Snake-like Chromatin Research Articles

The micronucleus cytome assay – A fast tool for DNA damage screening in human conjunctival epithelial cells

PurposeTo assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva. MethodsImpression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface. ResultsThe mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls. ConclusionsMicronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.

Nuclear parameters and chromatin remodeling in epithelial cells and lymphocytes from the palpebral conjunctiva of dogs with keratoconjunctivitis sicca.

To study parameters related to nuclear morphology and chromatin remodeling in epithelial cells and lymphocytes from the inferior palpebral conjunctiva of dogs with and without keratoconjunctivitis sicca (KCS). Thirty-two dogs (64 eyes) were included in the study. Based on the tear production measured by Schirmer tear test 1, the dogs were distributed into control and KCS groups. Epithelial cells and lymphocytes were collected by conjunctival brush cytology, fixed on glass slides, and subjected to the Feulgen reaction, a topochemical method specific for DNA/chromatin. Feulgen-stained cells were studied by microscopy and video image analysis to establish nuclear size (area and perimeter) and shape (relative nuclear roundness factor=RNRF), DNA content (ploidy), and compaction and texture of chromatin. Conjunctival samples in the KCS group showed infiltration of inflammatory and immune cells. Micronuclei, snake-like chromatin, aberrant chromosomes, and goblet cells were not detected. Compared with the controls, cells on the conjunctival surface of dogs with KCS showed altered nuclei. Conjunctival epithelial cells were more affected by KCS (changes in nuclear size, shape, DNA content, and chromatin compaction) than lymphocytes (changes in chromatin compaction, only). Significant chromatin decompaction was observed in both conjunctival epithelial cells and lymphocytes. Our results show that KCS promotes chromatin remodeling in epithelial cells and lymphocytes on the conjunctival surface of dogs. The changes described in this study are different from those reported for conjunctival cell nuclei of human KCS patients.

Snake-like Chromatin Cell Protein as an Indicator of Chronic Eye Rubbing in Patients with Keratoconus and Allergic Conjunctivitis

Snake-like Chromatin Cell Protein as an Indicator of Chronic Eye Rubbing in Patients with Keratoconus and Allergic Conjunctivitis

The Application of Autologous Serum Eye Drops in Severe Dry Eye Patients; Subjective and Objective Parameters Before and After Treatment

Aim: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment.Materials and methods: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients.Results: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes.Conclusions: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients’ subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.

Zur Evaluierung der lokalen Verträglichkeit von Antibiotika an der Augenoberfläche am praktischen Beispiel eines Aminoglykosides

The local application of drugs is an efficient method for treating diseases of the external eye. However, some active substances can also cause a chronic toxic reaction at the ocular surface. No results from clinical experiments on the local tolerance of kanamycin which would meet the requirements of evidence-based medicine are as yet available. Thus, the cytomorphological processes of the treatment with kanamycin were examined in a prospective randomised placebo-controlled double blind cross-over study. The effect of the topical application (5 times a day for 7 days) of Kanamycin POS® eye ointment on the conjunctival epithelial barrier was studied in comparison with an eye ointment base (placebo) in 25 healthy subjects. To detect cytopathological microchanges of the epithelium, we carried out a standardised impression cytology (primary compatibility parameter). Biomicroscopy, Schirmer II test and non-invasive measurement of the break-up-time (tearscope) were also examined. The subjective compatibility parameters (itching, tearing, foreign body sensation, burning sensation, mucus) were measured on a 0 - 10 visual analogue scale. The cytological findings of 23 subjects were included in the final evaluation of the study. The impression cytology did not reveal a different effect of kanamycin ophthalmic ointment on the conjunctival ocular surface in comparison with the placebo. Only in one subject was a marked shift found in the nuclear-cytoplasmic ratio after application of verum. No signs of increased apoptosis ("snake-like chromatin") were found at the conjunctival epithelial barrier. The mean goblet cell count was 109 goblet cells per mm (2). No significant reduction of the goblet cell density was found in any treatment group. Among the subjective compatibility parameters, only the mean for itching constantly remained above the placebo group after application of kanamycin. Statistically significant differences were not found by the Wilcoxon test. In contrast to the clinical experience with the systematic application of kanamycin, the standardised evaluation of this study confirmed the very good topical tolerability of the tested kanamycin eye ointment. In view of the continuing clinically relevant aspect of the availability of a wide spectrum of antibiotics to avoid the development of resistance while at the same time keeping within what is reasonable economically, Kanamycin POS® for topical application supplements the options of ophthalmological antibacterial chemotherapy.

Effects of Different Contact Lens Materials and Contact Lens–Wearing Periods on Conjunctival Cytology in Asymptomatic Contact Lens Wearers

To investigate the conjunctival cytologic changes induced by different contact lens materials and contact lens-wearing periods in asymptomatic contact lens wearers. Seventy-five patients (150 eyes) who were to wear contact lenses for the first time were evaluated in this study. Forty eyes of 20 soft hydroxyethylmethacrylate (vinyl pyrrolidone copolymer) contact lens wearers, 70 eyes of 35 soft Polymacon contact lens wearers, and 40 eyes of 20 hard gas-permeable contact lens wearers used the lenses on a daily-wear basis. Conjunctival cytologic changes were investigated by using impression cytology. Impression cytologic samples were obtained before contact lens fitting and after contact lens wear (mean evaluation time, 6.9 +/- 2.6 months; range, 4-12 months). The samples were appropriately stained and were assessed by using the Nelson grading system and for the presence of snakelike chromatin. The Kruskal-Wallis test, the Wilcoxon test, and logistic regression were used for statistical analysis. There were statistically significant differences in impression cytologic findings for all three materials before versus after contact lens wear (P=0.00, P=0.00, P=0.00, Wilcoxon test). The contact lens-wearing period produced a statistically significant effect on conjunctival cytologic changes and snakelike chromatin formation (P=0.00, P=0.00, logistic regression analysis), but the effects of different contact lens materials were insignificant (P=0.88, P=0.62). Conjunctival cytologic changes occurred in asymptomatic contact lens wearers and were related to the duration of lens wear. For this reason, asymptomatic contact lens wearers may be followed up because of conjunctival cytologic changes.

Presence of Snake-like Chromatin in Epithelial Cells of Keratoconjunctivitis Sicca Followed by a Large Number of Micronuclei

To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity.Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted.The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3.We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.

Morphological and immunocytochemical characterization of snake-like chromatin cells.

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.

Microscopie confocale et pathologies de la surface oculaire : corrélations anatomo-cliniques

To describe corneal and conjunctival epithelial changes in various ocular surface disorders with a new in vivo confocal microscope (Corneal HRT II module) and to compare these results with those obtained by immunohistology on impression cytology specimens. In this study, we investigated 36 eyes of 18 patients with ocular surface diseases such as corneal epitheliopathy induced by ocular rosacea (five patients), keratoconjunctivitis sicca related to tear evaporative condition in Meibomian gland dysfunction (eight patients), or tear-deficient dry eye in Sjögren's syndrome (five patients). In vivo confocal microscopy imaging (Heidelberg Retina Tomograph II Rostock Cornea Module) and impression cytology of the cornea and/or conjunctiva were performed. The images were analyzed for epithelial cell morphology at nuclear and cytoplasmic levels, in the cornea and the conjunctiva, number of goblet cells and their possible presence within the corneal epithelium, corneoconjunctival junction at the limbus, and density and presence of inflammatory cells within both epithelia. Images obtained consisted of two-dimensional 400x400 microm optical sections oriented parallel to the surface of the eye and compared with impression cytology. Conjunctival and corneal epithelia showed dramatic changes in ocular surface disease. In keratoconjunctivitis sicca, we found squamous metaplasia, inflammatory cell infiltration, goblet cell depletion, as well as a nuclear snake-like chromatin pattern. In rosacea associated with corneal epitheliopathy, a corneal conjunctivalization (goblet cells together with conjunctival epithelial cells within the corneal epithelium layer) was found. The images provided by this technique are in excellent correlation with the findings using impression cytology. The HRT II in vivo confocal microscope facilitates the study of both central and peripheral ocular surface epithelia compared to first-generation confocal microscopy devices. In vivo confocal microscopy, showing corneal and conjunctival epithelial structures in the live eye at the cellular level, is likely to open up a new promising way to investigate ocular surface involvement in complex diseases, providing a new insight on corneal and conjunctival disorders in the future.

Effects of soft contact lenses on conjunctival surface.

To examine the effects of soft contact lenses on tear breakup time (TBUT), basal Schirmer test result, and the conjunctival surface in patients wearing contact lenses. In this study, conjunctival cytologic changes, TBUT, and Schirmer test function alterations of soft contact lens wearers were evaluated by impression cytology. The study included 100 eyes of 50 soft contact lens wearers who were followed up in the Department of Ophthalmology of Dicle University and 80 eyes of 40 subjects as a control group. After TBUT and basal Schirmer test, conjunctival surface epithelial morphology was investigated using impression cytology. After the materials were appropriately stained, they were evaluated according to the Nelson grading scale. Contact lens wearers were divided into three groups according to the duration of contact lens wear. When these groups were evaluated according to the Nelson grading method, 21% of cases were grade 0; 32% were grade 1; and 28% were grade 3. There were statistically significant differences in epithelial cell morphology, goblet cell density, snakelike chromatin changes, TBUT, and basal Schirmer test result between the control and study groups. We suggest that TBUT and Schirmer test result be carefully monitored in contact lens wearers. Impression cytology may be used as a safe, simple, and noninvasive method in the diagnosis of ocular surface alternations in patients with contact lens intolerance.

Effects of Extended-Wear Soft Contact Lenses on the Ocular Surface and Central Corneal Thickness

Purpose: New contact lens materials with high oxygen permeability are said to be convenient for the extended-wear (EW) schedule. We evaluated the short-term effect of EW soft contact lens usage on ocular surface and central corneal thickness. Patients and Methods: Fifty-two eyes of 26 patients were included in the study. Sixteen of the patients used the high oxygen-permeable lenses for a 30-day extended-wear schedule (group I). Ten patients used the lenses on a daily wear basis (DW, group II). All patients were evaluated for visual acuity and central corneal thickness by corneal and conjunctival biomicroscopy, Schirmer and tear break-up time (BUT) tests. Impression cytology was performed in the superior/temporal, inferior/nasal quadrants of the conjunctiva, 1 mm from the limbus, with cellulose acetate filter paper. The specimens were graded under light microscopy according to Nelson’s classification. Results: Three patients in the EW group were excluded from the study as they discontinued lens usage due to slight discomfort and hyperemia. Biomicroscopic findings and tear function tests indicated no increased pathological results. Central corneal thickness decreased from 554 ± 9.55 to 546 ± 9.30 µm (mean ± SEM) in the EW group (p = 0.002), whereas no significant change occurred in the DW group. Cytological evaluation of the EW group showed an 88% increase in metaplasia (p = 0.007), especially close to the limbal areas and in the superior quadrants; this ratio was 30% in the DW group. Snake-like chromatin changes were found to be increased, from 4 to 6 patients, in the EW group while it remained the same in the DW group (p > 0.05). Individual comparisons of the initial and final pathological conditions of the ocular surfaces in the two groups showed that there was an increase in pathology in the EW group while there was no significant difference in the DW group. Conclusions: On a 30-day extended-wear basis with high oxygen-permeable lenses, the possible development and increase of conjunctival cytologic changes secondary to mechanical trauma is one of the main remaining problems. Conjunctival and related immunologic pathologies should not be overlooked when prescribing an extended-wear lens schedule.

Snake-like chromatin in conjunctival cells of a population aged 30-60 years from Copenhagen City.

To investigate the frequency of snake-like chromatin in the conjunctival epithelial cells (SLC) of a population from Copenhagen City. A questionnaire was mailed to 504 persons aged 30-60 years. Persons with symptoms of ocular/oral dryness and a control group without symptoms were examined by the conjunctival imprint technique. According to this material the frequency of SLC in persons 30-60 years living in Copenhagen may be estimated to 47%, which is considerably higher than earlier reported. In most cases SLC was, however, occupying less than 1% of the total cell area. No significant association was found to abnormal results of the Schirmer test, tear film break up time, rose bengal score or tear film lipid layer thickness. The frequency of SLC in persons with KCS was 53%, however, only few cases of KCS were found in the material. Results indicate that SLC is a quite frequent and unspecific finding, probably reflecting chronic irritation of the ocular surface.

Conjunctival Impression Cytology in Patients Wearing Contact Lenses

Evaluation of contact lens-induced cytologic changes on the conjunctival surface. Fifty eyes of 25 patients wearing soft or rigid gas-permeable contact lenses, and 50 healthy eyes of 25 control subjects were examined with conjunctival impression cytology. Of the patients wearing contact lenses, 40% did not have any contact lens-related complaints, whereas 60% had some minor complaints related to contact lens intolerance. The material obtained by impression cytology was examined with regard to epithelial cell morphology, goblet cell density, and snake-like nuclear chromatin changes. When epithelial cell morphology was graded according to the system described by Nelson, specimens from the control group revealed 90% of the eyes to be grade 0 and 10% to be grade 1, whereas of the eyes wearing contact lenses, 8% were grade 0, 36% grade 1, 32% grade 2, and 24% grade 3. Thus statistically significant differences were observed between the control group and the contact lens group with regard to each grade (p < 0.05) as well as to the goblet cell densities (p < 0.05). Snake-like chromatin changes, on the other hand, were observed in 30 and 27% of the eyes wearing soft and rigid gas-permeable contact lenses, respectively, whereas these were not encountered in any eye in the control group. Epithelial changes were noted to be more frequent and more severe in symptomatic patients than in those without any complaints. No correlation was found between average duration of contact lens wear and the risk of contact lens intolerance or development of squamous metaplasia.

Rose bengal score—A possible key parameter when evaluating disease level and progression in primary Sjögren's syndrome

The rose bengal score is one of the most commonly used tests for evaluation of ocular surface epithelial damage. The test is used in most Sjögren's syndrome criteria. We examined 24 female and four male patients with primary Sjögren's syndrome (primary SS) in order to evaluate possible correlation between the various tests for keratoconjunctivitis sicca (KCS), and for possible correlations to xerostomia and p-IgG levels. Among the KCS tests a high rose bengal score appeared to be the key parameter, being correlated to low break-up time ( P < 0.01), low tear lysozyme ( P < 0.01), appearance of snake-like chromatin in conjunctival imprints ( P < 0.05), low sialometry ( P < 0.01) and high p-IgG ( P < 0.01). We followed another group of patients with primary SS (30 females and four males) for a mean period of 53 (range 27–76) months. The patients were divided according to their initial response to systemic treatment with bromhexine. KCS parametres and p-IgG were measured repeatedly during the observation period. Patients responding to and continuously treated with bromhexine ( 2 3 of patients) improved significantly ( P < 0.05) in rose bengal score, but had increasing levels of p-IgG. Non-responders kept their low tear-production rate and had also increasing p-IgG levels. However, when subdivided according to p-IgG level, the group of patients with relatively low p-IgG improved in rose begal score, whereas the high p-IgG-group increased in rose bengal score. The rose bengal score appears to be a useful key parameter when evaluating disease level and progression.

The polyclonal B cell activation is related to the exocrine manifestations of primary Sjögren's syndrome: [formula omitted], R Manthorpe, JU Prause. Department of Rheumatology TTA, Rigshospitalet and Eye Pathology Institute, University of Copenhagen, (Denmark). Department of Rheumatology, Malmö General Hospital, University of Malmö/Lund, (Sweden)

The rose bengal score is one of the most commonly used tests for evaluation of ocular surface epithelial damage. The test is used in most Sjögren's syndrome criteria.We examined 24 female and four male patients with primary Sjögren's syndrome (primary SS) in order to evaluate possible correlation between the various tests for keratoconjunctivitis sicca (KCS), and for possible correlations to xerostomia and p-IgG levels. Among the KCS tests a high rose bengal score appeared to be the key parameter, being correlated to low break-up time (P < 0.01), low tear lysozyme (P < 0.01), appearance of snake-like chromatin in conjunctival imprints (P < 0.05), low sialometry (P < 0.01) and high p-IgG (P < 0.01).We followed another group of patients with primary SS (30 females and four males) for a mean period of 53 (range 27–76) months. The patients were divided according to their initial response to systemic treatment with bromhexine. KCS parametres and p-IgG were measured repeatedly during the observation period.Patients responding to and continuously treated with bromhexine (23 of patients) improved significantly (P < 0.05) in rose bengal score, but had increasing levels of p-IgG. Non-responders kept their low tear-production rate and had also increasing p-IgG levels. However, when subdivided according to p-IgG level, the group of patients with relatively low p-IgG improved in rose begal score, whereas the high p-IgG-group increased in rose bengal score.The rose bengal score appears to be a useful key parameter when evaluating disease level and progression.