Comprehensive Grading System for Experimental Autoimmune Uveitis in Mice.

Experimental autoimmune uveitis (EAU) is the most commonly used animal model to study the progression of chronic uveitis and to test various therapies to treat the disease. However, to accurately evaluate the effectiveness of such treatments, a grading system that combines the latest imaging techniques with definitive quantitative grading thresholds is required. This study aimed to develop a comprehensive grading system that objectively evaluates EAU progression in C57BL/6J mice. EAU was induced following immunisation with interphotoreceptor retinoid-binding protein (IRBP) and pertussis toxin. Weekly fundus and optical coherence tomography (OCT) images were acquired over 12 weeks using a Micron IV imaging system. Each mouse was graded (between 0 to 4) based on changes seen on both the fundus (optic disc, retinal blood vessels and retinal tissue) and OCT (vitreous and retinal layers) images. A total EAU response (with a maximum score of 48) was calculated for each mouse based on the sum of the individual scores each week. Analysis of the clinical scores depicted a gradual increase in inflammatory signs including optic disc and vascular swelling, leukocyte infiltration in the vitreous, lesions in the retina and formation of granulomas and hyper-reflective foci in the retinal layers in EAU mice, with most signs reaching a plateau towards the end of the study period. Development of these signs into sight-threatening complications such as optic disc atrophy, structural damage to the retina and subretinal oedema were noted in 80-90% of mice suggesting consistent disease induction. Overall, a comprehensive and objective grading system encompassing all pathologies occurring in EAU mice was developed to enhance the preclinical evaluation of novel uveitis treatments.

NLRP3 Inflammasome as a Potential Therapeutic Target in Dry Eye Disease.

Dry eye disease (DED) is a multifactorial ocular surface disorder arising from numerous interrelated underlying pathologies that trigger a self-perpetuating cycle of instability, hyperosmolarity, and ocular surface damage. Associated ocular discomfort and visual disturbance contribute negatively to quality of life. Ocular surface inflammation has been increasingly recognised as playing a key role in the pathophysiology of chronic DED. Current readily available anti-inflammatory agents successfully relieve symptoms, but often without addressing the underlying pathophysiological mechanism. The NOD-like receptor protein-3 (NLRP3) inflammasome pathway has recently been implicated as a key driver of ocular surface inflammation, as reported in pre-clinical and clinical studies of DED. This review discusses the intimate relationship between DED and inflammation, highlights the involvement of the inflammasome in the development of DED, describes existing anti-inflammatory therapies and their limitations, and evaluates the potential of the inflammasome in the context of the existing anti-inflammatory therapeutic landscape as a therapeutic target for effective treatment of the disease.

Retinal inner nuclear layer thickness in the diagnosis of cognitive impairment explored using a C57BL/6J mouse model.

Major neurocognitive disorder (NCD) affects over 55 million people worldwide and is characterized by cognitive impairment (CI). This study aimed to develop a non-invasive diagnostic test for CI based upon retinal thickness measurements explored in a mouse model. Discrimination indices and retinal layer thickness of healthy C57BL/6J mice were quantified through a novel object recognition test (NORT) and ocular coherence tomography (OCT), respectively. Based on criteria from the Diagnostic and statistical manual of mental disorders 5th ed. (DSM-V), a diagnostic test was generated by transforming data into rolling monthly averages and categorizing mice into those with and without CI and those with a high or low decline in retinal layer thickness. Only inner nuclear layer thickness had a statistically significant relationship with discrimination indices. Furthermore, our diagnostic test was 85.71% sensitive and 100% specific for diagnosing CI, with a positive predictive value of 100%. These findings have potential clinical implications for the early diagnosis of CI in NCD. However, further investigation in comorbid mice and humans is warranted.

The central role of the NLRP3 inflammasome pathway in the pathogenesis of age-related diseases in the eye and the brain.

With increasing age, structural changes occur in the eye and brain. Neuronal death, inflammation, vascular disruption, and microglial activation are among many of the pathological changes that can occur during ageing. Furthermore, ageing individuals are at increased risk of developing neurodegenerative diseases in these organs, including Alzheimer's disease (AD), Parkinson's disease (PD), glaucoma and age-related macular degeneration (AMD). Although these diseases pose a significant global public health burden, current treatment options focus on slowing disease progression and symptomatic control rather than targeting underlying causes. Interestingly, recent investigations have proposed an analogous aetiology between age-related diseases in the eye and brain, where a process of chronic low-grade inflammation is implicated. Studies have suggested that patients with AD or PD are also associated with an increased risk of AMD, glaucoma, and cataracts. Moreover, pathognomonic amyloid-ß and α-synuclein aggregates, which accumulate in AD and PD, respectively, can be found in ocular parenchyma. In terms of a common molecular pathway that underpins these diseases, the nucleotide-binding domain, leucine-rich-containing family, and pyrin domain-containing-3 (NLRP3) inflammasome is thought to play a vital role in the manifestation of all these diseases. This review summarises the current evidence regarding cellular and molecular changes in the brain and eye with age, similarities between ocular and cerebral age-related diseases, and the role of the NLRP3 inflammasome as a critical mediator of disease propagation in the eye and the brain during ageing.

Orally Delivered Connexin43 Hemichannel Blocker, Tonabersat, Inhibits Vascular Breakdown and Inflammasome Activation in a Mouse Model of Diabetic Retinopathy.

Diabetic retinopathy (DR), a microvascular complication of diabetes, is associated with pronounced inflammation arising from the activation of a nucleotide-binding and oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome. Cell culture models have shown that a connexin43 hemichannel blocker can prevent inflammasome activation in DR. The aim of this study was to evaluate the ocular safety and efficacy of tonabersat, an orally bioavailable connexin43 hemichannel blocker, to protect against DR signs in an inflammatory non-obese diabetic (NOD) DR mouse model. For retina safety studies, tonabersat was applied to retinal pigment epithelial (ARPE-19) cells or given orally to control NOD mice in the absence of any other stimuli. For efficacy studies, either tonabersat or a vehicle was given orally to the inflammatory NOD mouse model two hours before an intravitreal injection of pro-inflammatory cytokines, interleukin-1 beta, and tumour necrosis factor-alpha. Fundus and optical coherence tomography images were acquired at the baseline as well as at 2- and 7-day timepoints to assess microvascular abnormalities and sub-retinal fluid accumulation. Retinal inflammation and inflammasome activation were also assessed using immunohistochemistry. Tonabersat did not have any effect on ARPE-19 cells or control NOD mouse retinas in the absence of other stimuli. However, the tonabersat treatment in the inflammatory NOD mice significantly reduced macrovascular abnormalities, hyperreflective foci, sub-retinal fluid accumulation, vascular leak, inflammation, and inflammasome activation. These findings suggest that tonabersat may be a safe and effective treatment for DR.

Extracellular Vesicles as Biomarkers and Therapeutics for Inflammatory Eye Diseases.

Extracellular vesicles (EVs) are a group of cell-derived membrane vesicles of varying sizes that can be secreted by most cells. Depending on the type of cell they are derived from, EVs may contain a variety of cargo including proteins, lipids, miRNA, and DNA. Functionally, EVs play important roles in physiological and pathological processes through intercellular communication. While there has already been significant literature on the involvement of EVs in neurological and cardiovascular disease as well as cancer, recent evidence suggests that EVs may also play a role in mediating inflammatory eye diseases. This paper summarizes current advancements in ocular EV research as well as new ways by which EVs may be utilized as novel biomarkers of or therapeutics for inflammatory eye diseases.

Characterization of NLRP3 Inflammasome Activation in the Onset of Diabetic Retinopathy.

The aim of this study was to characterize the role of nucleotide-binding oligomerization domain- (NOD-) like receptor (NLR) protein 3 (NLRP3) inflammasome activation in the onset of diabetic retinopathy (DR) using retina and vitreous from donors without diabetes mellitus (CTL), with diabetes mellitus alone (DM), and with DR. Retinal expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the key markers of retinal inflammation, connexin43 (Cx43) which is involved in upstream inflammasome regulation, as well as NLRP3 and cleaved caspase-1, the main markers of inflammasome activation, were evaluated using immunohistochemistry and Western blotting. Vitreous interleukin (IL)-1ß and IL-18, biomarkers of the activated inflammasome, were measured using a Luminex multiplex assay. Results showed a significant increase in the number and size of Iba-1+ cells and NLRP3 expression in DM, while a significant increase in GFAP, Cx43, cleaved caspase-1 and vitreous IL-18, as well as a further increase in Iba-1 and NLRP3 was found in DR. This suggests that the inflammasome is already primed in DM before its activation in DR. Furthermore, IL-18 may act as the major effector of inflammasome activation in DR while nuclear translocation of cleaved caspase-1 may play a role in gene transcription contributing to DR onset.

Correlation between the progression of diabetic retinopathy and inflammasome biomarkers in vitreous and serum - a systematic review.

Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway has been implicated in Diabetic retinopathy (DR) pathogenesis, but its impact on DR development and progression remains unclear. Therefore, the primary aim of this systematic literature review was to determine the role of the inflammasome in DR development. Furthermore, the secondary aim was to determine whether systemic inflammasome activity can be used to predict DR progression. Studies measuring vitreous and/or serum inflammasome biomarkers in DR patients with Type 2 Diabetes Mellitus (T2DM) were searched systematically using online databases EMBASE, PubMed and Web of Science with the last search conducted on 29th of September 2021. The risk of bias was assessed using the Newcastle Ottawa Scale and 20 studies were eligible for narrative analysis. Limitations included the heterogeneity in detection assays used, the small and uneven sample size, a lack of vitreous data in earlier disease stages, and not accounting for patients with other systemic co-morbidities. Analysis showed that inflammasome biomarkers IL-1ß and IL-18 increased significantly from non-proliferative DR to proliferative DR in both vitreous and serum, suggesting the inflammasome pathway is activated as DR progresses and that serum inflammasome levels could be explored as potential biomarkers for DR progression.

The NLRP3 inflammasome in age-related eye disease: Evidence-based connexin hemichannel therapeutics.

The inflammasome pathway is a fundamental component of the innate immune system, playing a key role especially in chronic age-related eye diseases (AREDs). The inflammasome is of particular interest because it is a common disease pathway that once instigated, can amplify and perpetuate itself leading to chronic inflammation. With aging, it becomes more difficult to shut down inflammation after an insult but the common pathway means that a shared solution may be feasible that could be effective across multiple disease indications. This review focusses on the NLRP3 inflammasome, the most studied and characterized inflammasome in the eye. It describes the two-step signalling required for NLRP3 inflammasome complex activation, and provides evidence for its role in AREDs. In the final section, the article gives an overview of potential NLRP3 inflammasome targeting therapies, before presenting evidence for connexin hemichannel regulators as upstream blockers of inflammasome activation. These have shown therapeutic efficacy in multiple ocular disease models.

Blocking connexin43 hemichannels prevents TGF-ß2 upregulation and epithelial-mesenchymal transition in retinal pigment epithelial cells.

Epithelial-mesenchymal transition (EMT) occurs when polarised epithelial cells change to a mesenchymal phenotype. EMT plays a role in several chronic conditions, including ocular diseases with retinal pigment epithelium (RPE) EMT associated with retinal diseases such as diabetic retinopathy (DR). Here, EMT results in breakdown of the blood-retinal barrier (BRB) leading to sub-retinal fluid deposition and retinal detachment. Previous studies have shown that blocking connexin43 (Cx43) hemichannels can protect against RPE BRB breakdown, but the underlying mechanism is unknown. To determine whether open Cx43 hemichannels may enable EMT of RPE cells and thus result in BRB breakdown, ARPE-19 cells were either challenged with high glucose plus the inflammatory cytokines IL-1ß and TNF-α (HG + Cyt) to simulate DR or treated with the Cx43 hemichannel blocker tonabersat alongside the HG + Cyt challenge. HG + Cyt induced a morphological change in RPE cells to a fibroblastic phenotype with a corresponding decrease in epithelial zonular occludens-1 and an increase in the fibroblastic marker α-SMA. The HG + Cyt challenge also induced loss of transepithelial electrical resistance while increasing dye passage between RPE cells. All of these changes were significantly reduced with tonabersat treatment, which also prevented HG + Cyt-induced transforming growth factor-ß2 (TGF-ß2) release. In conclusion, Cx43 hemichannel block with tonabersat attenuated both TGF-ß2 release and RPE EMT under disease-mimicking conditions, offering the potential to ameliorate the progression of EMT-associated retinal diseases.

Cell transdifferentiation in ocular disease: Potential role for connexin channels.

Cell transdifferentiation is the conversion of a cell type to another without requiring passage through a pluripotent cell state, and encompasses epithelial- and endothelial-mesenchymal transition (EMT and EndMT). EMT and EndMT are well defined processes characterized by a loss of epithelial/endothelial phenotype and gain in mesenchymal spindle shaped morphology, which results in increased cell migration and decreased apoptosis and cellular senescence. Such cells often develop invasive properties. Physiologically, these processes may occur during embryonic development and can resurface, for example, to promote wound healing in later life. However, they can also be a pathological process. In the eye, EMT, EndMT and cell transdifferentiation have all been implicated in development, homeostasis, and multiple diseases affecting different parts of the eye. Connexins, constituents of connexin hemichannels and intercellular gap junctions, have been implicated in many of these processes. In this review, we firstly provide an overview of the molecular mechanisms induced by transdifferentiation (including EMT and EndMT) and its involvement in eye diseases. We then review the literature for the role of connexins in transdifferentiation in the eye and eye diseases. The evidence presented in this review supports the need for more studies into the therapeutic potential for connexin modulators in prevention and treatment of transdifferentiation related eye diseases, but does indicate that connexin channel modulation may be an upstream and unifying approach for regulating these otherwise complex processes.

Blocking the inflammasome: A novel approach to treat uveitis.

Uveitis is a complex ocular inflammatory disease often accompanied by bacterial or viral infections (infectious uveitis) or underlying autoimmune diseases (non-infectious uveitis). Treatment of the underlying infection along with corticosteroid-mediated suppression of acute inflammation usually resolves infectious uveitis. However, to develop more effective therapies for non-infectious uveitis and to better address acute inflammation in infectious disease, an improved understanding of the underlying inflammatory pathways is needed. In this review, we discuss the disease aetiology, preclinical in vitro and in vivo uveitis models, the role of inflammatory pathways, as well as current and future therapies. In particular, we highlight the involvement of the inflammasome in the development of non-infectious uveitis and how it could be a future target for effective treatment of the disease.

Characterization of a Novel Human Organotypic Retinal Culture Technique.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants. After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, Müller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium. H&E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved Müller cell integrity. The Luminex assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h. We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

Effect of therapeutic UVC on corneal DNA: Safety assessment for potential keratitis treatment.

PURPOSE: Antimicrobial ultraviolet C (UVC) has proven efficacy in vitro against keratitis isolates and has potential to treat corneal infection if safety can be confirmed. METHOD: Safety of 265 nm, 1.93 mW/cm2 intensity UVC (15-300 s exposures) was investigated in vitro via cyclobutane pyrimidine dimer (CPD) formation in DNA of human cultured corneal epithelial cells; ex vivo, by evaluating UVC transmissibility as a function of porcine corneal thickness; and in vivo, by evaluating CPD induction in the mouse cornea following UVC exposure. RESULTS: A single exposure of 15 s UVC did not induce significant CPD formation (0.92 ± 1.45%) in vitro relative to untreated control (p = 0.93) whereas 300 s exposure caused extensive CPD formation (86.8 ± 13.73%; p < 0.0001). Cumulative exposure to 15 s UVC daily for 3 days induced more CPD (14.6 ± 8.2%) than a single equivalent 45 s exposure (8.3 ± 4.0%) (p < 0.001) but levels returned to baseline within 72 h (p = 0.29), indicating highly efficient DNA repair. Ex vivo, UVC transmission decreased sharply with increasing corneal thickness, confirming UVC effects are limited to the superficial corneal layers. In vivo evaluations demonstrated no detectable CPD after three consecutive daily 15 s UVC exposures, whereas a single 300 s exposure induced extensive CPD formation in superficial corneal epithelium. CONCLUSION: Up to three daily doses of 15 s UVC, in vivo, appear safe with respect to CPD formation. Ongoing research exploring UVC as a possible treatment for microbial keratitis is warranted.

Connexin therapeutics: blocking connexin hemichannel pores is distinct from blocking pannexin channels or gap junctions.

Compounds that block the function of connexin and pannexin protein channels have been suggested to be valuable therapeutics for a range of diseases. Some of these compounds are now in clinical trials, but for many of them, the literature is inconclusive about the molecular effect on the tissue, despite evidence of functional recovery. Blocking the different channel types has distinct physiological and pathological implications and this review describes current knowledge of connexin and pannexin protein channels, their function as channels and possible mechanisms of the channel block effect for the latest therapeutic compounds. We summarize the evidence implicating pannexins and connexins in disease, considering their homeostatic versus pathological roles, their contribution to excesive ATP release linked to disease onset and progression.

Connexin43 hemichannel block inhibits NLRP3 inflammasome activation in a human retinal explant model of diabetic retinopathy.

Diabetic retinopathy (DR), the most common ocular complication associated with diabetes, is a chronic vascular and inflammatory disease that leads to vision loss. The inflammasome pathway, a key part of the innate immune system, is required to activate chronic inflammation in DR. Unfortunately, current therapies for DR target pathological signs that are downstream of the inflammasome pathway, making them only partly effective in treating the disease. Using in vitro and in vivo DR models, it was discovered that connexin43 hemichannel blockers can inhibit activation of the inflammasome pathway. However, those studies were conducted using in vitro cell culture and in vivo animal disease models that are predictive but do not, of course, like any model, completely replicate the human condition. Here, we have developed an addition to our armamentarium of useful models, an ex vivo human organotypic retinal culture model of DR by exposing human donor retinal explants to a combination of high glucose (HG) and pro-inflammatory cytokines, interleukin-1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α). We hypothesized that in this model, connexin43 hemichannel block would protect against NLRP3 inflammasome complex assembly which would in turn decrease signs of inflammation characteristic of DR. To test our hypothesis, molecular changes in the inflammatory and inflammasome pathway were assessed using immunohistochemistry and a Luminex cytokine release assay. Our results showed that the human retinal explant DR model was associated with increased inflammation and activation of the inflammasome pathway, characteristic of the human condition. Furthermore, we showed that by blocking connexin43 hemichannels with the hemichannel modulator, tonabersat, we were able to prevent NLRP3 inflammasome complex assembly, Müller cell activation, as well as release of pro-inflammatory cytokines and VEGF. This further supports the possible use of connexin43 hemichannel blockers as potential new therapies for DR.

The influence of hyperglycemia on the safety of ultrasound in retinal pigment epithelial cells.

Ultrasound (US) assisted drug delivery is receiving interest in treating posterior eye diseases, such as diabetic retinopathy due to its ability to maximize drug penetration into difficult to reach tissues. Despite its promise, the technique has only been investigated using healthy cell and tissue models, with no evidence to date about its safety in active disease. As a result, the aim of this study was to evaluate the safety of US administration in vitro in retinal pigment epithelial cells under normal and high glucose conditions. US protocols within the presently accepted safety threshold were applied and their influence on cell membrane and tight junction integrity as well as intracellular inflammation was evaluated using lactate dehydrogenase (LDH), zona occludens-1 (ZO-1), fluorescein isothiocyanate (FITC)-dextran dye leak and nuclear factor-kappaB (NF-κB) assays, respectively. Under high glucose conditions, US application increased LDH release and resulted in loss of ZO-1 labeling at 2 h; however, normal levels were restored within 24 h. US within its safety parameters did not induce any FITC-dextran dye leak or NF-κB nuclear translocation in normal or high glucose conditions. In conclusion, our results suggest that while high glucose conditions increase cell susceptibility to US-mediated stress, basal conditions can be restored within 24 h without long-lasting cell damage.

Characterization of Zinc Oxide Nanoparticle Cross-Linked Collagen Hydrogels.

Collagen is the most abundant protein in mammals and possesses high biocompatibility and low antigenicity. These biological properties render it one of the most useful biomaterials for medical applications. This study investigated the mechanical and physical characteristics of collagen hydrogels cross-linked with different ratios of polyvinylpyrrolidone capped zinc oxide nanoparticles (ZPVP). Fourier transform infrared spectroscopy indicated molecular interactions between collagen fibers and ZPVP. Texture analysis revealed a significant increase in gel hardness, adhesiveness, and viscosity after cross-linking with ZPVP. Rheological measurements showed that as the ratio of ZPVP increased, stronger hydrogels were formed which in turn resulted in more sustained release of the model drug, dexamethasone sodium phosphate. We can therefore conclude that the mechanical properties of collagen hydrogels can be modified by controlling the ratio of ZPVP used for cross-linking, offering the potential to develop biocompatible sustained release drug delivery systems.

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy.

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1ß and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1ß, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.

Connexin43 hemichannel block protects against retinal pigment epithelial cell barrier breakdown.

AIMS: The retinal pigment epithelium (RPE) is an important component of the outer blood-retinal barrier (BRB) that separates the choroid from the rest of the retina. Loss of RPE-mediated BRB integrity is a key feature of diabetic macular oedema (DME), a chronic pathology resulting from diabetic retinopathy (DR). Recent studies have shown that connexin43 hemichannel opening mediates key inflammatory pathways in DR, though its effect on the barrier properties of RPE cells remains unknown. Therefore, RPE breakdown was induced by exposing a monolayer of ARPE-19 cells to high glucose (HG) and 10 ng/mL each of the pro-inflammatory cytokines IL-1ß and TNF-α. The role of connexin43 hemichannels was assessed using a connexin43 hemichannel blocker, Peptide5. METHODS: Transepithelial resistance (TEER) and FITC-dextran dye leak across the ARPE-19 monolayer were used to measure RPE layer permeability. Immunohistochemistry was used to assess changes in connexin43, collagen IV and ZO-1 expression. ATP and lactate dehydrogenase (LDH) release were measured using commercially available kits. RESULTS: Connexin43 hemichannel block with Peptide5 prevented TEER reduction and FITC-dextran dye leak induced by a combination of HG and inflammatory cytokines. Peptide5 also blocked LDH and ATP release induced by the addition of HG and inflammatory cytokines. ZO-1 and connexin43 disruption and internalisation as well as upregulated secretion of collagen IV following HG and inflammatory cytokine exposure were also prevented. The addition of exogenous ATP into the culture medium was able to reverse Peptide5 protection against LDH release and change in connexin43 localisation, indicating that the initiating pathway in RPE disruption is connexin43 hemichannel-mediated ATP release. CONCLUSION: These findings support the idea that connexin43 hemichannels may mediate RPE disruption (and its role within the BRB) that occurs in DME through an ATP release/inflammasome pathway activation dependent manner. Connexin43 hemichannels are therefore a potential therapeutic target for the treatment of DME.