Domenico Puzzolo

Impression cytology of the conjunctival epithelium in patients with vernal conjunctivitis.

The alterations in the conjunctival epithelium during the course of vernal conjunctivitis were examined by conjunctival impression cytology. The study was carried out on 53 patients with vernal conjunctivitis and 20 normal subjects as control. The results of impression cytology demonstrated that all cytological parameters were significantly modified in vernal conjunctivitis patients; the earliest alterations were found in the distribution of goblet cells, in the intercellular junctions, in the chromatin morphology and in the degree of keratinisation. The morphometric comparison showed that in vernal conjunctivitis patients the mean number of goblet cells per field was significantly higher than in controls. Moreover the mean diameter of goblet cells was smaller in patients than in controls. Impression cytology can, therefore, be a simple, non-invasive and cheap method for the study of the ocular surface in vernal conjunctivitis.

Matrix Metalloproteinase 9 and Transglutaminase 2 Expression at the Ocular Surface in Patients with Different Forms of Dry Eye Disease

OBJECTIVE To evaluate the expression of matrix metalloproteinase 9 (MMP9) and transglutaminase 2 (TG2) in different forms of dry eye. DESIGN Case control study. PARTICIPANTS Seventy-five female subjects divided into 3 groups: group 1, 15 healthy controls; group 2, 30 subjects with Sjögren syndrome (SS); and group 3, 30 subjects with Meibomian gland dysfunction (MGD). METHODS A clinical assessment was carried out and impression cytologic specimens were processed for immunoperoxidase staining for MMP9 and TG2 and real-time polymerase chain reaction analyses were carried out for MMP9, TG2, interleukin-6, interferon-γ, B-cell lymphoma 2, and caspase 3. To study MMP9 and TG2 expression after anti-inflammatory treatment, patients were divided into 2 subgroups, one treated with saline and the other treated with saline plus topical corticosteroid eye drops (0.5% loteprednol etabonate) 4 times daily for 15 days. For statistical analysis, Student t test, Mann-Whitney U test, and Spearmans correlation coefficient were used as appropriate. MAIN OUTCOME MEASURES Conjunctival expression of MMP9 and TG2. RESULTS MMP9 and TG2 expression were higher in both patient groups than in controls (P < 0.0001). Group 2 patients showed higher expression than group 3 (P < 0.0001). The Spearmans correlation coefficient showed in group 2 a positive correlation between MMP9 and TG2 expression (ρ = 0.437; P = 0.01), but no correlation in group 3 (ρ = 0.143; P = 0.45). Corticosteroid treatment significantly reduced MMP9 and TG2 expression in both groups, ameliorating symptoms and signs. A much higher percentage reduction was observed in SS. CONCLUSIONS The pathogenic mechanisms of the 2 forms of dry eye give an account for the different MMP9 and TG2 expressions in the 2 groups of patients. The higher expression in SS is determined by the direct autoimmune insult to the ocular surface epithelia, whereas in MGD patients, with an epithelial damage due to an unbalanced tear secretion, the molecules expression is significantly lower, although higher than in controls. The corticosteroid treatment induced a reduction of both molecules, although higher in SS than in MGD, because of its direct inhibitory effect on inflammation.

Morphological changes of the conjunctival epithelium in contact lens wearers evaluated by impression cytology

Purpose Conjunctival changes induced by rigid, gas-permeable and soft contact lenses (CL) were investigated using impression cytology. A scoring system based on seven parameters (specimen cellularity, cell-to-cell contacts, nucleus/cytoplasm ratio, chromatin, goblet cell distribution, keratinisation, inflammatory cells) was used to evaluate the morphological results.Methods One hundred and seventy-two CL-wearing eyes and 40 control eyes were examined. The population was divided into three groups: group 1, control eyes; group 2, asymptomatic CL-wearing eyes; group 3, CL-wearing eyes with intolerance problems. Impression cytology was carried out and all specimens were evaluated according to a scoring system.Results Group 1 specimens always showed normal features in the conjunctival epithelium. In group 2, rigid and gas-permeable lenses produced greater changes in conjunctival morphology than soft lenses, especially as regards cellularity, nucleus/ cytoplasm ratio, goblet cell distribution and total score. On the contrary, in group 3 wearers of soft lenses showed higher partial and total scores than wearers of gas-permeable and rigid lenses.Conclusions In asymptomatic CL wearers conjunctival morphology was better preserved in wearers of soft lenses. In patients with intolerance problems, wearers of soft lenses showed the worst cytological features. Impression cytology, evaluated with this scoring system, can be used to demonstrate epithelial damage occurring in CL-wearing patients.

ROS-Mediated NLRP3 Inflammasome Activation in Brain, Heart, Kidney, and Testis Ischemia/Reperfusion Injury

Ischemia and reperfusion (I/R) causes a reduction in arterial blood supply to tissues, followed by the restoration of perfusion and consequent reoxygenation. The reestablishment of blood flow triggers further damage to the ischemic tissue through reactive oxygen species (ROS) accumulation, interference with cellular ion homeostasis, and inflammatory responses to cell death. In normal conditions, ROS mediate important beneficial responses. When their production is prolonged or elevated, harmful events are observed with peculiar cellular changes. In particular, during I/R, ROS stimulate tissue inflammation and induce NLRP3 inflammasome activation. The mechanisms underlying the activation of NLRP3 are several and not completely elucidated. It was recently shown that NLRP3 might sense directly the presence of ROS produced by normal or malfunctioning mitochondria or indirectly by other activators of NLRP3. Aim of the present review is to describe the current knowledge on the role of NLRP3 in some organs (brain, heart, kidney, and testis) after I/R injury, with particular regard to the role played by ROS in its activation. Furthermore, as no specific therapy for the prevention or treatment of the high mortality and morbidity associated with I/R is available, the state of the art of the development of novel therapeutic approaches is illustrated.

Safety and efficacy of 0.1% clobetasone butyrate eyedrops in the treatment of dry eye in Sjögren syndrome

Purpose To study the effects of a low administration rate and low concentration (0.1%) of clobetasone butyrate eyedrops in patients with Sjögren syndrome (SS). Methods This prospective, double-masked, randomized, placebo-controlled study included 40 subjects divided into 2 treatment groups: group 1 (2% polyvinylpyrrolidone eyedrops and placebo) and group 2 (2% polyvinylpyrrolidone and 0.1% clobetasone butyrate, 1 drop BID). The treatment lasted for 30 days, with visits at enrollment, baseline, day 15, day 30, and after 15 days of treatment discontinuation. At each visit, symptoms questionnaire, tear film break-up time, corneal fluorescein stain, lissamine green stain, conjunctival impression cytology for human leukocyte antigen–DR (HLA-DR) expression, intraocular pressure (IOP) measurement, and fundus examination were performed. Results No changes in IOP or fundus examination were observed in either group at each time point. Group 1 patients showed at day 30 a statistically significant amelioration of symptoms and reduction of HLA-DR expression. No changes in other parameters were detected. Group 2 patients showed at day 15 a statistically significant improvement of corneal and conjunctival stain versus baseline values and group 1 at the same time; after 30 days the symptoms score was statistically significantly better than baseline values and group 1 at the same time. The HLA-DR expression and the epithelial cells area were statistically significantly reduced versus baseline and group 1 at the same time. Conclusions Anti-inflammatory therapy is critical for the treatment of SS dry eye. Clobetasone butyrate, at low dosage, proved to be safe and effective in treating this condition.

Morphologic and Confocal Investigation on Salzmann Nodular Degeneration of the Cornea

PURPOSE To investigate the ultrastructure of advanced Salzmann nodular degeneration (SND) and to correlate it to confocal in vivo findings. METHODS The corneal degenerative nodules from four patients with SND were examined with confocal microscopy and then removed and processed for light microscopy (LM) and transmission electron microscopy (TEM). RESULTS The confocal examination revealed elongated basal epithelial cells and activated keratocytes in the anterior stroma near the nodules. Occasional subbasal nerves and tortuous stromal nerve bundles were observed. With LM and TEM, five zones were described: one internodular and four pertaining the nodule, each characterized by peculiar aspects of the epithelium and stroma. As also confirmed by the morphometry, in the zones corresponding to the nodules, the epithelium was lower and with fewer cell layers than the peripheral zones. Over the nodules, the basement membrane was discontinuous or absent and the Bowmans layer, when present, had a granular-filamentous appearance. The nodular stroma was formed by many activated keratocytes and irregular lamellae. Subbasal nerves were always isolated and had degenerative changes in the center of the nodule. CONCLUSIONS This work illustrates the confocal microscopic findings associated with LM and TEM observations in patients with advanced SND. Our data revealed milder changes of the epithelium together with more pronounced changes of the basement membrane and Bowmans layer, which are aspects of increased keratocyte activity and an altered nerve pattern. All of these structures seem to contribute to the characteristic corneal disorganization of SND.

Effects of amino acids enriched tears substitutes on the cornea of patients with dysfunctional tear syndrome.

Purpose:  To evaluate the effect of aminoacid enriched artificial tears on the ocular surface of patients with dysfunctional tear syndrome (DTS).

Ultrastructural Study of the Conjunctival Epithelium in the Mongolian Gerbil (Meriones unguiculatus)

The conjunctival epithelium of the Mongolian gerbil (Meriones unguiculatus) was studied using the transmission and scanning electron microscopy. The superficial cells of the conjunctival epithelium were isoprismatic in shape and were covered, on their luminal surface, with microplicae. They were connected with the adjacent cells by junctional complexes and desmosomes. Some of the superficial cells were partially or completely detached from the underlying intermediate cells: this morphological pattern was interpreted as an expression of cellular renewal of the conjunctival surface. Goblet cells were either isolated or gathered in clusters: in any case, they were firmly connected with the adjacent epithelial cells. The apical part of the goblet cells was covered with a great number of long microvilli: they showed a variety of morphological aspects, which were interpreted as occurring in a sequential pattern during the secretory process. The Mongolian gerbil can be considered among the laboratory animals used for ophthalmic research.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood–Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -β3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-β3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

Phenotype-genotype correlation in patients with Schnyder corneal dystrophy.

Purpose: The aim of this study was to analyze the corneal morphology features and define mutations in the UbiA prenyltransferase domain–containing 1 (UBIAD1) gene in patients with Schnyder corneal dystrophy from a Polish population. Methods: Five affected and 15 unaffected members originating from 3 families with Schnyder corneal dystrophy were included in the study. Phenotype analysis consisted of visual acuity, slit-lamp biomicroscopy with photography, time domain optical coherence tomography, spectral domain optical coherence tomography, and confocal microscopy. Three patients underwent a penetrating keratoplasty. Corneal buttons obtained from the penetrating keratoplasty were processed for light microscopy. Results: A novel mutation I245N of the UBIAD1 gene was revealed in 1 proband and associated with the phenotype without central corneal opacities. The analysis of the other patients showed the N102S mutation. In vivo corneal morphology analysis using optical coherence tomography and confocal microscopy confirmed the presence of multiple crystalline corneal deposits in all affected corneas. The histological examination revealed multiple empty widenings of the corneal lamellae that could represent lipids removed from the specimen. Conclusions: N102S may also be a mutation hotspot in the Polish population, as in other previously reported populations. Corneal crystals formed a characteristic pattern on optical coherence tomography scans.