The usefulness of lutein/trypan blue vital dye for the staining of corneal endothelium: a pilot study on DMEK pretreated tissues.

PURPOSE: The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium-Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK). METHODS: Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-ß1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB. RESULTS: Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-ß1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts. CONCLUSION: Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting.

Ex vivo evaluation of retinal cytotoxicity after the use of multiple medical devices in pars plana vitrectomy in porcine eyes.

This study aimed to evaluate viability of retinal cells after the use of multiple intraoperative devices, namely a vitreal dye (triamcinolone acetonide,TA), a ERM/ILM dye (solution of trypan blue 0.15% and brilliant blue 0.025%), and two intraocular tamponades, namely perfluoro-n-octane, (PFO) and silicone oil (SO 1000 cSt), with minimal and maximal removal of their residues, during a simulated pars plana vitrectomy (PPV) in porcine eyes ex-vivo. The in vitro cytotoxicity of each of these compounds was verified on ARPE-19 cells by direct tests according to the ISO 10993-5 (2009). Pars plana vitrectomy was performed on 25 enucleated porcine eyes divided in five groups according to the following conditions: Group A) No surgery control: eye bulbs were kept at room temperature for 40 min; Group B) Sham surgery: PPV with the sole use of BSS for 40 min; Group C) Cytotoxic control: PPV with BSS infusion (20 min) followed by intravitreal injection of 1H-PFO (contact time: 20 min); Group D) Surgery with residues: PPV with BSS infusion and sequential intravitreal injection of TA, ERM/ILM dye, PFO and SO, with minimal removal of each compound after a specified contact-time (overall duration: 40 min); Group E) Surgery with minimal residues: PPV performed as in group D, but with maximal removal of each compound (overall duration: 40 min). All the experimental procedures were performed at room temperature. Immediately after surgery, the retina was extracted from each eye bulb and samples of 3-mm diameter were prepared. Retinal viability was determined for each sample by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. A cell viability <70% was considered the cytotoxicity threshold. Kruskal-Wallis test was used to evaluate the differences in retinal viability between groups. No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The tested conditions indicated that the combined use of TA, ERM/ILM dye, PFO and SO during PPV does not affect retinal cells viability if all the devices are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability due to a cumulative and/or synergistic cytotoxic effect between them, supporting the crucial role of an optimal removal of the intraoperative medical devices to ensure a safe vitrectomy to the patient.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Trimanual vitrectomy for severe proliferative diabetic retinopathy.

PURPOSE: To describe and evaluate a novel technique of pars plana vitrectomy (PPV) under chandelier illumination which is aided with the vital dyes and perfluorocarbon liquids for the management of the complex diabetic vitrectomy cases. METHODS: We conducted a prospective interventional comparative study on 40 eyes of 36 patients with advanced diabetic eye disease requiring PPV. The study was conducted in a single tertiary referral center. Eyes were divided on 1:1 basis by stratified randomization into two groups. Group 1 had trimanual vitrectomy done assisted with chandelier illumination, perfluorocarbon liquid (PFCL) and vital dyes. Group 2 had the conventional bimanual vitrectomy done assisted with chandelier illumination only. All patients were followed up for a minimum of 6 months after the surgery. RESULTS: Forty eyes of 36 patients with the mean age of 51.42 years (range 28-69) were evaluated. The anatomical success at 6 months could be achieved in all the eyes in both groups. The complete removal of the pre-retinal proliferations could be accomplished in all the eyes in the trimanual PPV group, and only in 85% of the eyes in the bimanual PPV group. Operative time was significantly shorter in the trimanual PPV group (p < 0.001). More eyes in the trimanual PPV group (55.0%) could achieve better vision (> 6/60) 6 months after the operation compared to the bimanual PPV group (50.0%), but this difference was not statistically significant. CONCLUSION: Trimanual PPV is a novel, safe and effective technique that can improve the results of the complex diabetic PPV.

Metformin and rapamycin protect cells from vital dye-induced damage in retinal pigment epithelial cells and in vivo.

PURPOSE: To evaluate the effect of autophagy inducers on damage caused by vital dye in adult human RPE (ARPE) cells and in a rat model. METHODS: ARPE-19 cells were exposed to ICG or BBG (0.05 mg/ml) with rapamycin (200 nM) or metformin (2 mM) for 30 min and treated with or without 20 µM chloroquine (CQ) to identify the protein levels of LC3 and SQSTM1 by immunoblotting. In vivo study was performed by injecting 10 µl 0.05% ICG and 0.25% BBG into the subretinal space of the rat eyes, and/or co-treated them with metformin and rapamycin. The retinas were used to determine autophagy with the LC3-II level and apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay. RESULTS: In this study, both ICG and BBG inhibited autophagy flux in adult human retinal pigment epithelium cells (ARPE-19), whereas only ICG consistently reduced autophagy in the retina of rats. Moreover, rapamycin and metformin induced autophagic flux in ARPE-19 cells and increased the LC3-II level in retinal tissues exposed to vital dyes. Both ICG and BBG increased apoptosis in the retina of rats. However, both rapamycin and metformin induced autophagy and reduced the apoptosis caused by vital dyes. CONCLUSION: Taken together, these results suggest that rapamycin and metformin may diminish vital dye-induced retinal damage in vivo through activation of autophagy.

Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.

Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.

Current Practice and Emerging Molecular Imaging Technologies in Oral Cancer Screening.

Oral cancer is one of the most common cancers globally. Survival rates for patients are directly correlated with stage of diagnosis; despite this knowledge, 60% of individuals are presenting with late-stage disease. Currently, the initial evaluation of a questionable lesion is performed by a conventional visual examination with white light. If a lesion is deemed suspicious, a biopsy is taken for diagnosis. However, not all lesions present suspicious under visual white light examination, and there is limited specificity in differentiating between benign and malignant transformations. Several vital dyes, light-based detection systems, and cytology evaluation methods have been formulated to aid in the visualization process, but their lack of specific biomarkers resulted in high false-positive rates and thus limits their reliability as screening and guidance tools. In this review, we will analyze the current methodologies and demonstrate the need for specific intraoral imaging agents to aid in screening and diagnosis to identify patients earlier. Several novel molecular imaging agents will be presented as, by result of their molecular targeting, they aim to have high specificity for tumor pathways and can support in identifying dysplastic/cancerous lesions and guiding visualization of biopsy sites. Imaging agents that are easy to use, inexpensive, noninvasive, and specific can be utilized to increase the number of patients who are screened and monitored in a variety of different environments, with the ultimate goal of increasing early detection.

The Combination of Trypan Blue and Brilliant Blue G-Assisted Vitrectomy for Macular Pucker: Histopathological Findings.

PURPOSE: To report on the combined use of trypan blue (TB) and brilliant blue G (BBG) for staining the epiretinal membrane (ERM) and internal limiting membrane (ILM) during vitrectomy and to describe the histopathological findings. METHODS: 10 surgical specimens were removed from 10 eyes with macular pucker during vitrectomy using a commercially available combination of TB and BBG for ERM and ILM staining and peeling. Specimens were evaluated using light and transmission electron microscopy. RESULTS: In all cases the combination of TB and BBG was useful for identifying and delineating ERM and ILM. No complications related to the use of the dye were observed during or after surgery. Glial cells were present in all specimens. Hyalocytes were observed in 6 cases and myofibroblasts in 3 of them. In 7 cases native vitreous collagen fibrils were found on the ILM, while in 5 specimens newly formed collagen was present. No clinical evidence of toxicity was observed during the 3-month follow-up. CONCLUSION: The combined use of TB and BBG appeared to be very useful intraoperatively to improve the visualization of ERM and ILM, thus facilitating their complete removal. Anatomical and histopathological findings demonstrated the safety and the efficacy of this vital dye.

Ultrasound-Guided Intrauterine Labeling of Rat Fetuses.

AIM: To compare intra-amniotic versus fetal subcutaneous injections for selective fetal labeling in multifetal rat pregnancies. METHODS: A total of 14 pregnant rats were randomized to receive intra-amniotic injections of dyes (including Fluorescein, Indigo Carmine, or Evans Blue) or fetal subcutaneous injections (of commercial tattoo ink) both guided by ultrasound at 15-17 days of gestation. Survival, injection, and labeling success rates of both techniques were compared. RESULTS: Survival rates (84.4% for intra-amniotic injections vs. 90.9% for fetal subcutaneous injections) and injection success rates (94% for intra-amniotic injections vs. 100% for fetal subcutaneous injections) were similar among both groups. None of the neonates from the intra-amniotic injections group were labeled at birth, while 93% of the neonates from fetal subcutaneous injections group were tagged, showing a visible spot in the skin at birth. CONCLUSION: Our results suggest that ultrasound-guided fetal subcutaneous injections might be an adequate strategy for selectively labeling fetuses in multifetal pregnant animals.

Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown.

RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs.

Lid wiper epitheliopathy: Topical review of current identification strategies and future perspectives.

Lid Wiper Epitheliopathy (LWE) is a recognized ocular condition affecting visual quality and ocular comfort, characterized by the damage of the marginal conjunctiva of the upper eyelid. This review critically examines the current strategies for LWE identification and explores the multifactorial etiology of LWE and its association with various ocular surface diseases. A literature review conducted on PubMed identified 67 relevant studies, which were analyzed to enhance understanding of LWE's diagnostic criteria and its clinical implications, offering future perspectives on improving diagnostic accuracy and patient outcomes. The association between LWE and contact lens-related discomfort is examined, with studies reporting conflicting findings regarding its prevalence and severity among soft contact lens wearers. Moreover, LWE has been linked to tear film abnormalities, altered tear composition, and eyelid margin abnormalities in non-contact lens wearers, further complicating diagnosis and management. Current LWE grading systems, including subjective methods and semi-objective approaches, are reviewed, highlighting challenges such as subjectivity, complexity, and variability. Recent advances in LWE grading, including semi-automated methods and photographic scales, show promise but face implementation challenges in clinical settings. The review concludes by proposing areas for further research, including investigating factors contributing to LWE onset and development, developing more sensitive diagnostic tools, and exploring the impact of LWE on ocular surface health and visual function. Standardization, validation, and innovation in grading systems are emphasized to enhance diagnostic accuracy and improve clinical outcomes for patients with LWE. Ongoing research in LWE grading is essential for addressing diagnostic challenges and optimizing assessment methodologies in clinical practice.

Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate.

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25-200 µg L-1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 µg L-1) and lethal (200 µg L-1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 µg L-1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 µg L-1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

[Selective vital dyes in macular surgery : Do they increase the probability of intraoperative identification of the internal limiting membrane also for an experienced surgeon?] / Selektive Vitalfarbstoffe in der Makulachirurgie : Erhöhen sie die Wahrscheinlichkeit der intraoperativen ILM-Identifizierung auch bei einem erfahrenen Operateur?

BACKGROUND: Various vital dyes exist on the market for intraoperative internal limiting membrane (ILM) identification. The aim of this study was to verify the added value of these dyes for ILM identification and in the difficulty of ILM peeling during pars plana vitrectomy (ppV) by a single surgeon highly experienced in this operation. MATERIAL AND METHODS: In this study 400 ppV surgical reports involving ILM peeling were retrospectively analyzed. Intraoperative assessment of identification or difficulty of intraoperative ILM peeling had to be documented in the surgical report. The total group consisted of 2 cohorts each with 200 surgical reports (first cohort without selective vital dyes, period 2004-2006; second cohort with vital dyes in the majority of ppVs, period 2013-2020). RESULTS: The difference between both groups in terms of intraoperative identification of ILM was statistically significant (p < 0.001); however, no statistically significant difference (p = 0.951) was found between the two groups in terms of difficulty of ILM peeling. In logistic regression analysis neither patient gender, age, eye side, lens status nor posterior vitreous limiting membrane status were significantly associated with ILM identification. CONCLUSION: The introduction of intravital dyes represents a decisive advancement in retinal surgery. In the investigated sample this benefit was evident from two precisely defined surgical cohorts of a single highly experienced surgeon. This underlines the additional benefit of using selective vital dyes to identify ILM in macular surgery for less experienced surgeons.

Vital Dye Uptake of YO-PRO-1 and DASPEI Depends Upon Mechanoelectrical Transduction Function in Zebrafish Hair Cells.

PURPOSE: Vital dyes allow the visualization of cells in vivo without causing tissue damage, making them a useful tool for studying lateral line and inner ear hair cells in living zebrafish and other vertebrates. FM1-43, YO-PRO-1, and DASPEI are three vital dyes commonly used for hair cell visualization. While it has been established that FM1-43 enters hair cells of zebrafish and other organisms through the mechanoelectrical transduction (MET) channel, the mechanism of entry into hair cells for YO-PRO-1 and DASPEI has not been established despite widespread use. We hypothesize that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel uptake dependent similar to FM1-43. METHODS: To test this hypothesis, we used both genetic and pharmacologic means to block MET channel function. Genetic based MET channel assays were conducted with two different mechanotransduction defective zebrafish lines, specifically the myo7aa-/- loss of function mutant tc320b (p.Y846X) and cdh23-/- loss of function mutant (c.570-571del). Pharmacologic assays were performed with Gadolinium(III) Chloride (Gad(III)), a compound that can temporarily block mechanotransduction activity. RESULTS: Five-day post fertilization (5dpf) myo7aa-/- and cdh23-/- larvae incubated with FM1-43, YO-PRO-1, and DASPEI all showed nearly absent uptake of each vital dye. Treatment of wildtype zebrafish larvae with Gad(III) significantly reduces uptake of FM1-43, YO-PRO-1, and DASPEI vital dyes. CONCLUSION: These results indicate that YO-PRO-1 and DASPEI entry into zebrafish hair cells is MET channel dependent similar to FM1-43. This knowledge expands the repertoire of vital dyes that can be used to assess mechanotransduction and MET channel function in zebrafish and other vertebrate models of hair cell function.

Selective ILM Staining and Safety of Two Vital Dyes During a Human-Like Pars Plana Vitrectomy Ex Vivo in Porcine Eyes.

PURPOSE: An ideal dye for intraocular use should effectively stain the target tissue while being easy to apply and remove. Additionally, it should not have any adverse effects resulting from prolonged contact with the retinal tissue. Recently, concerns have been raised about the safety of some vital dyes during surgical procedures as they may cross the internal limiting membrane and deposit on the retina. In this study, we aimed to investigate whether commercially available vital dyes, VIEW-ILM® and TWIN® (AL.CHI.MI.A. S.r.l., Ponte San Nicolò, Padova, Italy), have the potential to cross the internal limiting membrane during vitreoretinal surgery and deposit on the retina. Furthermore, we evaluated their safety in vitro and in vivo. METHODS: A human-like pars plana vitrectomy was performed on porcine eyes ex vivo, with VIEW-ILM® or TWIN® used to stain the internal limiting membrane either with or without subsequent internal limiting membrane peeling. The two dyes were then extracted from retinal punches with or without internal limiting membrane, and quantified using high performance liquid chromatography. Safety was evaluated through in vitro cytotoxicity tests and in vivo skin sensitization and irritation tests according to ISO standards. RESULTS: High performance liquid chromatography analyses demonstrated that VIEW-ILM® and TWIN® effectively stained the internal limiting membrane without crossing the membrane. No residual dyes were found in the retinal layers after internal limiting membrane removal. Furthermore, both in vitro and in vivo safety tests confirmed the absence of cytotoxicity, skin sensitization, and irritation. CONCLUSION: The results of this study support the safety and efficacy of VIEW-ILM® and TWIN® for internal limiting membrane staining. The experimental protocol described in this study could be utilized to gain a comprehensive understanding of the characteristics of vital dyes.

Ocular surface staining: Current concepts and techniques.

The health of the ocular surface is vital for clear vision and comfort. Various factors can adversely influence the ocular surface and tear film homeostasis, and these include procedures like cataract and corneal refractive surgery. It is, therefore, important to assess the integrity of the ocular surface in a rapid, predictable, and consistent manner in the clinic. Various tests and devices have been described, and while these are useful, this article highlights the importance of using fluorescein staining of the ocular surface in detecting changes. This is a simple, inexpensive, rapidly performed test that is available in most eye clinics. However, a proper technique of dye instillation and assessment is important to recognize the changes that can occur. Once detected, these changes can be quantified, and the location and patterns can be used to diagnose the diseases that are present; these changes can also be used to monitor treatment outcomes and disease progression. The article discusses the technique, assessment, and interpretation of fluorescein staining of the ocular surface, along with the role of the two other vital dyes - rose bengal and lissamine green.

Modified perfluorocarbon liquid/internal limiting membrane interface staining in myopic macular hole retinal detachment.

PURPOSE: To demonstrate a modified technique of perfluorocarbon liquid (PFCL)/internal limiting membrane (ILM) interface staining in patients affected by macular hole retinal detachment (MHRD) in the setting of high myopia. METHODS: Two-surgeon retrospective case series and review of surgical videos with step-by-step technique analysis. RESULTS: Our modified technique was proficiently employed to treat 9 highly myopic patients affected by MHRD. Successful staining and peeling of the ILM with the creation of an inverted flap was achieved in all cases. A limited number of dye injections required to stain the ILM was noted. No subretinal dye migration or other intra- and postoperative complications were recorded. CONCLUSION: Modified PFCL/ILM interface staining is a surgically efficient technique potentially reducing the risk of iatrogenic damage, including the toxicity of vital dyes to the retinal pigment epithelium (RPE) in myopic MHRD.

Membrane Blue Dual Protects Retinal Pigment Epithelium Cells/Ganglion Cells-Like through Modulation of Mitochondria Function.

Although recent data highlight the greater protective effects exerted by Membrane Blue Dual (MBD), a precise analysis of the mechanisms of action is missing. We examined the effects of MBD with/without polyethylene glycol (PEG) on both human retinal pigment epithelial cells (ARPE-19) and retinal ganglion cells-like (RGC-5) cultured in the presence/absence of ultraviolet B (UVB) treatment on mitochondria function, oxidants, and apoptosis. In ARPE-19/RGC-5 cells either treated or not with UVB, the effects of MBD with/without PEG were evaluated by specific assays for viability, mitochondrial membrane potential and mitochondrial reactive oxygen species (mitoROS) release. Annexin V was used to detect apoptosis, whereas trypan blue and the scratch assay were used for proliferation/migration. In both physiologic conditions and in the presence of UVB, MBD with/without PEG increased cell viability, mitochondrial membrane potential, proliferation and migration in both ARPE-19 and RGC-5 cells. In general, the effects of MBD with PEG were greater than those caused by MBD without PEG. Our results suggest that, in particular, MBD with PEG is a safe and effective dye for vitreoretinal surgery through the modulation of mitochondrial function.

Spotlight on the Internal Limiting Membrane Technique for Macular Holes: Current Perspectives.

Pars plana vitrectomy has become the standard procedure for primary macular holes (MHs) repair, including the removal of the posterior cortical vitreous, the stripping of eventual epiretinal membranes, and finally an intraocular gas tamponade. During this procedure, peeling the internal limiting membrane (ILM) has been proven to increase closure rates and avoid postoperative reopening in several researches. In fact, even in large MHs more than 400 µm, the advantage of peeling off the ILM was highlighted by better anatomical closure rates. Nevertheless, some authors suggested that ILM peeling is not always essential, because it generates various side effects in retinal structure and function. Furthermore, the ideal amount of ILM peeling and the most effective strategies for removing the ILM are still subject of research. Different surgical modifications have been reported as alternatives to traditional peeling in certain clinical settings, including ILM flaps, ILM scraping, and foveal sparing ILM peeling. As regards large MHs, the introduction of ILM inverted flap appeared as a game changer, offering a significantly higher >90% closure rate when compared to traditional ILM peeling. Modifications to inverted ILM flap procedures have been claimed in recent years, in order to define the best area and direction of ILM peeling and its correlation with functional outcomes. Moreover, several innovations saw the light in the setting of recurrent MHs, such as ILM free flap transposition, inverted ILM flap combined autologous blood clot technique, neurosensory retinal flap, and human amniotic membrane (HAM) plug, claiming higher anatomical success rate also in those complex settings. In conclusion, the aim of this review is to report how the success rate of contemporary macular surgery has grown since the turn of the century, especially for big and chronic MHs, analyzing in which way ILM management became a crucial point of this kind of surgery.

Cryptosporidium spp. and Giardia spp. (oo)cysts as target-organisms in sanitation and environmental monitoring: A review in microscopy-based viability assays.

Cysts and (oo)cysts are the infective forms of parasitic protozoa, as Giardia and Cryptosporidium, which are widespread and associated to worldwide waterborne diseases outbreaks. These microorganisms pose a challenge to public health, as they are resistant to conventional disinfection methods, which make them important parameters when evaluating inactivation efficiency. However, when (oo)cysts are targets, it is challenging to infer inactivation efficacy, as it may require infectivity tests that are not often an option for laboratory routine analysis. In this scene, (oo)cyst viability based on induced excystation, membrane integrity and enzyme activity evaluated by dye inclusion and/or exclusion, as well as fluorescence reduction consist on microscopy-based techniques that may be options to estimate inactivation in the environmental context. This scoping review presents applications, advantages and limitations of these methodologies for viability assessment, in order to shed light on the (oo)cyst viability topic and provide insight strategies for choosing protocols in the environmental and sanitation field, in laboratory applications and novel research.