dye staining of the ocular surface, scraping and impression conjunctival cytology. Data were statistically evaluated by using SPSS software and Mann-Whitney analysis on unpaired data. Results. Data show that the subjective symptoms score, tear production, tear turnover, corneal sensitivity and ocular surface integrity are affected in SS I patients, with a statistically significant difference when matched to the other two groups. Conclusions. Our results suggest to enlarge the spectrum of ocular surface analysis, to support and orient a differential diagnosis among the autoimmune diseases