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Purpose: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. Methods: Twenty‐five Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 μm meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. Results: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with β3‐tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001). Conclusions: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach. [ABSTRACT FROM AUTHOR]

Aims/Purpose: Juvenile form of neuronal ceroid lipofuscinosis is a is a rare neurological disease that manifests in childhood, by progressive visual dysfunction. The progression and timeline of the retinal degeneration in the eye has not been comprehensively characterized. We performed a 15‐month follow‐up study aiming to reveal the progression of retinal pathology in CLN3 ex7/8 mice modelling juvenile neuronal ceroid lipofuscinosis. Methods: ERG, OCT, and fundus imaging was performed in CLN3 ex7/8 mice every three months at 3, 6, 9, 12 and 15 months. Scotopic electroretinography was performed with Espion ERG using silver electrodes. Mice were kept overnight in dark room before testing. Eyes were exposed to flashes of light periodically increasing in intensity (0,0001–1 Cd × s/m2). Responses to multiple flashes were averaged to obtain a single waveform at each intensity. OCT and fundus images were captured with the Phoenix MICRON® Image‐Guided OCT. Results: In scotopic ERG responses lower amplitudes of a‐ and b‐wave were observed CLN3 ex7/8 mice compared to WT mice at all time points. A progressive, almost 2‐fold decrease of b‐wave amplitude (at 0.03 Cd × s/m2) was seen in CLN3 ex7/8 mice between 3 and 15 months, compared to WT. Most notable decline of b‐wave amplitude (~70% of the decrease, at 0.03 Cd × s/m2) was seen from age of 6 to 12 months. A‐wave amplitudes in CLN3 ex7/8 mice were consistently lower but not progressively reduced when compared to WT mice. The accumulation of drusen was observed in the fundus images of CLN3 ex7/8 mice. In OCT images we observed signs of retinal outer segment atrophy. These effects were not observed in WT animals to the same degree. Conclusions: We observed age‐dependent retinal outer segment atrophy with drusen‐like deposit accumulation visually with OCT and fundus imaging in CLN3 ex7/8 mice, and the results correlate well with ERG results. According to the progressive decline of b‐wave amplitude, the decrease of vision seems to be most prominent between the ages of 6 and 12 months. This result indicates progressive loss of function in inner retinal neurons. The present results reveal the progression of the pathology in CLN3 ex7/8 mice as well as the time window for testing novel disease‐modifying interventions in CLN3 ex7/8 mice. [ABSTRACT FROM AUTHOR]

Purpose: Disturbed proteostasis and oxidative stress have a key role in Alzheimer's disease and age‐related macular degeneration pathology. Constant oxidative stress and disturbed proteostasis lead to intracellular protein aggregation and possibly the activation of secretory autophagy. In this study we assessed the oxidative stress and secretory autophagy related biomarker changes in the retinal pigment epithelium (RPE) of the 5xFAD mice model expressing abundant levels of the human beta‐amyloid. Methods: The eyes of 3‐, 6‐ and 12‐month‐old WT (n = 5) and 5xFAD (n = 5) mice were enucleated, paraffin fixed and examined by immunomicroscopy. The paraffin sections were immunostained for beta‐amyloid and the key secretory autophagy markers Sec22b, TRIM16, galectin 8, IL‐1β, HMGB1 and ferritin. Moreover, the sections were stained for oxidative stress marker 4‐HNE and antioxidant enzyme catalase. Protein degradation related markers ubiquitin (Ub) and SQSTM1/p62 were analysed from 6‐ and 12‐months timepoints. Results: Immunohistochemistry analysis revealed that the oxidative stress and protein degradation related markers were upregulated in the RPE of 6 and 12‐months old 5xFAD mice suggesting presence of oxidative stress and protein aggregation. TRIM16 and galectin 8 were increased in the RPE of the 6‐ and 12‐months old 5xFAD mice, while the increased expression of Sec22b was observed in 3‐ and 6‐months timepoint. IL‐1β, HMGB1 and ferritin were accumulated at the basal side of the RPE cells/Bruch's membrane in 6‐months old 5xFAD mice. The high accumulation of beta‐amyloid was detected in drusen like structures. Electrophysiological analysis revealed decreased scotopic and photopic signalling compared to WT mice. Conclusions: 5xFAD mouse model seems to be a good model to study protein aggregation and activation of the secretory autophagy in response to degenerative retina processes. [ABSTRACT FROM AUTHOR]

Aims/Purpose: We are developing new eye drop treatment for glaucoma with dosing frequency once a week by utilizing drug binding to ocular melanin. Carbonic anhydrase (CA) II enzyme inhibitors decrease intraocular pressure (IOP) and thus halt the progression of retinal damage by reducing the secretion of aqueous humour in the ciliary body. Since ciliary body is a melanin‐rich tissue, we hypothesize that long‐lasting CAII inhibition and IOP‐lowering effect may be achieved due to gradual drug release from melanin‐bound drug depot. Methods: Melanin binding affinity for about 30 novel CAII inhibitors was studied with microscale thermophoresis, and three structurally rather similar and equipotent molecules with different melanin binding properties were selected for in vivo efficacy studies. IOP‐lowering effect of these new molecules was studied with albino and pigmented rabbits with iCare® PRO rebound tonometer. For pharmacokinetic studies, the high melanin binding new CAI (molecule 184) was labelled with tritium, and the study was conducted with albino and pigmented rats with conventional methods. Bound drug was analysed from iris‐ciliary body pellet and unbound fraction from supernatants. Results: Melanin‐binding affinity of new CAII inhibitors varies from low or non‐binders (Kd > 650 μM) to high melanin binders (Kd < 65 μM). In vivo efficacy results show that the molecules decrease IOP in both albino and pigmented eyes at the level comparable with dorzolamide (Trusopt®). In pigmented eyes, melanin binding prolongs the effect of compound 184 even up to 2 weeks after single eye drop. In the pharmacokinetic rat study, molecule 184 shows remarkably prolonged tissue binding and high iris‐ciliary body exposure in pigmented eyes. Conclusions: After topical administration IOP‐lowering effect of high melanin‐binding CA II inhibitor is significantly prolonged in pigmented eyes. [ABSTRACT FROM AUTHOR]

Purpose: Chronic oxidative stress, disturbed proteostasis and increased protein aggregation are key hallmarks in Alzheimer's disease and age‐related macular degeneration pathology. In this study, we assessed the protein aggregation and secretory autophagy related biomarker changes in 5xFAD mice model expressing abundant levels of human beta‐amyloid (Aβ42). Methods: The eyes of 6‐ and 12‐month‐old WT (n = 5) and 5xFAD (n = 5) were examined by immunomicroscopy. The paraffin sections were immunostained with the key regulators of secretory autophagy HMGB1, Aβ42, IL‐1β, and ferritin as well as protein aggregation markers ubiquitin protein and SQSTM1/p62. Results: Immunohistochemistry analysis revealed that ubiquitin protein conjugates and Aβ42 were increased in 6 and 12 months old 5xFAD mice. SQSTM1/p62 was decreased in 12 months old 5xFAD mice. In 12‐month‐old time point, they located basolateral side of the retinal pigment epithelial (RPE) cells but also in extracellular space. Concurrently, secretory autophagy markers were elevated in the RPE and extracellular space between RPE and Bruch's membrane. Conclusions: Secretary autophagy may be a novel regulator in drusen biogenesis. 5xFAD mouse is a good model to study protein aggregation and secretory autophagy mechanism in response to degenerative retina processes. [ABSTRACT FROM AUTHOR]

Purpose: Disturbed proteostasis and protein aggregation have a key role in Alzheimer´s disease and age‐related macular degeneration pathology. In this study, we assessed the electrophysiological and protein aggregation related biomarker changes in 5xFAD mouse model. Methods: Scotopic and photopic electroretinograms (ERG) were recorded at 3, 6, 9 and 12‐ months old animals followed by optical coherent tomography (OCT) (WT; n = 12, 5xFAD; n = 12). The eyes of 3‐ and 6‐month‐old WT (n = 5) and 5xFAD (n = 5) were used in immunohistochemistry. The paraffin sections were immunostained with the key regulators of lysosomal and proteasomal clearance. Results: 5xFAD mice showed decreased scotopic and photopic ERG signaling over time compared to WT mice. However, there were no clear changes in OCT between groups. Immunohistochemistry results revealed that proteasomal clearance was disturbed in 6‐month‐old 5xFAD mice compared to WT. Interestingly, there were no changes in autophagy biomarkers. Conclusions: 5xFAD mouse is a good model to study visual function and protein aggregation mechanism in response to degenerative retina processes. [ABSTRACT FROM AUTHOR]

Purpose: Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age‐related macular degeneration (AMD). We have recently developed a dry AMD mice model with a nuclear factor‐erythroid 2‐related factor‐2 (NFE2L2) knock‐out (KO). Pinosylvin, a polyphenol abundant in bark waste, improves retinal pigment epithelial cell (RPE) cell viability in vitro. Therefore, one can estimate that pinosylvin slow down the progression of dry AMD. Methods: We investigated the effects of commercial natural pinosylvin extract, Retinari™, on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild type (WT) and NFE2L2 KO mice were raised in 12:12 until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow (50–250 mg/kg/day of pinosylvin) for 10 weeks before the assays. Results: Retinari™ treatment preserved significant retinal function in scotopic and photopic ERG and prevented thinning of the retina in the NFE2L2 KO mice. The NFE2L2 KO mice showed reduced ubiquitin‐tagged protein accumulation in addition to upregulation of the antioxidant enzymes superoxide dismutase 1 and catalase. Furthermore, the treatment seemed to reduce inflammation in NFE2L2 KO mice by causing a climb in the concentration of complement factor H. Conclusions: Our results demonstrate a strong protective effect of Retinari™ on the AMD disease model. We believe that pinosylvin in the Retinari™ elicits the positive effects. Thus, pinosylvin could potentially lower the risk of AMD onset and slow down its progression for which clinical trials with the compound are highly anticipated. [ABSTRACT FROM AUTHOR]

Purpose: Rats and rabbits are widely used in preclinical ocular pharmacodynamics and pharmacokinetics. Despite the wide application in the evaluation of intravitreally injected drugs, there is no information about scaling of vitreal kinetics between rats and rabbits. In this study, the vitreal kinetics of macromolecules in rats and rabbits were evaluated using in vivo fluorophotometry. Methods: In order to elucidate the pharmacokinetics of macromolecules and drug delivery systems we used ocular fluorophotometry that is a noninvasive technique for non‐invasive and quantitative measurement of fluorescence signals from the eye. Molecules with different molecular weights (peptide 4.5 kD, FITC‐hyaluronic acid 770 kD, FITC‐dextran 500 kD and fluorescein 330 D, FITC‐dextran 10 kD and FITC‐dextran 150 kD) were injected to the vitreous of rabbits and rats. The concentrations of these molecules were quantified in the vitreous at various time. Results and Discussion: The data were used to calculate kinetic parameter, such as vitreal elimination half‐life and clearance. For example, the vitreal elimination half‐lives of FITC‐hyaluronic acid 770 kD in rabbits and rats were about 280 hr and 9 hr, respectively. Overall the vitreal elimination half‐lives of the compounds in the rats were much faster than in the rabbits. Since rats are widely used in the retinal pharmacodynamics studies, the fast elimination of macromolecules from the rat vitreous should be taken into account when scaling the doses from rats to rabbits and man. [ABSTRACT FROM AUTHOR]

Purpose: Axons in the optic nerve are arranged in bundles and conducting action potential with resistance related to their membrane. Here, we aim to characterize the function of newly identified gap junctions coupling optic nerve axons with each other's. Methods: Proximal and distal rat optic nerve stumps were processed for immunostainings, electron microscopy (EM), RT‐PCR and western blots (WB). Additional 20 animals were deeply anesthetized, and stainless‐steel screw electrodes were drilled in skulls in standard locations to record fVEPs. Recordings were performed in untouched animals, after retrobulbar injection of saline (negative control) or meclofenamic acid solution (gap junctions' blocker). Human paraffin cross‐sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research, University of Toronto, Canada. Results: Immunostaining of rat and human samples revealed presence of Cx45 and 36 in axonal membranes, colocalizing with β3‐tubulin, but not with GFAP. In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 ‐ proximal stump; 0.44 ± 0.02 ‐ distal stump), Cx45 showed higher levels (0.68 ± 0.0003 ‐ proximal stump; 0.9 ± 0.07 ‐ distal stump). Additionally, we detected transcript of Cx45 and Cx36 in RT‐PCR analysis of optic nerve homogenates. In immunogold‐EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) that are directly coupling neighboring axons. In fVEPs, blocking of gap junctions' with meclofenamic acid resulted in prolongation of the latency of P1 wave up to 140% after 5 min from retrobulbar injection of this blocker (p < 0.001) and 170% after 30 min (p < 0.001). Conclusions: Optic nerve axons do not form absolutely independent conductive channels. They are directly coupled by gap junctions formed in majority by neuronal Cx45. Coupling of axons allows to reduce axonal membrane resistance and accelerates transduction of visual signal. [ABSTRACT FROM AUTHOR]

Purpose: Circadian clocks in the eye are critical for normal visual function and they relate to periodical renewal of photoreceptor outer segments as well as oxidative stress. Impaired rhythm can thus contribute to the pathogenesis of age‐related macular degeneration (AMD). Chronic oxidative stress leads eventually to protein aggregation in combination with impaired autophagy. We hypothesise that pinosylvin, a polyphenolic compound, slows down the progression of AMD through autophagy induction. Methods: We investigated the relative change in electroretinogram (ERG) metrics of mice raised in 12:12 h light‐dark cycle and after a 24 h two‐week dark period in wild type (WT; n = 10), PGC1‐α knock‐out (KO; n = 7) and Nrf2 KO (n = 7) mice aged 9.7 ± 3.4 months. From these, 9 WT, 5 PGC1‐α and 7 Nrf2 KO mice continued to be raised to an age of 17.3 ± 3.8 months and assigned to a treatment and control group and fed for 2 months with pinosylvin‐feed or regular‐feed prior to recording ERG. Results: The two‐week dark period resulted in significantly greater b‐wave amplitudes and shorter b‐wave latencies in WT mice in photopic ERG. Nrf2 KO and PGC1‐α/Nrf2 dKO mice exhibited b‐wave latency changes in the opposite. The dKO also had diminished b‐wave amplitudes. Scotopic ERG yielded no significant changes. Pinosylvin treatment in Nrf2 KO and PGC1‐α KO mice caused significantly increased b‐wave amplitudes in photopic ERG. In scotopic ERG, the pinosylvin treated mice had larger a‐ and b‐wave amplitudes in WT and Nrf2 KO mice, but not in PGC1‐α KO mice. Conclusions: PGC1‐α KO and Nrf2 KO mice showed different circadian rhythmicity. Pinosylvin treatment showed improved ERG signalling in aged NRf2 KO and WT mice. [ABSTRACT FROM AUTHOR]

Purpose To describe impact of topical preservative-free (PF) prostaglandin analogues on inflammatory response within eyelid tarsus. Methods New Zealand rabbits' eyes (n = 12 animals) were exposed to topical PF prostaglandins for 8 weeks daily. Each right eye was treated and each left eye kept as a control - topical PBS application. As eye drops we used PF tafluprost (Taflotan, Santen), PF latanoprost (Monoprost, Thea) and PF bimatoprost (Lumigan, Allergan). After 8 weeks animals were sacrificed, eyelids were post-fixed in 4% paraformaldehyde and embedded in paraffin. Tarsal sections were immunostained for macrophage-specific Iba-1 and mucin. Tissue histology was evaluated by Masson trichrome staining. Quantitative analysis of macrophages ( MFs) infiltration within tarsal: conjunctiva, stroma and glands was performed. Results PF prostaglandin treatment for 8 weeks did not affect mucin expression within analyzed tissues. Total number of MFs forming infiltration in tarsal conjunctiva of PF tafluprost group was 3.5 ± 2.5 vs 2.2 ± 1.5 cells (p > 0.05, respectively for treated vs control eye), in PF latanoprost group was 11.6 ± 9.8 vs 3.8 ± 1.8 cells (p < 0.05) and in PF bimatoprost group was 6.8 ± 5.1 vs 3.9 ± 2.5 (p = 0.05). There was significantly different intensity of MFs infiltration within tarsal conjunctiva between groups: PF latanoprost> PF bimatoprost> PF tafluprost (p < 0.05, Kruskal-Wallis test). MFs infiltration within tarsal stroma and glands was slightly increased in PF latanoprost group when compared with control (p < 0.05), however it was not significantly different from other prostaglandins (p > 0.05). Conclusions Applied PF prostaglandin analogues affected MFs infiltration within tarsus. The strongest macrophage-associated inflammatory reaction was related to PF latanoprost application, however there is a need for further analyses of other inflammatory response pathways. [ABSTRACT FROM AUTHOR]