Sheep corneal endothelium morphology - evaluation with trypan blue and alizarin red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research.

Visualization of Arbuscular Mycorrhizal Fungal Extraradical Hyphae and Spores Vitality and Activity.

The hyphae and spores of arbuscular mycorrhizal (AM) fungi represent an essential component in the extraradical zone due to their role in nutrients and water uptake and as propagules that allow the perpetuation of the AM symbiosis over time, respectively. However, the attention of scientific literature is usually more focused on root colonization than on the study of the extraradical components of AM fungi, especially their vital, active, or functional fractions. This chapter presents some easy-to-use alternatives for staining vital, active, or functional structures of AM fungi for their subsequent microscopic visualization, such as the application of enzyme-based stains, NADPH formation, and also nucleus staining. Some modified methods for the extraction of mycelium from the soil are also presented.

Relationship between Ocular Surface Alterations and Concentrations of Aerial Particulate Matter.

PURPOSE: To evaluate ocular surface alterations in two populations at different exposure levels to particulate matter (PM) in their living and work environments. METHODS: A cross-sectional study was conducted, including 78 volunteers from Argentina who lived and worked under different pollution levels in an urban (U; n = 44) or industrial zone (I; n = 34). Mean exposure level to PM was evaluated. Responses to the Ocular Symptom Disease Index and McMonnies questionnaire were obtained from all subjects. Subsequently, an assessment through the Schirmer I test (ST), slit lamp microscopy, vital staining, and tear breakup time was conducted. Statistical analyses with Chi-square and Bartlett's tests, as well as Student's t-tests and principal component analysis (PCA), were performed. RESULTS: Particles of size < 2.5 µm (PM 2 . 5 ) level was significantly higher in the I group than the U group (P = 0.04). Ocular surface parameters including bulbar redness, eyelid redness, and the degree of vital staining with fluorescein (SF) and lissamine green (SLG) exhibited difference between the groups. With regards to the tear film, statistically significant differences in the ST value and meibomian gland dysfunction between the groups were detected (P = 0.003 and P = 0.02, respectively). Conjunctival SF and SLG, and ST values were identified as factors which could distinguish groups exposed to different PM levels. CONCLUSION: Subjects exposed to higher levels of PM in the outdoor air presented greater ocular surface alterations. Thus, ST, SF, and SLG values could be used as convenient indicators of adverse health effects due to exposure to air pollution.

Development of macaque secondary follicles exposed to neutral red prior to 3-dimensional culture.

PURPOSE: Neutral red (NR) may assist identification of preantral follicles in pieces of cortical tissue prior to cryopreservation in cancer patients requesting fertility preservation. This study is the first to analyze this effect by follicle growth rate after long-term culture in primates. METHODS: Ovarian cortex was obtained from adult rhesus macaques, was cut into fragments, and was incubated with NR. Secondary follicles were readily visualized following NR staining and then were encapsulated into alginate beads and cultured individually for 4 weeks in αMEM media supplemented with 10 ng/ml FSH at 5% O2. RESULTS: The survival rates of secondary follicles during culture were similar between those derived from control tissue (71 ± 13%) and those treated with NR (68 ± 9%). The proportion of surviving follicles that formed an antrum were also similar in both groups (70 ± 17% control; 48 ± 24% NR-treated). Follicle diameters were not different between control follicles (184 ± 5µm) and those stained with NR (181 ± 7 µm) on the day of isolation. The percentages of surviving follicles within three cohorts based on their diameters at week 4 of culture were similar between the control group and NR-stained tissue group, fast-grow follicles (24 ± 6% vs. 13 ± 10%), slow-grow follicles (66 ± 5% vs. 60 ± 9%), or no-grow (10 ± 9% vs. 27 ± 6%), respectively. There were no differences in follicle diameters between groups during the culture period. Pre-exposure of secondary follicles to NR diminished their capacity to produce both estradiol and androstenedione by week 4 of culture, when follicles are exhibiting an antrum. Inhibitory effects of NR on steroid production by slow-grow follicles was less pronounced. CONCLUSIONS: NR does not affect secondary follicle survival, growth, and antrum formation during long-term culture, but steroid hormone production by fast-grow follicles is compromised. NR can be used as a non-invasive tool for in situ identification of viable secondary follicles in ovarian cortex before tissue cryopreservation without affecting follicle survival and growth in vitro. Whether maturation or developmental competence of oocytes derived from antral follicles in 3D culture that were previously isolated from NR-stained tissue is normal or compromised remains to be determined. Likewise, the functional consequences of pre-exposure to NR prior to ovarian cortical tissue cryopreservation and transplantation are unknown.