Hyper-reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy.

The aim of this study is to correlate small dot hyper-reflective foci (HRF) observed in spectral domain optical coherence tomography (SD-OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD-OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba-1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba-1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia-mediated inflammatory component of progressive diseases showing HRF.

Adherence to home-based videogame treatment for amblyopia in children and adults.

Clinical relevance: Home-based videogame treatments are increasingly popular for amblyopia treatment. However, at-home treatments tend to be done in short sessions and with frequent disruptions, which may reduce the effectiveness of binocular visual stimulation. These treatment adherence patterns need to be accounted for when considering dose-response relationships and treatment effectiveness.Background: Home-based videogame treatments are increasingly being used for various sensory conditions, including amblyopia ('lazy eye'), but treatment adherence continues to limit success. To examine detailed behavioural patterns associated with home-based videogame treatment, we analysed in detail the videogame adherence data from the Binocular tReatment of Amblyopia with VideOgames (BRAVO) clinical trial (ACTRN12613001004752).Methods: Children (7-12 years), teenagers (13-17 years) and adults (≥ 18 years) with unilateral amblyopia were loaned iPod Touch devices with either an active treatment or placebo videogame and instructed to play for a total of 1-2 hours/day for six weeks at home. Objectively-recorded adherence data from device software were used to analyse adherence patterns such as session length, daily distribution of gameplay, use of the pause function, and differences between age groups. Objectively-recorded adherence was also compared to subjectively-reported adherence from paper-based diaries.Results: One hundred and five of the 115 randomised participants completed six weeks of videogame training. Average adherence was 65% (SD 37%) of the minimum hours prescribed. Game training was generally performed in short sessions (mean 21.5, SD 11.2 minutes), mostly in the evening, with frequent pauses (median every 4.1 minutes, IQR 6.1). Children played in significantly shorter sessions and paused more frequently than older age groups (p < 0.0001). Participants tended to over-report adherence in subjective diaries compared to objectively-recorded gameplay time.Conclusion: Adherence to home-based videogame treatment was characterised by short sessions interspersed with frequent pauses, suggesting regular disengagement. This complicates dose-response calculations and may interfere with the effectiveness of treatments like binocular treatments for amblyopia, which require sustained visual stimulation.

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.

Sustained Connexin43 Mimetic Peptide Release From Loaded Nanoparticles Reduces Retinal and Choroidal Photodamage.

Purpose: To evaluate the long-term effect on inflammation and inflammasome activation of intravitreally delivered connexin43 mimetic peptide (Cx43MP) in saline or incorporated within nanoparticles (NPs) for the treatment of the light-damaged rat eye. Methods: Light-induced damage to the retina was created by exposure of adult albino Sprague-Dawley rats to intense light for 24 hours. A single dose of Cx43MP, Cx43MP-NPs, or saline was injected intravitreally at 2 hours after onset of light damage. Fluorescein isothiocyanate (FITC)-labelled Cx43MP-NPs were intravitreally injected to confirm delivery into the retina. Electroretinogram (ERG) recordings were performed at 24 hours, 1 week, and 2 weeks post cessation of light damage. The retinal and choroidal layers were analyzed in vivo using optical coherence tomography (OCT) and immunohistochemistry was performed on harvested tissues using glial fibrillary acidic protein (GFAP), leukocyte common antigen (CD45), and Cx43 antibodies. Results: FITC was visualized 30 minutes after injection in the ganglion cell layer and in the choroid. Cx43MP and Cx43MP-NP treatments improved a-wave and b-wave function of the ERG compared with saline-injected eyes at 1 week and 2 weeks post treatment, and prevented photoreceptor loss by 2 weeks post treatment. Inflammation was also reduced and this was in parallel with downregulation of Cx43 expression. Conclusions: The slow release of Cx43MP incorporated into NPs is more effective at treating retinal injury than a single dose of native Cx43MP in solution by reducing inflammation and maintaining both retinal structure and function. This NP preparation has clinical relevance as it reduces possible ocular complications associated with repeated intravitreal injections.

Optical treatment of amblyopia in older children and adults is essential prior to enrolment in a clinical trial.

PURPOSE: Optical treatment alone can improve visual acuity (VA) in children with amblyopia, thus clinical trials investigating additional amblyopia therapies (such as patching or videogames) for children require a preceding optical treatment phase. Emerging therapies for adult patients are entering clinical trials. It is unknown whether optical treatment is effective for adults with amblyopia and whether an optical correction phase is required for trials involving adults. METHODS: We examined participants who underwent optical treatment in the Binocular Treatment for Amblyopia using Videogames (BRAVO) clinical trial (ANZCTR ID: ACTRN12613001004752). Participants were recruited in three age groups (7 to 12, 13 to 17, or ≥18 years), and had unilateral amblyopia due to anisometropia and/or strabismus, with amblyopic eye VA of 0.30-1.00 logMAR (6/12 to 6/60, 20/40 to 20/200). Corrective lenses were prescribed based on cycloplegic refraction to fully correct any anisometropia. VA was assessed using the electronic visual acuity testing algorithm (e-ETDRS) test and near stereoacuity was assessed using the Randot Preschool Test. Participants were assessed every four weeks up to 16 weeks, until either VA was stable or until amblyopic eye VA improved to better than 0.30 logMAR, rendering the participant ineligible for the trial. RESULTS: Eighty participants (mean age 24.6 years, range 7.6-55.5 years) completed four to 16 weeks of optical treatment. A small but statistically significant mean improvement in amblyopic eye VA of 0.05 logMAR was observed (S.D. 0.08 logMAR; paired t-test p < 0.0001). Twenty-five participants (31%) improved by ≥1 logMAR line and of these, seven (9%) improved by ≥2 logMAR lines. Stereoacuity improved in 15 participants (19%). Visual improvements were not associated with age, presence of strabismus, or prior occlusion treatment. Two adult participants withdrew due to intolerance to anisometropic correction. Sixteen out of 80 participants (20%) achieved better than 0.30 logMAR VA in the amblyopic eye after optical treatment. Nine of these participants attended additional follow-up and four (44%) showed further VA improvements. CONCLUSIONS: Improvements from optical treatment resulted in one-fifth of participants becoming ineligible for the main clinical trial. Studies investigating additional amblyopia therapies must include an appropriate optical treatment only phase and/or parallel treatment group regardless of patient age. Optical treatment of amblyopia in adult patients warrants further investigation.

Effectiveness of a Binocular Video Game vs Placebo Video Game for Improving Visual Functions in Older Children, Teenagers, and Adults With Amblyopia: A Randomized Clinical Trial.

Importance: Binocular amblyopia treatment using contrast-rebalanced stimuli showed promise in laboratory studies and requires clinical trial investigation in a home-based setting. Objective: To compare the effectiveness of a binocular video game with a placebo video game for improving visual functions in older children and adults. Design, Setting, and Participants: The Binocular Treatment of Amblyopia Using Videogames clinical trial was a multicenter, double-masked, randomized clinical trial. Between March 2014 and June 2016, 115 participants 7 years and older with unilateral amblyopia (amblyopic eye visual acuity, 0.30-1.00 logMAR; Snellen equivalent, 20/40-20/200) due to anisometropia, strabismus, or both were recruited. Eligible participants were allocated with equal chance to receive either the active or the placebo video game, with minimization stratified by age group (child, age 7 to 12 years; teenager, age 13 to 17 years; and adult, 18 years and older). Interventions: Falling-blocks video games played at home on an iPod Touch for 1 hour per day for 6 weeks. The active video game had game elements split between eyes with a dichoptic contrast offset (mean [SD] initial fellow eye contrast, 0.23 [0.14]). The placebo video game presented identical images to both eyes. Main Outcomes and Measures: Change in amblyopic eye visual acuity at 6 weeks. Secondary outcomes included compliance, stereoacuity, and interocular suppression. Participants and clinicians who measured outcomes were masked to treatment allocation. Results: Of the 115 included participants, 65 (56.5%) were male and 83 (72.2%) were white, and the mean (SD) age at randomization was 21.5 (13.6) years. There were 89 participants (77.4%) who had prior occlusion. The mean (SD) amblyopic eye visual acuity improved 0.06 (0.12) logMAR from baseline in the active group (n = 56) and 0.07 (0.10) logMAR in the placebo group (n = 59). The mean treatment difference between groups, adjusted for baseline visual acuity and age group, was -0.02 logMAR (95% CI, -0.06 to 0.02; P = .25). Compliance with more than 25% of prescribed game play was achieved by 36 participants (64%) in the active group and by 49 (83%) in the placebo group. At 6 weeks, 36 participants (64%) in the active group achieved fellow eye contrast greater than 0.9 in the binocular video game. No group differences were observed for any secondary outcomes. Adverse effects included 3 reports of transient asthenopia. Conclusions and Relevance: The specific home-based binocular falling-blocks video game used in this clinical trial did not improve visual outcomes more than the placebo video game despite increases in fellow eye contrast during game play. More engaging video games with considerations for compliance may improve effectiveness. Trial Registration: anzctr.org.au Identifier: ACTRN12613001004752.

Binocular treatment of amblyopia using videogames (BRAVO): study protocol for a randomised controlled trial.

BACKGROUND: Amblyopia is a common neurodevelopmental disorder of vision that is characterised by visual impairment in one eye and compromised binocular visual function. Existing evidence-based treatments for children include patching the nonamblyopic eye to encourage use of the amblyopic eye. Currently there are no widely accepted treatments available for adults with amblyopia. The aim of this trial is to assess the efficacy of a new binocular, videogame-based treatment for amblyopia in older children and adults. We hypothesise that binocular treatment will significantly improve amblyopic eye visual acuity relative to placebo treatment. METHODS/DESIGN: The BRAVO study is a double-blind, randomised, placebo-controlled multicentre trial to assess the effectiveness of a novel videogame-based binocular treatment for amblyopia. One hundred and eight participants aged 7 years or older with anisometropic and/or strabismic amblyopia (defined as ≥0.2 LogMAR interocular visual acuity difference, ≥0.3 LogMAR amblyopic eye visual acuity and no ocular disease) will be recruited via ophthalmologists, optometrists, clinical record searches and public advertisements at five sites in New Zealand, Canada, Hong Kong and Australia. Eligible participants will be randomised by computer in a 1:1 ratio, with stratification by age group: 7-12, 13-17 and 18 years and older. Participants will be randomised to receive 6 weeks of active or placebo home-based binocular treatment. Treatment will be in the form of a modified interactive falling-blocks game, implemented on a 5th generation iPod touch device viewed through red/green anaglyphic glasses. Participants and those assessing outcomes will be blinded to group assignment. The primary outcome is the change in best-corrected distance visual acuity in the amblyopic eye from baseline to 6 weeks post randomisation. Secondary outcomes include distance and near visual acuity, stereopsis, interocular suppression, angle of strabismus (where applicable) measured at baseline, 3, 6, 12 and 24 weeks post randomisation. Treatment compliance and acceptability will also be assessed along with quality of life for adult participants. DISCUSSION: The BRAVO study is the first randomised controlled trial of a home-based videogame treatment for older children and adults with amblyopia. The results will indicate whether a binocular approach to amblyopia treatment conducted at home is effective for patients aged 7 years or older. TRIAL REGISTRATION: This trial was registered in Australia and New Zealand Clinical Trials Registry ( ACTRN12613001004752 ) on 10 September 2013.

Connexin43 Mimetic Peptide Improves Retinal Function and Reduces Inflammation in a Light-Damaged Albino Rat Model.

PURPOSE: Drugs that regulate connexin43 (Cx43) gap junction channels can reduce the spread of injury and improve functional outcomes after nervous system trauma. In the eye, Cx43 expression increases in the choroid following light damage. The aim of this study was to investigate whether Cx43 hemichannel block could preserve retinal function postinjury. METHODS: Light damage was induced by exposure of adult albino Sprague-Dawley rats to 2700 Lux light for 24 hours. Intravitreal injections of a Cx43 mimetic peptide hemichannel blocker, Peptide5, or sham were administered 2 hours after the onset and at the end of the light damage period. Retinal function was assessed by electroretinogram and inflammatory responses in the choroid and retina were assessed using immunohistochemistry (ionized calcium binding adaptor molecule 1 [Iba-1], leukocyte common antigen [CD45], glial fibrillary acidic protein [GFAP]). RESULTS: Light-damaged rat eyes had (1) reduced neuronal responses in both the rod and cone pathways and (2) marked inflammatory responses in the choroid and retina. Peptide5 significantly preserved function of photoreceptoral and postphotoreceptoral neurons in these animals. This was evident 24 hours after injury and 2 weeks later, as shown by improved mixed a-wave and mixed b-wave amplitudes, isolated rod PII and PIII amplitudes, and cone PII responses when compared with sham-treated controls. Retinal thinning and inflammation were also significantly reduced in Peptide5-treated eyes when compared with sham-treated controls. CONCLUSIONS: Blocking Cx43 hemichannels after light damage can significantly improve functional outcomes of neurons in both the rod and cone photo-transduction pathways in the light-damaged animal model, likely by reducing choroid inflammation and suppressing the glial-mediated inflammatory response. These data may have relevance for the treatment of conditions such as diabetic retinopathy and age-related macular degeneration.

Vinpocetine modulates metabolic activity and function during retinal ischemia.

Vinpocetine protects against a range of degenerative conditions and insults of the central nervous system via multiple modes of action. Little is known, however, of its effects on metabolism. This may be highly relevant, as vinpocetine is highly protective against ischemia, a process that inhibits normal metabolic function. This study uses the ischemic retina as a model to characterize vinpocetine's effects on metabolism. Vinpocetine reduced the metabolic demand of the retina following ex vivo hypoxia and ischemia to normal levels based on lactate dehydrogenase activity. Vinpocetine delivered similar effects in an in vivo model of retinal ischemia-reperfusion, possibly through increasing glucose availability. Vinpocetine's effects on glucose also appeared to improve glutamate homeostasis in ischemic Müller cells. Other actions of vinpocetine following ischemia-reperfusion, such as reduced cell death and improved retinal function, were possibly a combination of the drug's actions on metabolism and other retinal pathways. Vinpocetine's metabolic effects appeared independent of its other known actions in ischemia, as it recovered retinal function in a separate metabolic model where the glutamate-to-glutamine metabolic pathway was inhibited in Müller cells. The results of this study indicate that vinpocetine mediates ischemic damage partly through altered metabolism and has potential beneficial effects as a treatment for ischemia of neuronal tissues.

Gap junction proteins in the light-damaged albino rat.

PURPOSE: Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. METHODS: Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. RESULTS: Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. CONCLUSIONS: The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.

Adenosine kinase inhibition in the cochlea delays the onset of age-related hearing loss.

This study was undertaken to determine the role of adenosine signalling in the development of age-related hearing loss (ARHL). We and others have shown previously that adenosine signalling via A(1) receptors is involved in cochlear protection from noise-induced cochlear injury. Here we demonstrate that enhanced adenosine signalling in the cochlea provides partial protection from ARHL in C57BL/6J mice. We targeted adenosine kinase (ADK), the key enzyme in adenosine metabolism, using a treatment regime with the selective ADK inhibitor ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the age of three months or six months. This treatment, intended to increase free adenosine levels in the cochlea, was maintained until the age of nine months and hearing thresholds were evaluated monthly using auditory brainstem responses (ABR). At nine months, when C57BL/6J mice normally exhibit significant ARHL, both groups treated with ABT-702 showed lower ABR threshold shifts at 10 and 16kHz compared to control animals receiving the vehicle solution. The better thresholds of the ABT-702-treated mice at these frequencies were supported by increased survival of hair cells in the apical region of the cochlea. This study provides the first evidence that ARHL can be mitigated by enhancing adenosine signalling in the cochlea.

Adenosine amine congener mitigates noise-induced cochlear injury.

Hearing loss from noise exposure is a leading occupational disease, with up to 5% of the population at risk world-wide. Here, we present a novel purine-based pharmacological intervention that can ameliorate noise-induced cochlear injury. Wistar rats were exposed to narrow-band noise (8-12 kHz, 110 dB SPL, 2-24 h) to induce cochlear damage and permanent hearing loss. The selective adenosine A(1) receptor agonist, adenosine amine congener (ADAC), was administered intraperitoneally (100 microg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses and the hair cell loss was evaluated by quantitative histology. Free radical damage in the organ of Corti was assessed using nitrotyrosine immunohistochemistry. The treatment with ADAC after noise exposure led to a significantly greater recovery of hearing thresholds compared with controls. These results were upheld by increased survival of sensory hair cells and reduced nitrotyrosine immunoreactivity in ADAC-treated cochlea. We propose that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute and extended noise exposures.

Role of adenosine kinase in cochlear development and response to noise.

Adenosine signalling has an important role in cochlear protection from oxidative stress. In most tissues, intracellular adenosine kinase (ADK) is the primary route of adenosine metabolism and the key regulator of intracellular and extracellular adenosine levels. The present study provides the first evidence for ADK distribution in the adult and developing rat cochlea. In the adult cochlea, ADK was localized to the nuclear or perinuclear region of spiral ganglion neurons, lateral wall tissues, and epithelial cells lining scala media. In the developing cochlea, ADK was strongly expressed in multiple cell types at birth and reached its peak level of expression at postnatal day 21 (P21). Ontogenetic changes in ADK expression were evident in the spiral ganglion, organ of Corti, and stria vascularis. In the spiral ganglion, ADK showed a shift from predominantly satellite cell immunolabelling at P1 to neuronal expression from P14 onward. In contrast to the role of ADK in various aspects of cochlear development, the ADK contribution to the cochlear response to noise stress was less obvious. Transcript and protein levels of ADK were unaltered in the cochlea exposed to broadband noise (90-110 dBSPL, 24 hr), and the selective inhibition of ADK in the cochlea with ABT-702 failed to restore hearing thresholds after exposure to traumatic noise. This study indicates that ADK is involved in purine salvage pathways for nucleotide synthesis in the adult cochlea, but its role in the regulation of adenosine signalling under physiological and pathological conditions has yet to be established.

Post exposure administration of A(1) adenosine receptor agonists attenuates noise-induced hearing loss.

Adenosine is a constitutive cell metabolite with a putative role in protection and regeneration in many tissues. This study was undertaken to determine if adenosine signalling pathways are involved in protection against noise injury. A(1) adenosine receptor expression levels were altered in the cochlea exposed to loud sound, suggesting their involvement in the development of noise injury. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea 6h after noise exposure. Auditory brainstem responses measured 48h after drug administration demonstrated partial recovery of hearing thresholds (up to 20dB) in the cochleae treated with adenosine (non-selective adenosine receptor agonist) or CCPA (selective A(1) adenosine receptor agonist). In contrast, the selective A(2A) adenosine receptor agonist CGS-21680 and A(3) adenosine receptor agonist Cl-IB-MECA did not protect the cochlea from hearing loss. Sound-evoked cochlear potentials in control rats exposed to ambient noise were minimally altered by local administration of the adenosine receptor agonists used in the noise study. Free radical generation in the cochlea exposed to noise was reduced by administration of adenosine and CCPA. This study pinpoints A(1) adenosine receptors as attractive targets for pharmacological interventions to reduce noise-induced cochlear injury after exposure.