To evaluate the clinical implications of the different trypan blue dyeing techniques used during liquid bubble (LBT) and manual peel (MPT) DMEK lenticule preparation techniques. This study retrospectively compared the degree to which endothelial cells are preserved using selective Descemet Membrane (DM) staining (LBT) versus bath-staining (MPT) when performed by a single surgeon, sourced from a single eye bank. Endothelial cell density measured after the 3-month follow-up was 1805 and 1916 cells/mm2 respectively, differing significantly (p = 0.012). A double-scroll graft formation was found and maintained until implantation in 94% of preparations with bath staining and 50% of preparations using selective DM staining. Preoperative visual acuity was comparable between preparation techniques at 0.4 logMAR as well as postoperatively, at an average of 0.1 logMAR. Reducing chemical stress on the endothelium by avoiding any contact with trypan blue allows for a significantly higher degree of cell preservation. However, achieving the often-desired double-scroll graft formation was possible less frequently. It remains unclear which factors define the differences graft scrolling behavior observed between LBT and MPT.

OBJECTIVE: To evaluate the use of vital dyes and light microscopy for assessing canine corneal endothelial morphology ex vivo.
METHODS: The corneas of 40 canine eyes (n = 20 dogs) enucleated <24 h following euthanasia or death were isolated and flat-mounted on a slide. Corneal endothelium was stained via 0.25% trypan blue followed by 0.5% alizarin red (pH 4.2), photographed, then the following morphological features were calculated using ImageJ: mean cell density (MCD), mean cell area (MCA), polymegathism (coefficient of variation of cell area), and pleomorphism (% hexagonality).
RESULTS: Mean ± standard deviation (range) outcomes were: MCD, 2544 ± 541 cells/mm2 (1750-3922 cells/mm2); MCA, 431 ± 97 μm2 (251-626 μm2); polymegathism, 17 ± 2% (14%-22%); pleomorphism, 84 ± 3% (80%-90%). No significant differences (p ≥ .122) were noted for any outcome between male versus female or brachycephalic versus non-brachycephalic dogs. Young dogs (<10 years) had lower MCA (p = .044), lower pleomorphism (p = .003), and higher MCD (p = .035) when compared to older dogs (≥10 years). Age was significantly (p ≤ .049) correlated with MCA (r = 0.467), MCD (r = -0.476), polymegathism (r = 0.444), and pleomorphism (r = 0.609).
CONCLUSIONS: The combination of vital dyes and light microscopy allowed for clear visualization and evaluation of the corneal endothelium in canine eyes ex vivo. Our findings can be used in future studies to deepen our understanding of the corneal endothelium in health and disease.

UNASSIGNED: Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes.
UNASSIGNED: We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates.
UNASSIGNED: The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2-associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and pro-apoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO.
UNASSIGNED: Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish.

To compare functional and anatomical outcomes between the inverted flap technique and conventional removal of the internal limiting membrane (ILM) in the surgical management of idiopathic macular hole (IMH).
We retrospectively evaluated the anatomical and functional results in 67 eyes of 65 patients operated on for IMH. The patients were operated on either using the conventional ILM peeling technique (first group) or with the inverted ILM flap technique (second group). 43 eyes of 41 patients were included in the first group, 24 eyes of 24 patients in the second group. We indicated for surgery only patients with IMH stage 2-4 according to the Gasse classification. Best corrected visual acuity (VA) was always determined before and two months after surgery. Furthermore, a comparison of both techniques was made according to the average letter gain after surgery, and the effect of surgery was evaluated using OCT with regard to whether IMH closure succeeded. For both techniques, 25G PPV with SF6 tamponade was performed.
Hole closure took place in 41 eyes with conventional ILM removal. In one eye, the hole did not close even after reoperation with the same technique. Median ETDRS letter gain was 7.0. VA remained the same in 2 eyes (4.7%), worsened in 7 cases (16.2%), and improved in all other cases (79.0%). In 16 eyes (37.2%), VA improved by 2 or more lines of ETDRS charts. Using the inverted flap technique, the hole was closed in all 24 monitored eyes. Median ETDRS letter gain was 9.5. VA remained the same in 2 eyes (8.3%), worsened in 2 cases (8.3%), and improved in all other cases (83.3%). In 12 eyes (50.0%), VA improved by 2 or more lines of ETDRS charts. There were no serious complications intraoperatively or postoperatively.
Our study demonstrated the safety and efficacy of both methods. Although the results were not statistically significant, the inverted flap technique recorded a greater ETDRS letter gain (9.5 vs. 7.0) and proportion of closed holes (100% vs. 95.3%) compared to the conventional ILM peeling technique in our set of eyes.

Solid tumors and tumor-derived cell lines commonly contain highly enlarged (giant) cancer cells that enter a state of transient dormancy (active sleep) after they are formed, but retain viability, secrete growth promoting factors, and exhibit the ability to generate rapidly proliferating progeny with stem cell-like properties. Giant cells with a highly enlarged nucleus or multiple nuclei are often called polyploid giant cancer cells (PGCCs). Although PGCCs constitute only a subset of cells within a solid tumor/tumor-derived cell line, their frequency can increase markedly following exposure to ionizing radiation or chemotherapeutic drugs. In this chapter we outline a simple and yet highly sensitive cell-based assay, called single-cell MTT, that we have optimized for determining the viability and metabolic activity of PGCCs before and after exposure to anticancer agents. The assay measures the ability of individual PGCCs to convert the MTT tetrazolium salt to its water insoluble formazan metabolite. In addition to evaluating PGCCs, this assay is also a powerful tool for determining the viability and metabolic activity of cancer cells undergoing premature senescence following treatment with anticancer agents, as well as for distinguishing dead cancer cells and dying cells (e.g., exhibiting features of apoptosis, ferroptosis, etc.) that have the potential to resume proliferation through a process called anastasis.

OBJECTIVE: To compare 3 capsulotomy centration methods.
METHODS: Private clinic, Zlin, Czech Republic.
METHODS: Prospective, consecutive case series.
METHODS: 180 eyes undergoing cataract surgery had anterior capsule staining with microfiltered 0.4% trypan blue solution before selective laser capsulotomy. The first 60 eyes (Group 1) had mydriatic dilated pupil centered capsulotomies. The next 60 eyes (Group 2) were centered on the trypan blue central landmark (TCL). The final 60 capsulotomies (Group 3) were centered on the patient fixated coaxial Purkinje reflex (CPR). Measurements between key anatomical landmarks and the TCL, CPR capsulotomies, and implanted intraocular lens (IOL) center were made.
RESULTS: The TCL, observed in >94% of eyes in the study, coincided with the CPR with a displacement of <0.1 ± 0.1 mm. Group 1 capsulotomies were noticeably decentered on the IOLs by 0.3 ± 0.2 mm. The Group 2 symmetrical IOL relationship was maintained with a decentration of 0.15 ± 0.1 mm. Group 3 had a similar decentration with the IOLs with 0.15 ± 0.1 mm. Verification with IOLMaster 700 data and CALLISTO Eye System showed that the CPR and the TCL were coincident with the measured visual axis.
CONCLUSIONS: The clearly visible TCL served as an alternate landmark to the patient fixated CPR, and being on the anterior capsule was not sensitive to tilt. Further patient compliance was not required. Both were superior to dilated pupil centration, to achieve symmetric IOL coverage. This has application for both capsulotomies and capsulorhexes.

Keratoglobus is a rare subset of noninflammatory corneal ectasia, which is a group of disorders characterized by corneal thinning, projection, and scarring. Patients with keratoglobus commonly present with poor vision. A case of advanced keratoglobus was managed by a modified phacoemulsification surgical technique using endoillumination and capsular staining with trypan blue. In this case, we present a 54-year-old man with keratoglobus. In January 2023, a modified phacoemulsification surgical technique using endoillumination was described with a video in a patient with bilateral corneal opacification, neovascularization, significant peripheral thinning, and moderate to severe corneal opacity in which cataract surgery had to be performed alone without considering penetrating keratoplasty. Postoperatively, the patient was doing well with no leaks. We may conclude that this method allows for better visualization during surgery and decreases the risk of intraoperative complications due to poor visualization in patients with severe corneal opacity.

Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern-central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at -20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.

Iron oxide nanoparticles (Fe2O3NPs) were synthesized utilizingMentha spicatasourced from Cyprus as a stabilizing agent. The study delved into assessing the antimicrobial, cytotoxic, anti-proliferative, and anti-migratory potential of Fe2O3 NPs through disc diffusion, trypan blue, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Characterization of the synthesized Fe2O3 NPs was conducted using Fourier-transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), UV-vis spectroscopy (UV-vis), scanning electron microscopy (SEM), and energy-dispersive x-ray spectroscopy (EDX). The FTIR, XRD, and SEM-EDX spectra confirmed the successful formation of Fe2O3 NPs. The analysis of UV-vis spectra indicates an absorption peak at 302 nm, thereby confirming both the successful synthesis and remarkable stability of the nanoparticles. The nanoparticles exhibited uniform spherical morphology and contained Fe, O, and N, indicating the synthesis of Fe2O3NPs. Additionally, the Fe2O3NPs formed through biosynthesis demonstrated antimicrobial capabilities againstEscherichia coliandBacillus cereus. The significant anti-migratory potential on MDA-MB 231 human breast cancer cells was observed with lower concentrations of the biosynthesized Fe2O3NPs, and higher concentrations revealed cytotoxic effects on the cells with an IC50of 95.7μg/ml. Stable Fe2O3NPs were synthesized usingMentha spicataaqueous extract, and it revealed antimicrobial activity onE. coliandB. cereus, cytotoxic, anti-proliferative and anti-migratory effect on highly metastatic human breast cancer cell lines.

OBJECTIVE: Endothelial cell loss (ECL) during Descemet membrane endothelial keratoplasty (DMEK) graft preparation has been shown to affect graft survival and the need for re-grafting. The purpose of this study was to quantitatively assess the impact of the plastic and glass mediums in contact with DMEK donor tissue during intra-operative graft staining on ECL.
METHODS: Retrospective study that included patients who underwent DMEK surgery between January 2019 and June 2021 at Hôpital Maisonneuve-Rosemont and the Jewish General Hospital in Montreal, Canada. DMEK grafts were stained with 0.06% Trypan blue ophthalmic solution (VisionBlue®, Dutch Ophthalmic, USA, Exeter, NH) for 120 s in either a plastic or glass medium prior to delivery into the recipient\'s eye. The ECL was compared between the two groups 12-30 months post-operatively.
RESULTS: ECL at 12-30 months was significantly less in the eyes that had received grafts stained in a plastic medium compared to those stained in a glass medium. Graft survival and re-bubbling was higher in the glass group however this difference was not statistically significant.
CONCLUSIONS: Staining of the DMEK graft in a plastic medium caused less ECL compared to the glass medium.