Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release.

INTRODUCTION: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds. METHODS: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1ß, ß3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury. RESULTS: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining. CONCLUSIONS: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Penetration rates into the stratum corneum layer: A novel quantitative indicator for assessing skin barrier function.

BACKGROUND: The stratum corneum (SC), the outermost layer of the skin epidermis, acts as an effective bi-directional barrier, preventing water loss (inside-outside barrier) and entry of foreign substances (outside-inside barrier). Although transepidermal water loss (TEWL) is a widely-used measure of barrier function, it represents only inside-outside protection. Therefore, we aimed to establish a non-invasive method for quantitative evaluation of the outside-inside barrier function and visually present a skin barrier model. MATERIALS AND METHODS: Skin barrier damage was induced by applying a closed patch of 1% sodium dodecyl sulfate to the forearms of eight participants; they were instructed to apply a barrier cream on a designated damaged area twice daily for 5 days. The SC barrier was evaluated by measuring TEWL and fluorescein sodium salt penetration rate before, immediately after, and 5 days after damage. The penetration rate was assessed using tape-stripping (TS) technique and fluorescence microscopy. RESULTS: The rates of fluorescein sodium salt penetration into the lower layers of SC differed significantly based on the degree of skin barrier damage. The correlation between penetration rate and TEWL was weak after two rounds of TS and became stronger after subsequent rounds. Five days after skin barrier damage, the penetration rate of all layers differed significantly between areas with and without the barrier cream application. CONCLUSION: Our findings demonstrated that the penetration rate was dependent on skin barrier conditions. The penetration rate and corresponding fluorescence images are suitable quantitative indicators that can visually represent skin barrier conditions.

Integrating Continuous Transepithelial Flux Measurements into an Ussing Chamber Set-Up.

Fluorescently labelled compounds are often employed to study the paracellular properties of epithelia. For flux measurements, these compounds are added to the donor compartment and samples collected from the acceptor compartment at regular intervals. However, this method fails to detect rapid changes in permeability. For continuous transepithelial flux measurements in an Ussing chamber setting, a device was developed, consisting of a flow-through chamber with an attached LED, optical filter, and photodiode, all encased in a light-impermeable container. The photodiode output was amplified and recorded. Calibration with defined fluorescein concentration (range of 1 nM to 150 nM) resulted in a linear output. As proof of principle, flux measurements were performed on various cell lines. The results confirmed a linear dependence of the flux on the fluorescein concentration in the donor compartment. Flux depended on paracellular barrier function (expression of specific tight junction proteins, and EGTA application to induce barrier loss), whereas activation of transcellular chloride secretion had no effect on fluorescein flux. Manipulation of the lateral space by osmotic changes in the perfusion solution also affected transepithelial fluorescein flux. In summary, this device allows a continuous recording of transepithelial flux of fluorescent compounds in parallel with the electrical parameters recorded by the Ussing chamber.

Influential Signs of Dry Eye-Related Ocular Symptoms in Participants With Unstable Tear Film.

PURPOSE: The aim of this study was to investigate the factors influencing dry eye disease (DED)-related ocular symptoms in participants with short fluorescein tear break-up time (FTBUT). METHODS: This cross-sectional study included 82 participants with short FTBUT (<10 seconds). Examinations included Ocular Surface Disease Index (OSDI), FTBUT, average noninvasive tear break-up time (NIBUTave), lid wiper epitheliopathy, lipid layer thickness, blink rate, partial blink, tear meniscus height, and meibomian gland (MG) evaluation which included ratio of residual MG area (RMGA) and MG grade in tarsal plates. One-way analysis of variance was used to detect differences between symptomatic tear film instability group (FTBUT <5 s, OSDI ≥13), asymptomatic tear film instability group (FTBUT <5 s, OSDI <13), and control group (FTBUT ≥5 s, OSDI <13). A bivariate correlation, partial correlation, and multiple linear regression analyses were used to identify major factors. Only the right eye was included. RESULTS: Among the participants with FTBUT <5 seconds, symptomatic group showed less upper RMGA ( P < 0.001) and NIBUTave ( P = 0.010). OSDI was negatively associated with upper RMGA ( r = -0.450, P < 0.001) and NIBUTave ( r = -0.414, P = 0.001), and positively associated with upper MG grade ( r = 0.277, P = 0.027). Linear regression analysis showed that the upper RMGA significantly affected OSDI (B = -41.895, P = 0.001), while not significantly correlated with age, upper MG grade, and NIBUTave. CONCLUSIONS: The upper RMGA might be the main factor affecting DED-related discomfort in participants with unstable tear film, indicating an early ocular change in DED.

Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

An enhanced model for environmental dry eye: Exploring pathological features and underlying factors.

This study aimed to develop an enhanced environmental dry eye (EDE) model that accurately reproduces the etiology of prolonged visual fatigue and investigates the underlying pathological features. A total of 40 adult SPF-grade Wistar rats were randomly assigned to control (n = 20) and model (n = 20) groups. Rats in the control group were maintained under normal conditions, while rats in the model group were exposed to a controlled frontal airflow of 2-4 m/s from a fan for 7.5 h daily while placed on a suspended cylindrical wire mesh frame. Various assessments were performed at different time points during the 14-day experiment, including blink frequency, tear secretion (phenol red thread test), tear film breakup time (BUT), fluorescein staining (FL), corneal epithelial status (light microscopy), ultrastructure of corneal epithelial cells (electron microscopy), and expression levels of inflammatory cytokines (IL-1ß, TNF-α) in tears (enzyme-linked immunosorbent assay). Additionally, mRNA and protein expression levels of MMP-9, IL1ß, IL6, TNF-α, IFN-γ, and caspase-3 in corneal tissues were quantified (real-time quantitative PCR and Western blotting). Compared to the control group, the model group rats exhibited significant decreases in blink frequency (P < 0.001), tear secretion (Schirmer I test) values (P < 0.001), and tear film breakup time levels (P < 0.001). There was also a significant increase in fluorescein staining scores (P < 0.001) in the model group. Histological examination revealed distinct differences of the corneal epithelium between groups. The corneal epithelium of the model group appeared thicker, with disorganized cell arrangement in the superficial and basal layers, partial defects or detachment of superficial epithelial cells, and a rough, uneven surface. Scanning electron microscopy observations showed a rough corneal epithelial surface with numerous cracks and scattered vesicular-like structures in the model group. Furthermore, the model group rats exhibited a significant increase in expression of IL-1ß and TNF-α in tears (P < 0.001), and upregulated expression levels of MMP-9, TNF-α, IL-1ß, caspase-3, IL-6, and IFN-γ at both the mRNA and protein levels in corneal tissues (P < 0.001). In conclusion, the modified "wire-meshing cylindrical board" model effectively overcomes the limitations of the traditional "jogging board " dry eye model and successfully simulates the etiology of prolonged visual fatigue. This innovative EDE model demonstrates a high degree of relevance to dry eye conditions resulting from prolonged visual tasks, with a high success rate of model induction. Moreover, it proves to be a simple, practical, and easily replicable model, making it highly suitable for further studies on prolonged visual fatigue and facilitating its widespread adoption in research and clinical applications.

Hypoxia-Induced miR-101 Impairs Endothelial Barrier Integrity Through Altering VE-Cadherin and Claudin-5.

Stroke is a life-threatening medical condition across the world that adversely affects the integrity of the blood-brain barrier (BBB). The brain microvascular endothelial cells are the important constituent of the BBB. These cells line the blood vessels and form a semipermeable barrier. Disruptions in adherens junction and tight junction proteins of brain microvascular endothelial cells compromise the integrity of BBB. The Vascular Endothelial (VE)-cadherin is an integral adherens junction protein required for the establishment and maintenance of the endothelial barrier integrity. This study aims to investigate the role of miRNA in hypoxia-induced endothelial barrier disruption. In this study, brain endothelial cells were exposed to hypoxic conditions for different time points. Western blotting, overexpression and knockdown of miRNA, real-time PCR, TEER, and sodium fluorescein assay were used to examine the effect of hypoxic conditions on brain endothelial cells. Hypoxic exposure was validated using HIF-1α protein. Exposure to hypoxic conditions resulted to a significant decrease in endothelial barrier resistance and an increase in sodium fluorescein migration across the endothelial barrier. Reduction in endothelial barrier resistance demonstrated compromised barrier integrity, whereas the increase in migration of sodium fluorescein across the barrier indicated the increase in barrier permeability. The present study revealed microRNA-101 decreases the expression of VE-cadherin and claudin-5 in brain endothelial cells exposed to the hypoxic conditions.

Role of Inhibiting Inflammation of LC3-Associated Phagocytosis in Dry Eye Disease.

PURPOSE: To confirm the expression and investigate the role of LC3-associated phagocytosis (LAP) in dry eye disease (DED). METHODS: The DED model of mice was established by scopolamine subcutaneous injection in a low-humidity environment chamber. Tear secretion test and corneal fluorescein sodium staining were used to evaluate the severity of DED. Expression levels of Rubicon, microtubule-associated protein light chain 3-II (LC3-II), Beclin-1 and autophagy-related gene-7 (Atg-7) in corneas of mice with DED were tested by western blot. Cell Counting Kit-8 (CCK-8) assay was used to detect the effects of different concentrations of hypertonic solutions on the proliferation activity of human corneal epithelial cells (HCECs). The expression levels of Dectin-1, IL-6 and IL-1ß in HCECs after stimulation with different concentrations of hypertonic solutions were tested. The expressions of Rubicon, LC3-II, Beclin-1 and ATG-7 in HCECs were detected by reverse transcription polymerase chain reaction (RT-PCR). After being pretreated with 10 µM si-Rubicon, the severity of the disease was documented by corneal fluorescein sodium staining. And the expression levels of IL-6 and IL-1ß were also tested by RT-PCR. RESULTS: Compared with the normal control group, the corneal fluorescein sodium staining scores and tear secretion were significantly reduced. Rubicon, LC3-II, Beclin-1 and ATG-7 were significantly elevated. CCK-8 showed that the 400 and 450 mOsM hypertonic solutions did not affect the proliferation activity of HCECs. The expression of Dectin-1, IL-1ß and IL-6 were elevated after stimulation with 450 mOsM solution. LC3-II, Rubicon, ATG-7 and Beclin-1 increased after stimulation with 450 mOsM hyperosmolar solution in HCECs. Corneal fluorescein staining showed that si-Rubicon increased the severity of DED in mice. Moreover, the mRNA expressions of inflammatory factors IL-1ß and IL-6 in the cornea of mice were significantly increased. CONCLUSION: DED increased the expression of proteins associated with LAP. LAP could play an anti-inflammatory effect in DED.

Fluorescein staining of chloroplast starch granules in living plants.

Chloroplast starch granules (cpSGs) store energy harvested through photosynthesis in plants, and cpSG dynamics have important roles in plant energy metabolism and stress responses. To date, cpSGs have been visualized using several methods, such as iodine staining; however, no method can be used to specifically visualize cpSGs in living cells from various plant species. Here, we report a simple method to visualize cpSGs in living plant cells in various species by staining with fluorescein, a commonly used fluorescent dye. We show that fluorescein is taken up into chloroplasts and interacts with cpSGs similarly to iodine. Fluorescein also interacts with refined starch in vitro. Using a fluorescein derivative for ultrabright cpSG imaging, we produced high-quality 3D reconstructions of cpSGs and evaluated their accumulation in multiple plant species. As fluorescein is well known and readily purchasable, our fluorescein-based staining method should contribute to all research regarding starch.

Atmospheric pollutant black carbon induces ocular surface damage in mice.

The threats of air pollution to human health have been gradually discovered, including its effects on eyes. The purpose of the study is to investigate the potential correlation between ocular surface exposure to black carbon and ocular surface structural damage as well as tear film dysfunction. To achieve this goal, 60 6-8-week-aged male BALB/C mice were randomly divided into 4 groups (n = 15). 0.5 mg/ml (group A), 1 mg/ml (group B), 5 mg/ml (group C) black carbon suspension droplets and PBS solution (group D) were used in the right eyes, 4 µl per time of three times per day. Tear break-up time, corneal fluorescein staining scores, and tear volume were assessed before treatment (day 0) and on days 4, 7, 10, and 14 after treatment. On day 14, the mice were sacrificed, and corneal and conjunctival tissues were collected for histological analysis. As the exposure time increased, there were no significant changes in the measured parameters from PBS-treated group of mice (P > 0.05). However, in the black carbon-treated group, there were significant decreases in tear film break-up time, significant increases in corneal fluorescein staining scores, and significant reductions in tear secretion (all P < 0.05). After 14 days, H&E staining of the corneal epithelium showed that in the PBS-treated group of mice, the corneal epithelial cells were neatly arranged, with no inflammatory cell infiltration, while in the black carbon-treated group, the corneal epithelium was significantly thickened, the basal cell arrangement was disrupted, the number of cell layers increased, and there was evidence of inflammatory cell infiltration. In the ultrastructure of the corneal epithelium, it could be observed that the black carbon-treated group had an increased amount of corneal epithelial cell detachment compared to the PBS-treated group, at the same time, the intercellular connections were looser, and there was a decrease in the number of microvilli and desmosomes in the black carbon-treated group. The results indicate that the ocular surface exposure to black carbon can result in a decrease in tear film stability and tear secretion in mice. Moreover, it can induce alterations in the corneal structure.

PFT-α protects the blood-brain barrier through the Wnt/ß-catenin pathway after acute ischemic stroke.

The dysfunction of blood-brain barrier (BBB) plays a pivotal role in brain injury and subsequent neurological deficits of ischemic stroke. The current study aimed to examine the potential correlation between p53 inhibition and the neuroprotective effect of on the BBB. Rat middle cerebral artery occlusion and reperfusion model (MCAO/R) and oxygen-glucose deprivation/re-oxygenation model (OGD/R) were employed to simulate cerebral ischemia-reperfusion (CI/R) injury occurrence in vivo and in vitro. mNSS and TTC staining were applied to evaluate neurological deficits and brain infarct volumes. Evans blue (EB) staining was carried out to examine the permeability of BBB. RT-qPCR and Western blot to examine the mRNA and protein levels. Cell viabilities were detected by CCK-8. Flow cytometry and ELISA assay were employed to examine apoptosis and neuroinflammation levels. TEER value and sodium fluorescein were carried out to explore the permeability of HBMEC cells. PFT-α inhibited P53 and promoted the expression of ß-catenin and cyclin D1, which were reversed by DKK1. PFT-α inhibited neurological deficits, brain infarct volume, neuroinflammation, apoptosis, and BBB integrity than the MCAO/R rats; however, this inhibition was reversed by DKK1. PFT-α promoted OGD/R-induced cell viability in NSCs, and suppressed inflammation and apoptosis, but DKK1 weakened the effect of PFT-α. PFT-α increased OGD/R-induced TEER values in cerebrovascular endothelial cells, inhibited sodium fluorescein permeability, and increased the mRNA levels of tight junction protein, but they were all attenuated by DKK1. PFT-α protects the BBB after acute ischemic stroke via the Wnt/ß-catenin pathway, which in turn improves neurological function.

Bacterial pathogens deliver water- and solute-permeable channels to plant cells.

Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells1,2. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants3-5. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria6. These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a ß-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Changes caused by fluorescein in the tear film evaluated with hybrid break-up time test as a new method - Part Two: Its effect on breakup locations and other quantitative values.

OBJECTIVES: To examine the changes caused by fluorescein in the tear film by analyzing the qualitative parameters such as breakup location and also detailed quantitative parameters. METHODS: After determining the break-up time (BUT) value and breakup locations by the Non-invasive break-up time (NI-BUT) method, we re-evaluated the changes in the tear film stained with fluorescein using the topographical method. We called the topographic evaluation of the tear film stained with fluorescein as the Hybrid-BUT test. The results for the parameters obtained for each participant by the NI-BUT and Hybrid-BUT tests were compared. RESULTS: Our study was conducted with 82 participants aged between 18 and 58 years (mean age 34.1 ± 11.1). The mean first break-up time value (BUT1) was 4.1 ± 2.7 s on the NI-BUT test versus 5.1 ± 3.2 s on the Hybrid-BUT test (p = 0.029). The mean average of all break-up time values (BUTAvg) for each participant was 7.2 ± 3.2 s on the NI-BUT test versus 8.4 ± 3.1 s on the Hybrid-BUT test (p = 0.004). After dividing the corneal surface into quadrants of 90°, there was no significant difference in the comparison of the locations of the first breakup (QUAD(First breakup)), the second breakup (QUAD(2nd breakup)) and the third breakup (QUAD(3rd breakup)) between the two tests (p>0.05). CONCLUSIONS: Fluorescein affects quantitative values rather than qualitative parameters in tear film. We observed that the change caused by fluorescein in tear film break-up time could be detected objectively and in a documented manner using Hybrid-BUT test.

Dietary Alcohol Consumption Elicits Corneal Toxicity Through the Generation of Cellular Oxidative Stress.

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.

Transdermal on-demand drug delivery based on an iontophoretic hollow microneedle array system.

Transdermal drug delivery has emerged as an alternative administration route for therapeutic drugs, overcoming current issues in oral and parenteral administration. However, this technology is hindered by the low permeability of the stratum corneum of the skin. In this work, we develop a synergic combination of two enhancing technologies to contribute to an improved and on-demand drug delivery through an iontophoretic system coupled with hollow microneedles (HMNs). For the first time, a polymeric HMN array coupled with integrated iontophoresis for the delivery of charged molecules and macromolecules (e.g. proteins) is devised. To prove the concept, methylene blue, fluorescein sodium, lidocaine hydrochloride, and bovine serum albumin-fluorescein isothiocyanate conjugate (BSA-FITC) were first tested in an in vitro setup using 1.5% agarose gel model. Subsequently, the ex vivo drug permeation study using a Franz diffusion cell was conducted, exhibiting a 61-fold, 43-fold, 54-fold, and 17-fold increment of the permeation of methylene blue, fluorescein sodium, lidocaine hydrochloride, and BSA-FITC, respectively, during the application of 1 mA cm-2 current for 6 h. Moreover, the total amount of drug delivered (i.e. in the skin and receptor compartment) was analysed to untangle the different delivery profiles according to the types of molecule. Finally, the integration of the anode and cathode into an iontophoretic hollow microneedle array system (IHMAS) offers the full miniaturisation of the concept. Overall, the IHMAS device provides a versatile wearable technology for transdermal on-demand drug delivery that can improve the administration of personalised doses, and potentially enhance precision medicine.

Changes in the meibomian glands in postmenopausal women with primary acquired nasolacrimal duct obstruction: a prospective study.

BACKGROUND: Primary acquired nasolacrimal duct obstruction (PANDO) is frequently encountered in perimenopausal women, causing tear flow stagnation and resulting in a variety of ocular discomfort symptoms. However, little is known about the alterations in the meibomian gland in postmenopausal women with PANDO. Hence, this study investigated the changes in the meibomian gland and ocular surface in postmenopausal women with PANDO. METHODS: This prospective study included 60 eyes of 60 postmenopausal women with PANDO (PANDO group) and 30 eyes of 30 postmenopausal women without PANDO (control group). The PANDO group was further subdivided into incomplete and complete PANDO groups, based on the degree of nasolacrimal duct obstruction. The patients' symptoms were evaluated using the ocular surface disease index questionnaire. The meibomian gland and ocular surface were assessed using the Keratograph 5 M. Other ophthalmologic examinations included the tear break-up time, corneal fluorescein staining, meibomian gland expression, and Schirmer I test. The correlations between the degree of nasolacrimal duct obstruction and other metrics were analyzed. RESULTS: The loss ratio of the upper eyelid was greater in the incomplete PANDO group than in the control group (p = 0.023). Meibomian gland distortion of the upper eyelid was more severe in the control group than in the complete PANDO group (p = 0.022). The non-invasive tear meniscus height was greater, whereas the intensity of corneal fluorescein staining was lower in the PANDO group than in the control group (all p < 0.05). The degree of nasolacrimal duct obstruction was positively associated with the non-invasive tear meniscus height and ocular surface disease index scores (p < 0.001 and p < 0.001, respectively). Corneal fluorescein staining and meibomian gland distortion of the upper eyelid were negatively correlated with the degree of nasolacrimal duct obstruction (p = 0.01 and p = 0.007, respectively). CONCLUSION: Postmenopausal women with PANDO exhibit significant morphological changes in the meibomian gland. More attention should be paid to meibomian gland loss in postmenopausal women with incomplete PANDO, as it is crucial for identifying meibomian gland impairments in patients with PANDO.

Transcorneal but not transpalpebral electrical stimulation disrupts mucin homeostasis of the ocular surface.

PURPOSE: Transcorneal electrical stimulation (TcES) is increasingly applied as a therapy for preserving and improving vision in retinal neurodegenerative and ischemic disorders. However, a common complaint about TcES is its induction of eye pain and dryness in the clinic, while the mechanisms remain unknown. METHOD: TcES or transpalpebral ES (TpES) was conducted in C57BL6j mice for 14 days. The contralateral eyes were used as non-stimulated controls. Levels of intracellular [Ca2+] ([Ca2+]i) were assessed by Fura-2AM. The conductance resistances of the eye under various ES conditions were measured in vivo by an oscilloscope. RESULTS: Although TcES did not affect tear production, it significantly induced damage to the ocular surface, as revealed by corneal fluorescein staining that was accompanied by significantly decreased mucin (MUC) 4 expression compared to the control. Similar effects of ES were detected in cultured primary corneal epithelium cells, showing decreased MUC4 and ZO-1 levels after the ES in vitro. In addition, TcES decreased secretion of MUC5AC from the conjunctiva in vivo, which was also corroborated in goblet cell cultures, where ES significantly attenuated carbachol-induced [Ca2+]i increase. In contrast to TcES, transpalpebral ES (TpES) did not induce corneal fluorescein staining while significantly increasing tear production. Importantly, the conductive resistance from orbital skin to the TpES was significantly smaller than that from the cornea to the retina in TcES. CONCLUSION: TcES, but not TpES, induces corneal epithelial damage in mice by disrupting mucin homeostasis. TpES thus may represent a safer and more effective ES approach for treating retinal neurodegeneration clinically.

Relationship between dry eye and expressions of CXCR3 and CCR5 after ocular acid burn.

OBJECTIVE: To investigate the manifestation of dry eye and its relationship with CXCR3 and CCR5 expression in patients with ocular acid burns. METHODS: This is a case-control study. A total of 27 eyes of 22 cases ocular with acid burns of I-V degrees from Jan.2020 to Feb.2021 in Jinshan Hospital of Fudan University were selected as observation group, and 8 eyes of 8 cases of normal people were selected as control group. The follow-up period was 3 months. The visual acuity, intraocular pressure (IOP), corneal fluorescein staining scores (CFS), breakup time of tear film (BUT), Schirmer I test, corneal thickness and tear meniscus height (TMH) were observed at 1 day, 1 and 3 months after injury. The protein expressions of CXCR3 and CCR5 were examined by ELISA and compared among groups at each time point. RESULTS: BUT and Schirmer I tests value in the observation group were lower than those in the control group 3 months after injury (BUT: Group I ~ IV p = 0.0266, p = 0.0222, p = 0.0003, p = 0.0059, respectively; Schirmer I test: Group I ~ IV p = 0.0027, p = 0.0033, p = 0.0016, p = 0.0032, respectively). CFS scores were higher than those in the control group at 1 day after injury (all p < 0.0001), but decreased gradually at 1 and 3 months after injury (Group I ~ IV p = 0.0042, p = 0.0096, p < 0.0001, p < 0.0001, respectively). The corneal thickness and TMH 1 day after injury were higher than those in the control group (corneal thickness: Group II ~ IV p = 0.0010, p < 0.0001, p < 0.0001, respectively; TMH: Group II ~ IV p = 0.0002, p < 0.0001, p < 0.0001, respectively), and also higher than those at 1 month and 3 months after injury (corneal thickness: Group II ~ IV p = 0.0010, p < 0.0001, p < 0.0001, respectively; TMH: Group II ~ IV p = 0.0345 and p = 0.0045, p = 0.0005 and p < 0.0001, p = 0.0114 and p = 0.0019, respectively). The expression levels of CXCR3 and CCR5 protein were significantly negatively correlated with BUT (all p < 0.0001), and CXCR3 and CCR5 were also significantly negatively correlated with Schirmer I test value (p < 0.0001, p = 0.0004, respectively). CONCLUSION: Ocular acid burns can cause dry eye, and the expression of CXCR3 and CCR5 protein in tears may be related to the occurrence of dry eye after ocular acid burn.

Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection.

BACKGROUND: Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host-pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction. METHODS: We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection. RESULTS: Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection. CONCLUSIONS: Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting.