Retinal Histology Research Articles

Impaired pericyte-Müller glia interaction via PDGFRβ suppression aggravates photoreceptor loss in a rodent model of light-induced retinal injury.

To investigate the involvement of pericyte-Müller glia interaction in retinal damage repair and assess the influence of suppressing the platelet-derived growth factor receptor β (PDGFRβ) signaling pathway in retinal pericytes on photoreceptor loss and Müller glial response. Sprague-Dawley rats were exposed to intense light to induce retinal injury. Neutralizing antibody against PDGFRβ were deployed to block the signaling pathway in retinal pericytes through intravitreal injection. Retinal histology and Müller glial reaction were assessed following light injury. In vitro, normal and PDGFRβ-blocked retinal pericytes were cocultured with Müller cell line (rMC-1) to examine morphological and protein expression changes upon supplementation with light-injured supernatants of homogenized retinas (SHRs). PDGFRβ blockage 24h prior to intense light exposure resulted in a significant exacerbation of photoreceptor loss. The upregulation of GFAP and p-STAT3, observed after intense light exposure, was significantly inhibited in the PDGFRβ blockage group. Further upregulation of cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin-1β (IL-1β) was also observed following PDGFRβ inhibition. In the in vitro coculture system, the addition of light-injured SHRs induced pericyte deformation and upregulation of proliferating cell nuclear antigen (PCNA) expression, while Müller cells exhibited neuron-like morphology and expressed Nestin. However, PDGFRβ blockage in retinal pericytes abolished these cellular responses to light-induced damage, consistent with the in vivo PDGFRβ blockage findings. Pericyte-Müller glia interaction plays a potential role in the endogenous repair process of retinal injury. Impairment of this interaction exacerbates photoreceptor degeneration in light-induced retinal injury.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

Age-related effects of optineurin deficiency in the mouse eye

Optineurin (OPTN) is a gene associated with familial normal tension glaucoma (NTG). While NTG involves intraocular pressure (IOP)-independent neurodegeneration of the visual pathway that progresses with age, how OPTN dysfunction leads to NTG remains unclear. Here, we generated an OPTN knockout mouse (Optn−/−) model to test the hypothesis that a loss-of-function mechanism induces structural and functional eye deterioration with aging. Eye anatomy, visual function, IOP, retinal histology, and retinal ganglion cell survival were compared to littermate wild-type (WT) control mice. Consistent with OPTN’s role in NTG, loss of OPTN did not increase IOP or alter gross eye anatomy in young (2–3 months) or aged (12 months) mice. When retinal layers were quantitated, young Optn−/− mice had thinner retina in the peripheral regions than young WT mice, primarily due to thinner ganglion cell-inner plexiform layers. Despite this, visual function in Optn−/− mice was not severely impaired, even with aging. We also assessed relative abundance of retinal cell subtypes, including amacrine cells, bipolar cells, cone photoreceptors, microglia, and astrocytes. While many of these cellular subtypes were unaffected by Optn deletion, more dopaminergic amacrine cells were observed in aged Optn−/− mice. Taken together, our findings showed that complete loss of Optn resulted in mild retinal changes and less visual function impairment, supporting the possibility that OPTN-associated glaucoma does not result from a loss-of-function disease mechanism. Further research using these Optn mice will elucidate detailed molecular pathways involved in NTG and identify clinical or environmental risk factors that can be targeted for glaucoma treatment.

Metabolic Deficits in the Retina of a Familial Dysautonomia Mouse Model.

Neurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber's hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients' serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson's, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (1H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30-P90, and dopamine levels were 25-35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD.

Protective efficacy of nafronyl in diabetic retinopathy through targeted inhibition of key enzymes.

Diabetic retinopathy is governed by abnormal apoptosis, increased capillary pressure, and other linked pathology that needs an efficient treatment by multitargeted approaches. Thus, the current study aimed to explore the potential of inhibition of targeted enzymes (DPP4, ACE-2, and aldose reductase) and free radical scavenging capabilities of selected compounds (nafronyl or naftidrofuryl) through in silico and in vivo investigations. Significant binding energies were observed in complexes of aldolase reductase, angiotensin type 1 receptor, and DPP4 against the nafronyl and sitagliptin more than -7.5kcal/mol. Further validation of free energy was confirmed by calculations of molecular mechanics Poisson-Boltzmann surface area (MMPBSA), and configurational stabilities examined by PCA (principal component analysis). Additionally, drug-likeness was examined by the Swiss ADME web tool, which showed significant findings. Consequently, in vivo experimentations showed significant inflammation and alterations in retinal layers of inner plexiform (inner limiting membrane, nerve fibers, and ganglionic cells), inner nuclear layer (bipolar cells and horizontal cells), and photoreceptors cells. Whereas the treatments (nafronyl and sitagliptin) caused significant improvements in the histoarchitecture of the retina. Additionally, the HOMA indices (IR-insulin resistance, sensitivity, and β cells functioning) and levels of free radicals were significantly altered in the diabetic control group in comparison to intact control. Nafronyl administration showed significant ameliorations in HOMA indices as well as antioxidant levels. Based on the results, it can be concluded that nafronyl efficiently interacts with target enzymes, which may result in potent inhibition and ameliorations in retinal histology as well as glucose homeostasis and antioxidants.

Light perception and related alternation of physiological performance in spotted knifejaw Oplegnathus punctatus

Light is one of the main environmental factors that affects the behavior and physiology of fish. In the present study, the behavioral responses, retinal histology, hematological parameters (white blood cell [WBC], red blood cell [RBC], hemoglobin [Hb] hematocrit [Hct]), hepatic antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSH-Px]) activity, aspartate-aminotransferase (AST) and alanine-aminotransferase (ALT) activities, malondialdehyde (MDA) content, histological alterations, apoptosis, and stress-related gene expression (hsp70, hsp90a, hsp90b) of spotted knifejaw were investigated under different light colors. The results showed that the residence time of spotted knifejaw plants significantly decreased under red and blue light stimuli but significantly increased under green light. Moreover, a positive and negative phototaxis of the spotted knifejaw under green and red lights was observed. However, retinal structure (relative thickness, cell number, and size) remained unchanged after red and green light treatment for 12 h. WBC and RBC numbers, Hb and Hct contents, hepatic SOD, CAT, GSH-Px, ALT and AST activities, and MDA levels were significantly increased after red and green light treatment for 12 h. Moreover, red and green light treatment caused hepatocyte vacuolization and nuclear migration and significantly increased the percentage of apoptotic cells. Hepatic hsp70, hsp90a, and hsp90b mRNA levels were significantly upregulated under red and green stimuli. Furthermore, the values of the above parameters under red light treatment were significantly greater than those under green light treatment. The aforementioned parameters nearly recovered to normal levels after the fish returned to normal light for 12 h. In conclusion, in spotted knifejaw, green light promoted attraction and red light avoidance, while red light could induce a more sensitive physiological response than green light. These findings expand the current knowledge on light perception and aid in the management of spotted knifejaw in captivity.

Early Choriocapillaris Loss in a Porcine Model of RPE Cell Debridement Precedes Pathology That Simulates Advanced Macular Degeneration.

No large-mammal surgical models exist for geographic atrophy (GA), choroidal neovascularization (CNV), and pachychoroidal vascular remodeling. Our goal was to develop a porcine RPE debridement model of advanced macular degeneration to study photoreceptor cell loss and choroidal remodeling. Seven 2-month-old female domestic pigs were used for this study. After 25G vitrectomy, the area centralis was detached via subretinal bleb. A nitinol wire (Finesse Flex Loop) was used to debride RPE cells across a 3- to 5-mm diameter region. Fluid-air exchange was performed, and 20% SF6 gas injected. Animals underwent fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT-angiography (OCTA) at 2 weeks, 1month, 2months, 3months, and 6 months postoperatively. Retinal histology was obtained at euthanasia, 2months (n = 3), 3months (n = 2), or 6months (n = 2) after surgery. RPE debridement resulted in GA with rapid loss of choriocapillaris, progressive loss of photoreceptors, and pachychoroidal changes in Sattler's and Haller's layers in all seven eyes undergoing debridement within 2 months. OCT and histological findings included subretinal disciform scar with overlying outer retinal atrophy; outer retinal tubulations and subretinal hyper-reflective material. OCTA revealed type 2 CNV (n = 4) at the edges of the debridement zone by 2 months, but there was no significant exudation noted at any time point. Surgical debridement of the RPE results in GA, CNV, and pachychoroid and reproduced all forms of advanced macular degeneration. This surgical model may be useful in examining the role of RPE and other cell replacement in treating advanced macular disease.

Aggravated visual toxicity in zebrafish larvae upon co-exposure to titanium dioxide nanoparticles and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate

The bioavailability and toxicity of organic pollutants in aquatic organisms can be largely affected by the co-existed nanoparticles. However, the impacts of such combined exposure on the visual system remain largely unknown. Here, we systematically investigated the visual toxicity in zebrafish larvae after single or joint exposure to titanium dioxide nanoparticles (n-TiO2) and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) at environmentally relevant levels. Molecular dynamics simulations revealed the enhanced transmembrane capability of the complex than the individual, which accounted for the increased bioavailability of both TBPH and n-TiO2 when combined exposure to zebrafish. Transcriptome analysis showed that co-exposure to n-TiO2 and TBPH interfered with molecular pathways related to eye lens structure and sensory perception of zebrafish. Particularly, n-TiO2 or TBPH significantly suppressed the expression of βB1-crystallin and rhodopsin in zebrafish retina and lens, which was further enhanced after co-exposure. Moreover, we detected disorganized retinal histology, stunted lens development and significant visual behavioral changes of zebrafish under co-exposure condition. The overall results suggest that combined exposure to water borne n-TiO2 and TBPH increased their bioavailability, resulted in severer damage to optic nerve development and ultimately abnormal visual behavior patterns, highlighting the higher potential health risks of co-exposure to aquatic vertebrates.

RNA‐binding protein ELAVL1‐mediated neuroprotection of retinal ganglion cells

Aims/Purpose: To evaluate the impact of ELAVL1 overexpression on survival and function of retinal ganglion cells (RGC) in rat glaucoma model.Methods: The study included ex vivo and in vivo approaches. Ex vivo, we used rat retinal explants to investigate, whether AAV‐ELAVL1 delivery affects the survival of RGC in the degeneration model induced by axotomy. In vivo approach investigated histology and function of RGC in rat glaucoma model. Rats received intravitreal injection of AAV‐ELAVL1, AAV‐ELAVL1(S202A) or AAV‐control. To induce chronic glaucoma, unilateral episcleral vein cauterization was performed. Animals underwent electroretinography tests (ERG) during the experiment. Collected retinas and optic nerves were processed for immunostainings, western blots, and stereology.Results: In ex vivo retinal explants culture, the RGC count was 162 ± 35, 148 ± 22 and 149 ± 28 for AAV‐ELAVL1, AAV‐ELAVL1(S202A) and control, respectively (p < 0.03). The IOP values were similar in all experimental groups, and on average, they were significantly higher than in healthy eyes (p < 0.001). In retinal histology, clear neuroprotective effect in the AAV‐ELAVL1 group was observed. After AAV therapy, both constructs (ELAVL1 and ELAVL1(S202A) showed a significant increase in protein expression in RGC, which was also associated with a significant increase in the expression of BDNF in the retinas, especially after AAV‐ELAVL1 treatment. In functional tests, the PhNR response was significantly reduced in AAV‐control glaucoma (p < 0.001). The application of AAV‐ELAVL1 treatment resulted in the preservation of RGC function at a level very similar to the situation before the induction of glaucoma.Conclusions: The AAV‐ELAVL1 approach showed a very strong neuroprotective effect in a rat glaucoma model. The neuroprotective effect is associated with antioxidant and anti‐inflammatory activity with the increase in BDNF expression, which intensifies the neuroprotective effect.

Retinopathy in Greyhound dogs: Prevalence, fundoscopic, and histopathological findings.

To determine the prevalence of retinal lesions and describe the fundoscopic findings of retinopathy in Greyhound dogs in the Manawatu/Whanganui region of New Zealand. To examine possible associations between sex, age, and racing variables with retinopathy in the study population. To describe retinal histologic findings in seven Greyhounds with retinopathy in New Zealand. Two hundred Greyhound dogs from the Manawatu/Whanganui region of New Zealand underwent fundoscopy and fundic photography to identify and score the degree of retinopathy. Associations between retinopathy and age, sex, as well as racing variables, were examined. Histologic examination of the retina was undertaken on the eyes of seven Greyhounds from the Manawatu and Canterbury regions previously diagnosed with retinopathy by fundoscopy. Fifty dogs (25.1%) were identified with retinopathy of varying degrees of severity. In at least one eye, 7.5% of dogs had mild retinopathy, 11.6% moderate retinopathy, and 6.0% severe retinopathy. Males were more likely to be affected in both eyes and with moderate or severe grades, than females. Increasing age was not associated with increased prevalence of retinopathy, nor increased grade of severity. Retinal histology identified multifocal retinal detachment in 5 of the 7 cases examined and other common lesions included choroidal necrosis and outer to full-thickness retinal atrophy in the absence of significant inflammation. Retinopathy is prevalent in Greyhounds in the Manawatu/Whanganui region of New Zealand, but more research is required to elucidate the etiopathogenesis. Consideration should be made to include mandatory eye health examination in racing Greyhound dogs.

A Novel and Low-cost Approach for Intravitreal Injection in an Experimental Model of Endophthalmitis.

Animal models are necessary in understanding the pathogenesis of endophthalmitis and are also necessary to assist the development of new therapeutics for this sight-threatening ocular inflammation. Hamilton syringes are usually preferred to inject pathogens when performing experiments on test subjects, however, this method has technical and financial disadvantages. In this study, we report the findings and assess the related benefits of applying a novel low-cost intravitreal injection technique to initiate endophthalmitis in a mouse model while using the Eppendorf tip and a 26G needle. The 18-hr culture of clinical isolates of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and fungus (Aspergillus flavus and Candida albicans) were resuspended to a final concentration of 10,000 colony forming units (CFU)/1 µL which were separately injected intravitreally into C57BL/6 mice (6-8 weeks) using a 0.1-2.5µL pipette attached to the modified Eppendorf tip with a 26G needle. The contralateral eye served as vehicle/uninjected control. Disease progression was determined by assessing the corneal haze, opacity, bacterial burden, and retinal histology of the eyes used in the model. Following euthanization, bacteria-infected mice were enucleated after 24 hr of the initial injection, and fungus-infected mice after 72 hr. Of the 50 mice injected, the modified technique was successful in 48 mice. Two mice were excluded due to cataract formed by accidental injury to the lens. The experimental endophthalmitis mice model successfully mimicked the natural clinical course. Clinical assessment and histopathology confirmed the influx of inflammatory cells into the posterior segment of the eye along with dissolution of retinal architecture. Our novel method of injection using a modified Eppendorf tip and 26G needle yielded a cost-effective mouse model of clinical endophthalmitis, resulting in reproducible infection for understanding various aspects of its pathobiology.

Survival of Neural Progenitors Derived from Human Embryonic Stem Cells Following Subretinal Transplantation in Rodents.

Purpose: To examine the survival of neural progenitors (NPs) cells derived from human embryonic stem cells (hESCs) following subretinal (SR) transplantation in rodents. Methods: hESCs engineered to express enhanced green fluorescent protein (eGFP) were differentiated in vitro toward an NP fate using a 4-week protocol. State of differentiation was characterized by quantitative-PCR. NPs in suspension (75,000/μl) were transplanted to the SR-space of Royal College of Surgeons (RCS) rats (n = 66), nude-RCS rats (n = 18), and NOD scid gamma (NSG) mice (n = 53). Success of engraftment was determined at 4 weeks post-transplant by in vivo visualization of GFP-expression using a properly filtered rodent fundus camera. Transplanted eyes were examined in vivo at set time points using the fundus camera, and in select cases, by optical coherence tomography imaging, and after enucleation, by retinal histology and immunohistochemistry. Results: In RCS rats, cell rejection was observed in 29% of eyes at 6 weeks, rising to 92% at 8 weeks. In the more immunodeficient nude-RCS rats, the rejection rate was still high reaching 62% of eyes at 6 weeks post-transplant. Following transplantation in highly immunodeficient NSG mice, survival of the hESC-derived NPs was much improved, with 100% survival at 9 weeks and 72% at 20 weeks. A small number of eyes that were followed past 20 weeks showed survival also at 22 weeks. Conclusions: Immune status of recipient animals influences transplant survival. Highly immunodeficient NSG mice provide a better model for studying long-term survival, differentiation, and possible integration of hESC-derived NPs. Clinical Trial Registration numbers: NCT02286089, NCT05626114.

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis

Introduction: Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology. Methods: Zebrafish larvae with a developed visual system at three days post-fertilization (dpf) were exposed to CSE for 4, 24 or 48 hours. Visual behaviour, hyaloid vasculature morphology, retinal histology, oxidative stress gene expression and outer segment phagocytosis were investigated using visual behavioural optokinetic and visual motor response assays (OKR and VMR), microscopy (light, fluorescence and transmission electron microscopy), and real-time PCR. Results: In zebrafish larvae, 48 hours of CSE treatment resulted in significantly reduced visual behaviour. Larvae treated with 10, 15 or 20 μg/mL CSE showed an average of 13.7, 10.7 or 9.4 saccades per minute, respectively, significantly lower compared with 0.05% DMSO controls (p=0.0093, p=0.0004 and p<0.0001, respectively) that exhibited 19.7 saccades per minute. The diameter of intraocular vessels increased from 4.12 μm in 0.05% DMSO controls to 5.69 μm in the 20 μg/mL CSE-treated larvae (p≤0.0001). Biometry analysis highlighted a significant axial length elongation in 20 μg/mL CSE-treated larvae (216.9 μm, p<0.0001) compared to 0.05% dimethyl sulfoxide (DMSO) controls (205.1 μm). Larvae exposed to 20 μg/mL CSE had significantly (p=0.0002) higher numbers of RPE phagosomes compared to vehicle controls (0.1425 and 0.093 phagosomes/μm RPE, respectively). Conclusions: Zebrafish larvae with a developed visual system display apparent defects in visual behaviour and retinal biology after acute exposure to CSE, establishing a valuable in vivo model to investigate ocular disorders related to cigarette smoke.

Intravitreal Injection of AAV for the Transduction of Mouse Retinal Ganglion Cells.

The injection of therapies into the eye is common practice, both clinically and pre-clinically. The most straightforward delivery route is via an intravitreal injection, which introduces the treatment into the largest cavity at the posterior of the eye. This technique is frequently used to deliver gene therapies, including those containing recombinant adeno-associated viral vectors (AAVs), to the back of the eye to enable inner retinal targeting. This chapter provides detailed methodology on how to successfully perform an intravitreal injection in mice. The chapter covers vector preparation considerations, advice on how to minimize vector loss in the injection device, and ways to reduce vector reflux from the eye when administering a therapy. Finally, a protocol is provided on common retinal histology processing techniques to assess vector-mediated expression in retinal ganglion cells. It is hoped that this chapter will enable researchers to carry out effective and consistent intravitreal injections that transduce the inner retinal surface while avoiding common pitfalls.

Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model

Blindness in Bardet-Biedl syndrome (BBS) is caused by dysfunction and loss of photoreceptor cells in the retina. BBS10, mutations of which account for approximately 21% of all BBS cases, encodes a chaperonin protein indispensable for the assembly of the BBSome, a cargo adaptor important for ciliary trafficking. The loss of BBSome function in the eye causes a reduced light sensitivity of photoreceptor cells, photoreceptor ciliary malformation, dysfunctional ciliary trafficking, and photoreceptor cell death. Cone photoreceptors lacking BBS10 have congenitally low electrical function in electroretinography. In this study, we performed gene augmentation therapy by injecting a viral construct subretinally to deliver the coding sequence of the mouse Bbs10 gene to treat retinal degeneration in a BBS10 mouse model. Long-term efficacy was assessed by measuring the electrical functions of the retina over time, imaging of the treated regions to visualize cell survival, conducting visually guided swim assays to measure functional vision, and performing retinal histology. We show that subretinal gene therapy slowed photoreceptor cell death and preserved retinal function in treated eyes. Notably, cone photoreceptors regained their electrical function after gene augmentation. Measurement of functional vision showed that subretinal gene therapy provided a significant benefit in delaying vision loss.

New model of complete retinal degeneration: A good tool for therapeutic essays?

AbstractPurpose: To characterize a mouse model of complete blindness after induction of retinal degeneration based on the degenerative capacity of NaIO3 on cell photoreceptors, rods and cones, and to test the effect of new pharmacological tools.Methods: Two doses of NaIO3 (40 mg/kg and 60 mg/kg) were intraperitoneally injected into a series of C57BL/6J mice with a mutation in the Opn4−/− gene. The effect of NaIO3 on the retina was assessed at 7, 14, 21 and 28 days after the injection by means of visual behavioural test, electroretinogram (ERG) recordings and immunohistochemical staining. To further assert the model's effectiveness, new optopharmacological agents were tested on the developed model, followed by visual testing.Results: Both doses of the degenerative agent induced a gradual reduction of the visual capacities of the animals. The different components of the ERG waves showed a marked decrease in their amplitude throughout the 28 days of the experiment, with the 60 mg/kg dose provoking a more pronounced functional loss. Similar results were observed in the behavioural tests, in which the animals' visual sensitivity, visual acuity, photomotor capacity were highly decreased. Same tests were performed under the effect of new optopharmacological therapies. Our results demonstrate not only the model's effectiveness, but also the usefulness of the new therapy for vision recover.Conclusions: NaIO3 induced murine model of retinal degeneration seems to be a good model for testing new pharmacological approaches to retinal diseases. Assessment of retinal function by electrophysiological tests, visual animal behaviour and retinal histology seem to be basic and reliable techniques for the evaluation of drug effect on this new animal model.

3K3A-Activated Protein C Prevents Microglia Activation, Inhibits NLRP3 Inflammasome and Limits Ocular Inflammation.

3K3A-Activated Protein C (APC) is a recombinant variant of the physiological anticoagulant APC with pleiotropic cytoprotective properties albeit without the bleeding risks. The anti-inflammatory activities of 3K3A-APC were demonstrated in multiple preclinical injury models, including various neurological disorders. We determined the ability of 3K3A-APC to inhibit ocular inflammation in a murine model of lipopolysaccharide (LPS)-induced uveitis. Leukocyte recruitment, microglia activation, NLRP3 inflammasome and IL-1β levels were assessed using flow cytometry, retinal cryosection histology, retinal flatmount immunohistochemistry and vascular imaging, with and without 3K3A-APC treatment. LPS triggered robust inflammatory cell recruitment in the posterior chamber. The 3K3A-APC treatment significantly decreased leukocyte numbers and inhibited leukocyte extravasation from blood vessels into the retinal parenchyma to a level similar to controls. Resident microglia, which underwent an inflammatory transition following LPS injection, remained quiescent in eyes treated with 3K3A-APC. An inflammation-associated increase in retinal thickness, observed in LPS-injected eyes, was diminished by 3K3A-APC treatment, suggesting its clinical relevancy. Finally, 3K3A-APC treatment inhibited inflammasome activation, determined by lower levels of NLRP3 and its downstream effector IL-1β. Our results highlight the anti-inflammatory properties of 3K3A-APC in ocular inflammation and suggest its potential use as a novel treatment for retinal diseases associated with inflammation.

The impact of obesity-related raised intracranial pressure in rodents

Elevated intracranial pressure (ICP) is observed in many brain disorders. Obesity has been linked to ICP pathogenesis in disorders such as idiopathic intracranial pressure (IIH). We investigated the effect of diet induced obesity (DIO) on ICP and clinically relevant sequelae. Rats were fed either a control or high fat diet. Following weight gain long term ICP, headache behavior, body composition and retinal outcome were examined. Post-hoc analysis of retinal histology and molecular analysis of choroid plexus and trigeminal ganglion (TG) were performed. DIO rats demonstrated raised ICP by 55% which correlated with the abdominal fat percentage and increased non-respiratory slow waves, suggestive of altered cerebral compliance. Concurrently, DIO rats demonstrated a specific cephalic cutaneous allodynia which negatively correlated with the abdominal fat percentage. This sensitivity was associated with increased expression of headache markers in TG. Additionally, DIO rats had increased retinal nerve fiber layer thickness in vivo associated with raised ICP with a subsequent post-hoc demonstration of neuroretinal degeneration. This study demonstrates for the first time that DIO leads to raised ICP and subsequent clinically relevant symptom development. This novel model of non-traumatic raised ICP could expand the knowledge regarding disorders with elevated ICP such as IIH.

Beneficial Actions of Essential Fatty Acids in Streptozotocin-Induced Type 1 Diabetes Mellitus.

The essential fatty acids (EFA), n3 alpha-linolenic acid (ALA), and n6 linoleic acid (LA) are of benefit in diabetes mellitus, but their mechanisms of action are unknown. We, therefore, examined the effects of EFAs on the metabolism, gut microbiota, and inflammatory and retinal histopathology indices in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) animals, and we assessed the levels of vitreal lipoxin A4 (LXA4)—derived from LA—in subjects with diabetic retinopathy (DR). STZ-induced T1DM rats received LA or ALA 100 μg/day intraperitoneally on alternate days for 21 days, and their blood glucose; lipid profile; plasma, hepatic, and retinal fatty acid profiles (by gas chromatography); retinal histology; activities of hepatic and retinal desaturases; and inflammatory markers (by qRT-PCR) were evaluated. Gut microbiota composition was assayed by 16S rDNA sequencing technology of the fecal samples, and their short-chain fatty acids and bile acids were assayed by gas chromatography, liquid chromatography coupled with tandem mass spectrometry, respectively. The human vitreal fatty acid profiles of subjects with proliferative DR and LXA4 levels were measured. LA and ALA significantly improved the plasma glucose and lipid levels; increased the abundance of Ruminococcaceae (the ALA-treated group), Alloprevotella, Prevotellaceae_Ga6A1_group, Ruminococcaceae_UCG_010, and Ruminococcus_1 (the LA-treated group) bacteria; enhanced acetate and butyrate levels; and augmented fecal and hepatic concentrations of cholic acid, chenodeoxycholic acid, and tauro ursodeoxycholic acid in ALA- and LA-treated animals. Significant STZ-induced decreases in plasma LA, gamma-linolenic acid, arachidonic acid, and ALA levels reverted to near normal, following LA and ALA treatments. Significant changes in the expression of desaturases; COX-2, 5-LOX, and 12-LOX enzymes; and cytokines in T1DM were reverted to near normal by EFAs. DR subjects also had low retinal LXA4 levels. The results of the present study show that ALA and LA are of significant benefit in reversing metabolism, gut microbiota, and inflammatory and retinal index changes seen in T1DM, suggesting that EFAs are of benefit in diabetes mellitus.

Retinal Pathophysiological Evaluation in a Rat Model.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.