Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans.

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

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.

The effect of time on grading corneal fluorescein and conjunctival lissamine green staining.

PURPOSE: To explore the effect of time on grading corneal fluorescein and conjunctival lissamine green staining in dry eye disease (DED). METHODS: Photographs of 68 subjects with non-Sjogren's DED (nSS DED) and 32 with Sjogren's DED (SS DED) were taken of corneal fluorescein staining, then conjunctival lissamine green staining every 30 s for at least 5 min. Photographs of one randomly selected eye were then randomly ordered and graded on a scale from 0 to 5 (severe staining) by two clinicians, masked to both site and subject. The average time required to reach the maximum grade of staining (Gmax) was calculated. RESULTS: The median time (upper and lower quartiles) to corneal fluorescein Gmax was 2.6 (1.3-5.3) minutes for nSS DED and 3.8 (2.6-5.4) minutes for SS DED, a statistically significant difference (Mann Whitney U test, p = 0.018). In contrast, the median time to the Gmax for lissamine green staining of the nasal and temporal conjunctiva was 0.5 (0.5-1.1 nasal, 0.5-0.8 temporal) minutes for nSS DED and 0.5 (0.5-0.8 nasal, 0.5-0.5 temporal) minutes for SS DED subjects, which was not statistically significant (p ≥ 0.383). CONCLUSIONS: The time required to reach the maximum grade of corneal fluorescein staining, but not conjunctival lissamine green staining, varied widely and was significantly longer in subjects with Sjögren's Syndrome. Early observation of corneal fluorescein staining can lead to under-grading, which may impact the diagnosis and assessment of treatment in DED. Further study of the best time to assess corneal fluorescein staining in various DED populations is warranted.

Strongly Basic Anion Exchange Resin Based on a Cross-Linked Polyacrylate for Simultaneous C.I. Acid Green 16, Zn(II), Cu(II), Ni(II) and Phenol Removal.

The adsorption ability of Lewatit S5528 (S5528) resin for C.I. Acid Green 16 (AG16), heavy metals (Zn(II), Cu(II) and Ni(II)) and phenol removal from single-component aqueous solutions is presented in this study to assess its suitability for wastewater treatment. Kinetic and equilibrium studies were carried out in order to determine adsorption capacities, taking into account phase contact time, adsorbates' initial concentration, and auxiliary presence (NaCl, Na2SO4, anionic (SDS) and non-ionic (Triton X100) surfactants). The pseudo-second-order kinetic model described experimental data better than pseudo-first-order or intraparticle diffusion models. The adsorption of AG16 (538 mg/g), phenol (14.5 mg/g) and Cu(II) (5.8 mg/g) followed the Langmuir isotherm equation, while the uptake of Zn(II) (0.179 mg1−1/nL1/n/g) and Ni(II) (0.048 mg1−1/nL1/n/g) was better described by the Freundlich model. The auxiliary's presence significantly reduced AG16 removal efficiency, whereas in the case of heavy metals the changes were negligible. The column studies proved the good adsorption ability of Lewatit S5528 towards AG16 and Zn(II). The desorption was the most effective for AG16 (>90% of dye was eluted using 1 mol/L HCl + 50% v/v MeOH and 1 mol/L NaCl + 50% v/v MeOH solutions).

Interaction of Commonly Used Oral Molecular Excipients with P-glycoprotein.

P-glycoprotein (P-gp) plays a critical role in drug oral bioavailability, and modulation of this transporter can alter the safety and/or efficacy profile of substrate drugs. Individual oral molecular excipients that inhibit P-gp function have been considered a mechanism for improving drug absorption, but a systematic evaluation of the interaction of excipients with P-gp is critical for informed selection of optimal formulations of proprietary and generic drug products. A library of 123 oral molecular excipients was screened for their ability to inhibit P-gp in two orthogonal cell-based assays. ß-Cyclodextrin and light green SF yellowish were identified as modest inhibitors of P-gp with IC50 values of 168 µM (95% CI, 118-251 µM) and 204 µM (95% CI, 5.9-1745 µM), respectively. The lack of effect of most of the tested excipients on P-gp transport provides a wide selection of excipients for inclusion in oral formulations with minimal risk of influencing the oral bioavailability of P-gp substrates.

Canonical Grading Scales of Corneal and Conjunctival Staining Based on Psychophysical and Physical Attributes.

Purpose: In this study, we apply psychophysical scaling principles based on physical (photometric) attributes of images to better understand the factors involved in clinician judgement of ocular surface staining and, using that knowledge, to develop photographic scales for the assessment of staining for dry eye (DE) and related conditions. Methods: Subjects with noninfectious ocular surface staining were enrolled at five clinical sites. Following instillation of fluorescein, photographs of corneal staining were taken every 30 seconds for at least 5 minutes. The same procedure was followed for conjunctival staining after instillation of 2 µl of 1% lissamine green. A subset of the best corneal and bulbar conjunctival staining images were anonymized and a spectroradiometer measured photometric attributes (luminance and chromaticity). The images were scaled psychophysically by study investigators, who participated in constructing grading scales based on physical and psychophysical analyses. The final grading scales were refined following consultation with outside DE experts. Results: Photographs were collected from 142 subjects (81% women), with an average age of 58 ± 17 years; 89% were diagnosed with DE. There was a monotonic relationship between between physical measurements and psychophysically scaled staining of both corneal (fluorescein) and bulbar (lissamine green) staining. Michelson contrast and u' (chromaticity) accounted for 66% and 64% of the variability in the psychophysically scaled images of fluorescein corneal and lissamine green conjunctival staining, respectively. Translational Relevance: This paper provides examples of the first ever clinically usable ocular surface staining scales validated using psychophysical scaling and the physical attributes (luminance and chromaticity) of the staining itself. In addition, it provides a generalizable method for the development of other clinical scales of ocular appearance.

Are there Clinical Ways to Assess Inflammation in Dry Eye Disease?

In the diagnostic process of dry eye disease, the detection of inflammatory activity is critical in order to evaluate the risk of progression and immunologic shift of the disease, to predict patient response to treatment, and to design an efficient therapeutic strategy, including artificial tear replacement, punctal occlusion or anti-inflammatory therapy.Even if it is difficult to quantify, some indicators of the presence of inflammation are collectible during the examination of the ocular surface in a first-line clinical setting. This review presents and critically discusses the assessment of inflammation in dry eye disease in clinical practice.

The food additive fast green FCF inhibits α-synuclein aggregation, disassembles mature fibrils and protects against amyloid-induced neurotoxicity.

α-Synuclein (α-syn) aggregates into cytotoxic amyloid fibrils, which are recognized as the defining neuropathological feature of Parkinson's disease (PD). Therefore, inhibiting α-syn fibrillogenesis and disrupting the preformed fibrils are both considered attractive strategies to cure PD. We discovered that a safe food additive, fast green FCF, is capable of inhibiting α-synuclein fibrillogenesis and reducing the related cytotoxicity. Thioflavin T fluorescence assays demonstrated that fast green FCF could inhibit the fibrillogenesis α-synuclein. In the presence of 100 µM fast green FCF, amorphous aggregates were formed and observed by atomic force microscopy. Toxicity assays in cell cultures revealed that fast green FCF significantly reduced the cytotoxicity of α-syn. Molecular dynamics simulations revealed the potential mechanism of the interactions between fast green FCF and α-synuclein. Fast green FCF greatly disrupted the α-synuclein pentamer and reduced the ß-sheet content by reducing both nonpolar and polar interactions. Furthermore, two binding sites were identified, named region I (Y39-K45) and region II (H50-Q62). Our data reveal that electrostatic interactions, hydrogen bonds, and π-π interactions synergistically contribute to the binding of fast green FCF to the α-synuclein pentamer. These results indicate that fast green FCF is a candidate prototype for the development of drugs against the aggregation of amyloid fibrils in PD.

Fast green FCF inhibits Aß fibrillogenesis, disintegrates mature fibrils, reduces the cytotoxicity, and attenuates Aß-induced cognitive impairment in mice.

Fast green FCF (FGF) is often used in foods, pharmaceuticals, and cosmetics. However, little is known about the interactions of FGF with amyloid-ß protein (Aß) associated with Alzheimer's disease. In this study, the inhibitory effects of FGF on Aß fibrillogenesis, the disruption of preformed Aß fibrils, the reduction of Aß-induced cytotoxicity, and the attenuation of Aß-induced learning and memory impairments in mice were investigated. FGF significantly inhibited Aß fibrillogenesis and disintegrated the mature fibrils as evidenced by thioflavin T fluorescence and atomic force microscopy studies. Co-incubation of Aß with FGF greatly reduced Aß-induced cytotoxicity in vitro. Moreover, FGF showed a protective effect against cognitive impairment in Aß-treated mice. Molecular dynamics simulations further showed that FGF could synergistically interact with the Aß17-42 pentamer via electrostatic interactions, hydrogen bonds and π-π interactions, which reduced the ß-sheet content, and disordered random coils and bend structures of the Aß17-42 pentamer. This study offers a comprehensive understanding of the inhibitory effects of FGF against Aß neurotoxicity, which is critical for the search of effective food additives that can combat amyloid-associated disease.

New Freeware for Image Analysis of Lissamine Green Conjunctival Staining.

PURPOSE: Lissamine green (LG) is often used in addition to fluorescein to assess the severity of conjunctival damage in dry eye syndrome, which is graded manually. Our purpose was to describe an algorithm designed for image analysis of LG conjunctival staining. METHODS: Twenty pictures of patients suffering from dry eye with visible LG conjunctival staining were selected. The images were taken by 2 different digital slit lamps with a white light source and a red filter transmitting over the wavelengths absorbed by LG. Conjunctival staining appeared in black on a red background. The red channel was extracted from the original image. Stained areas were then detected using a Laplacian of Gaussian filter and applying a threshold whose value was determined manually on a subset of images. The same algorithm parameters remained constant thereafter. LG-stained areas were also drawn manually by 2 experts as a reference. RESULTS: The delineation obtained by the algorithm closely matched the actual contours of the punctate dots. In 19 cases of 20 (95%), the algorithm found the same Oxford grade as the experts, even for confluent staining that was detected as a multitude of dots by the algorithm but not by the experts, resulting in a high overestimation of the total number of dots (without mismatching the Oxford grade estimated by the experts). The results were similar for the 2 slit-lamp imaging systems. CONCLUSIONS: This efficient new image-analysis algorithm yields results consistent with subjective grading and may offer advantages of automation and scalability in clinical trials.

Reactive Green 5-Decorated Polyacrylamide/Chitosan Cryogel: An Affinity Matrix for Catalase.

Acrylamide/chitosan-based cryogel was fabricated, and a triazine dye, Reactive Green 5, was attached to the cryogel by nucleophilic substitution to build a dye affinity support for adsorption of catalase enzyme. Characterization of cryogel was performed using FTIR, SEM, EDX, BET, and swelling test. Synthesized cryogel beared pores with ~ 200 µm in size and the surface area of 11.8 m2/g. Maximum catalase adsorption was (17.6 ± 0.29 mg/g) measured at pH 4.0 and 25 °C. The adsorption sites on the cryogel were saturated at 0.75 mg/mL enzyme concentration. Increased ionic strength caused a decrease in adsorption capacity. Desorption of catalase from cryogel was enabled using 0.5 M NaSCN solution. Consecutive adsorption experiments were carried out fifteen times to evaluate the reusability of the cryogel. Thermal, storage, and operational stabilities of immobilized catalase were higher than the free one. The data produced implicate that catalase-adsorbed dye-affinity cryogel may be used for H2O2 detection or removal when necessary. Graphical Abstract.

Foam Separation of Dyes Using Anionic, Cationic, and Amphoteric Surfactants.

Foam separation can selectively remove a target substance from a solution via adsorption of the substance with the surfactant at the surface of the bubble. A cationic dye, methylene blue, and an anionic dye, Fast Green FCF, were prepared as substances to be removed via foam separation. Anionic (sodium dodecyl sulfate, SDS), cationic (dodecyltrimethylammonium chloride, DTAC), and amphoteric (3-(dodecyldimethylammonio)propane-1-sulfonate, SB-12) surfactants were used in the foam separation process. The effectiveness of the surfactants for removing the cationic methylene blue increased as follows: DTAC < SB-12 < SDS. On the other hand, the effectiveness of the surfactants for removing the anionic Fast Green FCF was in the opposite order. The dyes were effectively adsorbed by the foams via electrostatic interactions between the oppositely charged surfactant and the dye molecules. Since amphoteric surfactants have both anionic and cationic charges in a molecule, they could effectively remove both dyes in the foam separation process. Therefore, it was found that the amphoteric surfactant was highly versatile. Analysis of the kinetics of the removal rate showed that the aqueous solutions of monomers could remove the dyes more effectively than micellar solutions in foam separation.

Conjunctival staining with lissamine green as a predictor of tear film deficiency in dogs.

OBJECTIVE: To evaluate a grading scheme for conjunctival staining patterns with lissamine green ocular dye in the diagnosis of tear film deficiencies in dogs. PROCEDURES: Client-owned and research colony dogs were enrolled in a prospective study between February and October 2018 in which slit-lamp biomicroscopy, Schirmer tear test (STT), tear film breakup time (TFBUT), conjunctival lissamine green staining (LGS), and intraocular pressure (IOP) measurement were performed in both eyes of all dogs. Lissamine green staining of the temporal bulbar conjunctiva was graded from 0-3, with a higher grade corresponding to an increased stain intensity. RESULTS: Fifty-four dogs (107 eyes), comprising 31 males and 23 females with a mean age of 5.0 ± 3.9 years (range 0.5-14.3), were enrolled in the study. STT was <15 mm/min in 21 eyes and ≥15 mm/min in 86 eyes. Lissamine green staining grade for eyes with a STT of <15 mm/min (2.0 ± 0.9) was significantly higher than for eyes with a STT ≥15 mm/min (0.2 ± 0.7) (P < .001). TFBUT for eyes with a STT <15 mm/min (6.5 ± 4.4 seconds) was significantly shorter than for eyes with a STT ≥ 15 mm/min (16.1 ± 3.6 seconds) (P < .001). As LGS grade increased, both STT (P < .001) and TFBUT (P < .001) significantly decreased. CONCLUSIONS: A higher LGS grade was significantly associated with a lower STT and more rapid TFBUT in dogs. Lissamine green ocular dye can be considered as an adjunctive diagnostic test when evaluating tear film deficiency in dogs.

Antimicrobial activity of topical dyes used in clinical veterinary ophthalmology.

OBJECTIVE: To evaluate in vitro the antibacterial effects of fluorescein, rose bengal, and lissamine green topical ophthalmic dyes against selected Gram-positive and Gram-negative bacteria, and to evaluate whether preserved or preservative-free fluorescein solutions are able to inhibit or potentiate bacterial growth. PROCEDURES: Susceptibility testing was performed using the Kirby-Bauer disk diffusion method plated with clinical ocular isolates of Staphylococcus aureus, Staphylococcus pseudintermedius, Streptococcus spp., Escherichia coli, and Pseudomonas aeruginosa. Bacterial growth inhibition was evaluated 24 hours following the addition of commercially available fluorescein, rose bengal, and lissamine green sterile strips. Antimicrobial effectiveness testing was performed by inoculation of compounded 1% dye solutions, both with and without preservatives (fluorescein and lissamine contained thiomersal, and rose bengal contained nipagin and nepazol), with the five previously mentioned bacteria. Growth was evaluated at days 7, 14, and 28. RESULTS: All dyes showed antibacterial activity against Gram-positive organisms. Preservative-free compounded 1% fluorescein solution inhibited growth of Gram-positive organisms but not of Gram-negative organisms. Preservative-free rose bengal and lissamine green inhibited growth of both types of organisms. CONCLUSIONS: Preferably, ocular surface samples for antimicrobial culture should be taken prior to the administration of topical dyes, due to their potential antibacterial activity, particularly if undiluted strips are applied directly or commercial fluorescein solutions are used and not immediately rinsed. Ophthalmic dye solutions containing preservative are safe from bacterial growth for up to 28 days if properly handled and stored. The use of preservative-free fluorescein solutions should be avoided and preservative-free rose bengal and lissamine green should be handled carefully.

Nanomolar Potency Aminophenyltriazine CFTR Activator Reverses Corneal Epithelial Injury in a Mouse Model of Dry Eye.

Purpose: Dry eye disorders are a major health care burden. We previously reported the identification of N-methyl-N-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine [cystic fibrosis transmembrane conductance regulator (CFTR)act-K267], which activated human wild-type CFTR chloride conductance with EC50 ∼ 30 nM. Here, we report in vivo evidence for CFTRact-K267 efficacy in an experimental mouse model of dry eye using a human compatible ophthalmic vehicle. Methods: CFTR activation in mice in vivo was demonstrated by ocular surface potential difference (OSPD) measurements. Ocular surface pharmacodynamics was measured in tear fluid samples obtained at different times after topical administration of CFTRact-K267. Dry eye was produced by lacrimal duct cautery (LDC) and corneal epithelial injury and was assessed by Lissamine green (LG) staining. Results: OSPD measurements demonstrated a hyperpolarization of -8.6 ± 3 mV (standard error of the mean, 5 mice) in response to CFTRact-K267 exposure in low chloride solution that was reversed by a CFTR inhibitor. Following single-dose topical administration of 2 nmol CFTRact-K267, tear fluid CFTRact-K267 concentration was >500 nM for more than 6 h. Following LDC, corneal surface epithelial injury, as assessed by LG staining, was substantially reversed in 10 of 12 eyes receiving 2 nmol CFTRact-K267 3 times daily starting on day 2, when marked epithelial injury had already occurred. Improvement was seen in 3 of 12 vehicle-treated eyes. Conclusion: These studies provide in vivo evidence in mice for the efficacy of a topical, human use compatible CFTRact-K267 formulation in stimulating chloride secretion and reversing corneal epithelial injury in dry eye.

Comprehensive Clinical, Diagnostic, and Advanced Imaging Characterization of the Ocular Surface in Spontaneous Aqueous Deficient Dry Eye Disease in Dogs.

PURPOSE: To perform a comprehensive clinical, diagnostic, and imaging characterization of the ocular surface in West Highland White Terriers (WHWTs) diagnosed with aqueous deficient dry eye (ADDE) disease. METHODS: Six ADDE-affected and 13 ADDE-unaffected WHWT dogs were enrolled and underwent clinical assessment and disease scoring, tear osmolarity, phenol red thread test, Schirmer tear test, tear film breakup time, fluorescein staining, Rose bengal and lissamine green vital dye staining, meibometry, corneal esthesiometry, ultrasound pachymetry, optical coherence tomography, in vivo confocal microscopy, and conjunctival biopsy. Subjective assessment of their condition was provided by owner-reported surveys. RESULTS: ADDE-affected WHWT dogs had higher median clinical disease (conjunctiva: 5.75 vs. 0.00; cornea: 14.00 vs. 5.00; total: 17.50 vs. 5.00), vital staining (Rose bengal: 2.25 vs. 1.50; lissamine green: 2.00 vs. 1.00), and histologic disease (conjunctiva: 2 vs. 0) scores when compared with the controls. In addition, ADDE-affected WHWTs had significantly lower phenol red thread test (5.0 vs. 17.5, mm/15 s), Schirmer tear test (3 vs. 20, mm/min), tear film breakup time (3.6 vs. 13.9, s) values and higher area under the curve values for meibometry (394 vs. 245, meibometry units [MU]). There were no significant differences in other tear film tests performed. Advanced imaging revealed decreased tear meniscus height (optical coherence tomography) and variable pigment deposition within corneal epithelial cells (in vivo confocal microscopy). CONCLUSIONS: This comprehensive assessment of ADDE-affected WHWTs depicts the ocular surface changes associated with quantitative lacrimal gland dysfunction. Importantly, ADDE-affected WHWTs may prove a valuable naturally occurring ADDE model for investigating underlying pathophysiological mechanisms and the development of novel therapeutics.

Randomised trial of the clinical utility of an eyelid massage device for the management of meibomian gland dysfunction.

PURPOSE: To compare the single application and two week treatment effects of device-applied (Eyepeace) and manually-applied eyelid massage techniques, as an adjunct to warm compress therapy, on ocular surface and tear film parameters. METHODS: Twenty participants (11 females, 9 males; mean age, 27 ±â€¯11 years) with dry eye symptoms were recruited in a two week, investigator-masked, randomised, contralateral-eye trial. Following 10 min of warm compress therapy application (MGDRx EyeBag®) on both eyes, eyelid massage therapy was applied to one eye (randomised) by device, and to the fellow eye by manual eyelid massage, once daily for 14 days. Ocular surface and tear film measurements were conducted at baseline, and 15 min post-application by a clinician, then again after 14 days of self-administered daily treatment at home. RESULTS: Baseline clinical measurements did not differ between the treatment groups (all p > 0.05). Following two weeks of treatment, tear film lipid layer grade improved significantly with device massage (p = 0.008), and was marginally greater than manual massage by less than 1 grade (p = 0.03). Although immediate post-treatment improvements in tear film stability were observed in both groups (both p < 0.05), no significant long-term cumulative effects or inter-treatment differences in stability measures were detected (all p > 0.05). Visual acuity, tear meniscus height, conjunctival hyperaemia, ocular surface staining, and meibomian gland dropout did not change during the treatment period (all p > 0.05). CONCLUSIONS: Two weeks of treatment with the eyelid massage device, as an adjunct to warm compress therapy, effected marginally greater improvements in tear film lipid layer thickness than the conventional manual technique, which were statistically but not clinically significant. Future parallel group trials with longer treatment periods and a greater range of disease severity are required.

Bilateral Effect of the Unilateral Corneal Nerve Cut on Both Ocular Surface and Lacrimal Gland.

Purpose: This study investigated the effect of a unilateral cut of the corneal nerve on the bilateral ocular surface and tear secretory function. Methods: Seven-week-old female BALB/c mice were divided into control and nerve-cutting (NC) groups (n = 60). The left cornea was partially incised with a 2-mm circular trephine through the upper half of the stromal layer. Lissamine green corneal staining and tear volume measurements were conducted, and corneal whole-mount staining using class III ß-tubulin antibody was performed to assess corneal nerves. Flow cytometric analyses for dendritic cells (DCs), CD4+/CD8+ and regulatory T cells and ELISA for neuropeptides were performed. Results: The grading of corneal staining increased in the NC group, while the tear volume decreased over the 4 weeks. The nerve density decreased in bilateral corneas over 2 weeks. At day 14, CD11b+ or CD11c+ DCs and the mature DCs expressing CD86 or MHCII increased in bilateral cornea/conjunctiva. At day 28, CD11c+CD86hi, CD11c+MHCIIhi, Th17 and IFN-γ-secreting CD8+ T cells highly increased in bilateral draining lymph nodes. CD4+CD25hiFoxp3hi and CD8+CD25hiFoxp3hi regulatory T cells notably increased in the spleen. In ELISA, neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide were generally suppressed in the extraorbital lacrimal glands at day14. Conclusions: The unilateral corneal nerve severing resulted in activation of the immune cells on the ocular surface and dysregulated lacrimal secretion bilaterally through the bidirectional neuronal signals. It suggests that the unilateral corneal nerve damage may alter immune homeostasis and mechanistically participate in the development of bilateral inflammatory disorders such as dry eye.

The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca2+ Signaling Events During the Early Development of Zebrafish (Danio rerio).

We have visualized many of the Ca2+ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca2+ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca2+ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca2+ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca2+ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca2+ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca2+ channel antagonists, Ca2+ chelators, fluorescent dyes, RNA, and DNA.

Lid wiper epitheliopathy: The influence of multiple lid eversions and exposure time.

PURPOSE: To investigate the effect of multiple lid eversions on lid wiper epitheliopathy (LWE), along with the effect of cumulative lid exposure time and the patterns of associated staining. METHODS: The increase in area of lid wiper staining with lissamine green was compared by everting both the upper eyelids of each subject (i.e. contralateral design), with one eye being everted once for 45 s and the fellow eyelid everted three times, each time for 15 s. This pattern of contralateral eversion was repeated with a total of three eversions in one eye and nine eversions in the fellow eye, with each eye totalling 135 s cumulative exposure to eversion over about 9 min. The LWE area of staining was objectively quantified from slit lamp photography images captured at every lid eversion by 2 masked observers. Two-way repeated measures ANOVAs were used to determine the effect of number of lid eversions and cumulative exposure time on the amount of staining caused. Each image was also categorized into its primary LWE staining pattern, by a masked observer. RESULTS: The multiple eversions condition caused significantly greater LWE than the single eversion condition (p < 0.001), while cumulative exposure time did not have a significant effect on LWE (p = 0.137). Classification of the primary staining patterns revealed that with more eyelid eversions there was a shift from mostly 'no staining' to minor patterns ('short horizontal bands' and 'vertical streaks') and then to more extensive patterns ('broad horizontal bands' and 'comb-shaped'). CONCLUSIONS: The number of eyelid eversions is a confounding factor that should be controlled when investigating LWE, in particular when considering the link with dry eye or contact lens discomfort. However the cumulative exposure time did not appear to influence the LWE magnitude.