Rat Eyes Research Articles

Paclitaxel and Urolithin A Prevent Histamine-Induced Neurovascular Breakdown Alike, in an Ex Vivo Rat Eye Model.

Neurovascular eye problems are better prevented than managed or treated. Despite growing concern of occurrence in aging populations and development secondary to diseases such as diabetes and hypertension, we currently have very few options to tackle this global problem. Creating effective and high-throughput screening strategies is as important as the intervention itself. Here, we present for the first time a robust ex vivo rat eye model of histamine-induced vascular damage for investigating the therapeutic potential of paclitaxel (PTX) and urolithin A (UA) as alternatives to dexamethasone for preventing vascular damage in the retina. Extensive loss of vascularization and apoptosis were observed in the histamine-challenged group and successfully prevented in the intervention groups, more significantly in the PTX and UA. These important early results indicate that PTX and UA could be developed as potential preventive strategies for a wide variety of retinal diseases.

Human Umbilical Mesenchymal Stem Cell Xenografts Repair UV-Induced Photokeratitis in a Rat Model.

Most patients with a corneal injury are administered anti-inflammatory medications and antibiotics, but no other treatments are currently available. Thus, the corneal injury healing is unsatisfactory, affects the vision, and has a risk of blindness in severe cases. Human umbilical mesenchymal stem cells exhibit pluripotent and anti-inflammatory properties and do not cause immunological rejection in the host. Rats were irradiated with type B ultraviolet (UVB) light to generate a stable animal model of photokeratitis. After irradiation-induced photokeratitis, human umbilical mesenchymal stem cells were implanted into the subconjunctival space of the lateral sclera, and the changes in the corneal pathology were evaluated. Three weeks after implantation, many mesenchymal stem cells were visible in the subconjunctival space. These mesenchymal stem cells effectively reduced the extent of injury to the adjacent corneal tissue. They accelerated the epithelial layer repair, reduced the inflammatory response and neovascularization, and improved the disorganization of collagen and fibronectin in the corneal stroma caused by the injury. In conclusion, xenografted human umbilical mesenchymal stem cells can survive in rat eye tissues for a long time, effectively support the structural integrity of injured corneal tissues, restore corneal permeability, and reduce abnormal neovascularization. This study provides a new approach to the treatment of photokeratitis.

Intravitreal and subretinal syngeneic bone marrow mononuclear stem cell transplantation improves photoreceptor survival but does not ameliorate retinal function in two rat models of retinal degeneration.

To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.

Sodium Hyaluronate-Induced Ocular Hypertension in Rats Damages the Direction-Selective Circuit and Inner/Outer Retinal Plexiform Layers.

PurposeTo assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension.MethodsIntraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry.ResultsIOP was higher in HYA-treated eyes than in sham-operated eyes along the 10-week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina.ConclusionsSerial HYA treatment in the rat anterior eye chamber results in mild-to-moderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma.

IMMUNOSTIMULANT POTENTIAL OF OYSTER MUSHROOM NUGGET (Pleourotus ostreatus)

Oyster mushrooms are widely known as having high beta glucan content. Beta glucan is a water insoluble fiber that indicates an immunomodulator with increasing the activity of NK cells in the bloodstream which provides immune effect. Oyster mushroom nuggets are processed oyster mushrooms that can be proven containing the highest beta glucans. This study aims to observe the immunostimulant potential of oyster mushroom nuggets in BALB/c mice by analyzing its white blood cell profile, spleen and liver histopathology. The research method is conducted through orbital sinus of the rat's eye using a microhematocrit to count total leukocytes, lymphocytes, and granulocytes with the Delayed Type Hypersensitivity test. The 18mg/kg body weight oyster mushroom nugget can increase lymphocytes cells 7.2 ± 1.22 103/µl. It is significantly different from control group. Immunostimulatory activity also was proven with the widening of the white pulp of the spleen with the growth of young lymphocytes after being given oyster mushroom nuggets of 18 mg/kg body weight.

Melatonin prevents experimental central serous chorioretinopathy in rats.

Central serous chorioretinopathy (CSC) is a vision-threatening disease with no validated treatment and unclear pathogenesis. It is characterized by dilation and leakage of choroidal vasculature, resulting in the accumulation of subretinal fluid, and serous detachment of the neurosensory retina. Numerous studies have demonstrated that melatonin had multiple protective effects against endothelial dysfunction, vascular inflammation, and blood-retinal barrier (BRB) breakdown. However, the effect of melatonin on CSC, and its exact pathogenesis, is not well understood thus far. In this study, an experimental model was established by intravitreal injection of aldosterone in rats, which mimicked the features of CSC. Our results found that melatonin administration in advance significantly inhibited aldosterone-induced choroidal thickening and vasodilation by reducing the expression of calcium-activated potassium channel KCa2.3, and attenuated tortuosity of choroid vessels. Moreover, melatonin protected the BRB integrity and prevented the decrease in tight junction protein (ZO-1, occludin, and claudin-1) levels in the rat model induced by aldosterone. Additionally, the data also showed that intraperitoneal injection of melatonin in advance inhibited aldosterone-induced macrophage/microglia infiltration, and remarkably diminished the levels of inflammatory cytokines (interleukin-6 [IL-6], IL-1β, and cyclooxygenase-2), chemokines (chemokine C-C motif ligand 3, and C-X-C motif ligand 1), and matrix metalloproteinases (MMP-2and MMP-9). Luzindole, as the nonselective MT1 and MT2 antagonist, and 4-phenyl-2-propionamidotetraline, as the selective MT2 antagonist, neutralized the melatonin-induced inhibition of choroidal thickening and choroidal vasodilation, indicating that melatonin might exert the effects via binding to its receptors. Furthermore, the IL-17A/nuclear factor-κB signaling pathway was activated by intravitreal administration of aldosterone, while it was suppressed in melatonin-treated in advance rat eyes. This study indicates that melatonin could serve as a promising safe therapeutic strategy for CSC patients.

Design and synthesis of endocannabinoid enzyme inhibitors for ocular indications

A small library of FAAH and dual FAAH/MAGL inhibitors designed for peripheral selectivity were targeted. Of these compounds, three were identified to have desirable FAAH inhibition and reduced permeability in a PAMPA assay. Those three compounds were advanced into a MAGL inhibitor assay and one was found to be a relative selective FAAH inhibitor, FAAH to MAGL IC50 ratio of 1:27, and one was found to be more characteristic of a true dual enzyme inhibitor, FAAH to MAGL IC50 ratio of 1:4. Both compounds showed activity in an ABPP assay, blockage of TAMRA-FP labeling of FAAH and MAGL in rat eye homogenate.

Therapeutic Properties of Ayahuasca Components in Ischemia/Reperfusion Injury of the Eye.

Ischemic eye diseases are major causes of vision impairment. Thus, potential retinoprotective effects of N’N-dimethyltryptamine (DMT) were investigated. To inhibit its rapid breakdown by monoamine-oxidase A (MAO-A) enzyme, DMT was co-administered with harmaline, a β-carboline in the Amazonian Ayahuasca brew. Using ligation, 60 min of ischemia was provoked in eyes of rats, followed by 7 days of reperfusion whilst animals received harmaline alone, DMT + harmaline, or vehicle treatment. After 1 week of reperfusion, electroretinographical (ERG) measurements, histological analysis, and Western blot were performed. Harmaline alone exhibited retinoprotection in ischemia–reperfusion (I/R) which was, surprisingly, counterbalanced by DMT in case of co-administration. As both MAO-A inhibition and DMT increase serotoninergic tone synergistically, communicated to be anti-ischemic, thus, involvement of other pathways was investigated. Based on our experiments, DMT and harmaline exert opposite effects on important ocular proteins such as PARP1, NFκB, MMP9, or HSP70, each having a critical role in a different mechanism of eye-ischemia-related pathologies, e.g., cell death, inflammation, tissue destruction, and oxidative stress. Since DMT is proclaimed to be a promising drug candidate, its potentially undesirable effect on eye-ischemia should be further investigated. Meanwhile, this experiment revealed the potential therapeutic effect of MAO-A inhibitor harmaline in I/R-related eye diseases.

Forkhead Domain Inhibitor-6 Suppresses Corneal Neovascularization and Subsequent Fibrosis After Alkali Burn in Rats

PurposeThe purpose of this study was to investigate the effects of Forkhead Domain Inhibitor-6 (FDI-6) on regulating inflammatory corneal angiogenesis and subsequent fibrosis induced by alkali burn.MethodsA corneal alkali burn model was established in Sprague Dawley rats using NaOH and the rat eyes were topically treated with FDI-6 (40 µM) or a control vehicle four times daily for 7 days. Corneal neovascularization, inflammation and epithelial defects were observed on days 1, 4, and 7 under a slit lamp microscope after corneal alkali burn. Analysis of angiogenesis-, inflammation-, and fibrosis-related indicators was conducted on day 7. Murine macrophages (RAW264.7 cells) and mouse retinal microvascular endothelial cells (MRMECs) were used to examine the effects of FDI-6 on inflammatory angiogenesis in vitro.ResultsTopical delivery of FDI-6 significantly attenuated alkali burn-induced corneal inflammation, neovascularization, and fibrosis. FDI-6 suppressed the expression of angiogenic factors (vascular epidermal growth factor, CD31, matrix metalloproteinase-9, and endothelial NO synthase), fibrotic factors (α-smooth muscle actin and fibronectin), and pro-inflammatory factor interleukin-6 in alkali-injured corneas. FDI-6 downregulated the expression of monocyte chemotactic protein-1, pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-alpha), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3, and vascular endothelial growth factor in RAW264.7 cells and inhibited the proliferation, migration, and tube formation of MRMECs in vitro.ConclusionsFDI-6 can attenuate corneal neovascularization, inflammation, and fibrosis in alkali-injured corneas.

Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

Evaluation of butyric acid as a potential supportive treatment in anterior uveitis

Background: The aim of the study was to evaluate the anti-inflammatory effect of topically administered aqueous sodium butyrate solution in an endotoxin-induced uveitis rat model and compare the results with corticosteroid treatment. Material and methods: Forty female Lewis rats were randomly divided into five groups. Uveitis was induced by a single lipopolysaccharide (LPS) injection into each footpad of each LPS+ rat. Group I (naive) received saline injected into the footpad of each rat at a dose of 0.1 mL/each footpad; Group II (LPS+) received saline solution topically. Group III (LPS+ Dex) — an aqueous dexamethasone sodium phosphate solution topically; Group IV (LPS+ But 0.5 mM) — 0.5 mM aqueous sodium butyrate solution topically, Group V (LPS+ But 1 mM) — 1.0 mM aqueous sodium butyrate solution topically. Clinical scoring of inflammation in rat eyes was evaluated before LPS injection and after 24 hours. The iris involvement, posterior synechiae presence, and insight into the eye fundus were clinicallyassessed. A histopathological examination was also performed. The rats were euthanatized 24 hours after LPS injection, and aqueous humor (AqH) was collected from the eyes by anterior chamber puncture. Levels of inflammatory cytokines and chemokines in the AqH were determined with commercially available Luminex assays. Results: Development of iris hyperemia associated with miosis and poor visibility of fundus details occurred 24 hours after LPS injection. Compared to the LPS+ group, the clinical scores were strongly suppressed in rats treated with Dexamethasone and moderately diminished in LPS+ But 0.5 mM. These clinical features were not observed in the controls (Group 1 — naive). Data from inflammatory cytokines evaluation indicates no significant differences between the LPS+ group (Group 2) and the LPS+ But groups (Groups 4 and 5). Histopathological examinations suggest that hyperemia, corneal stratification, and lesions were less common in the group of animals treated with BA in a lower concentration. Conclusion: Topical administration of sodium butyrate as a therapeutic agent might alleviate the severity of intraocular inflammation in eyes with uveitis. The effect of sodium butyrate was slight but clinically significant in 0.5 mM dose, so other doses of topically administered sodium butyrate should be considered and evaluated in further research.

Evolving Patterns of Hyperfluorescent Fundus Autofluorescence Accompany Retinal Atrophy in the Rat and Mimic Atrophic Age-Related Macular Degeneration

PurposeComplex two-dimensional (2D) patterns of hyperfluorescent short-wave fundus autofluorescence (FAF) at the border of geographic atrophy (GA) can predict its expansion in patients with late non-exudative “dry” AMD. However, preclinical models do not phenocopy this important feature of disease. We sought to describe the spatiotemporal changes in hyperfluorescent FAF patterns that occur following acute oxidative stress, potentially in association with GA expansion.MethodsSprague Dawley rats (n = 54) received systemic sodium iodate (25–45 mg/kg, n = 90 eyes) or saline (n = 18 eyes) and underwent serial full fundus imaging by confocal scanning laser ophthalmoscopy, including blue FAF and delayed near-infrared analysis. Composite images of the fundus were assembled, and the 2D patterns were described qualitatively and quantitatively. A subset of eyes underwent tissue analysis, and four underwent optical coherence tomography (OCT) imaging.ResultsReproducibly changing, complex patterns of hyperfluorescent FAF emerge at the borders of toxin-induced damage; however, in the absence of GA expansion, they percolate inward within the region of retinal pigment epithelium loss, evolving, maturing, and senescing in situ over time. Unexpectedly, the late FAF patterns most closely resemble the diffuse tricking form of clinical disease. A five-stage classification system is presented.ConclusionsLongitudinal, full-fundus imaging of outer retinal atrophy in the rat eye identifies evolving, complex patterns of hyperfluorescent FAF that phenocopy aspects of disease.Translational RelevanceThis work provides a novel tool to assess hyperfluorescent FAF in association with progressive retinal atrophy, a therapeutic target in late AMD.

Biodistribution of Cy5-labeled Thiolated and Methylated Chitosan-Carboxymethyl Dextran Nanoparticles in an Animal Model of Retinoblastoma.

PurposeThe use of more potent medicine for local chemotherapy of retinoblastoma in order to minimize local and systemic adverse effects is essential. The main goal of this investigation was to assess the biodistribution of thiolated and methylated chitosan-carboxymethyl dextran nanoparticles (CMD-TCs-NPs and CMD-TMC-NPs) following intravitreal (IVT) injection into rat eyes with retinoblastoma.MethodsAn ionic gelation method was used to fabricate Cy5-labelled CMD-TCs-NPs and CMD-TMC-NPs. The NPs were characterized. Cellular internalization of Cy5-labelled NPs was investigated using confocal microscopy and the absorption of labeled NPs was quantified by flow cytometry in human retinoblastoma (Y79) cells. In addition, the Cy5-labeled distribution of nanoparticles in the posterior segment of the eye was histologically imaged by confocal microscopy after IVT injection of NPs into the eyes of rats with retinoblastoma.ResultsCMD-TCs-NPs and CMD-TMC-NPs showed a mean diameter of 34 3.78 nm and 42 4.23 nm and zeta potential of +11 2.27 mV and +29 4.31mV, respectively. The in vivo study of intraocular biodistribution of Cy5-labeled CMD-TCs-NPs and CMD-TMC-NPs revealed that there is more affinity of CMD-TCs-NPs to the retina and retinoblastoma tumor after IVT administration while methylated chitosan nanoparticles are immobilized in the vitreous and are not able to reach the retina even after 24 hr.ConclusionThe ionic gelation technique was efficient in synthesizing a biocompatible polymeric nanosystem for drug delivery into the posterior segment of the eye. The current study demonstrated increased ocular bioavailability of CMD-TCs-NPs relative to CMD-TMC-NPs in retinoblastoma induced rat eyes.

Changes in Thiols Indicators in the Lens of the Eye of White Rats under the Exposure of Radiation during Operation of a Cellular Mobile Phone

The results of previous work carried out at the Laboratory of Biophysics of Cellular Metabolism of the Institute of Physiology named after A.I. Karaev, which concerned the study of the effect of EMR on LPO processes and the antioxidant system in visual structures, pointed to the oxidative nature of the implementation of EMR exposure. Since thiol compounds in living tissues primarily interact with peroxide products, it was assumed that the effect of EMR should be reflected in the thiol content of the lens. For this purpose, a group of white outbred rats (40 species, half of which were identified as the control group) were studied, which were daily irradiated with EMR from a working cell phone. On the 7th, 14th, 21st and 28th day of the experiment, 5 animals from the experimental group and 5 animals from the control group were slaughtered. The lens of the eye was removed from the animals, in which the level of thiols was determined according to the modified Lindsey-Sedlak method based on the Ellman method. The results of the study showed that common and readily available (cytoplasmic) thiols in the nucleus of the lens of the eyes of rats irradiated with mobile phone EMR, in general, have similar dynamics of quantitative changes depending on the duration of exposure. Hidden thiols behave similarly. In the lens cortex, the nature of changes in thiols is not so pronounced, but generally similar to the results in the nucleus. The authors believe that these results may indicate the antioxidant properties of thiols in LPO reactions.

Glial cell response to constant low light exposure in rat retina.

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

Investigating the effects of docetaxel on rat eye

AbstractPurposeDocetaxel (DTX) is a widely used chemotherapeutic agent in breast, prostate and lung cancers. Approximetly 60% of patients face ocular disorders including epiphora, stye, blepharedema, dacryostenosis, corneal and conjunctival disorders, entropion and blurred vision. The aim of this study is to investigate the effects of DTX on rat eyeMethodsThis study mainly planned to assess the alopecia effect of DTX, but the remained eye tissues have been evaluated to decrease the animal number used in medical researches. 6‐8 months old albino male Wistar rats (n = 7) were given 10 mg/kg intraperitoneally (i.p) DTX treatment three times at the 0th, 8th, 15th days. Control group (n = 6) was administered serum physiologic i.p once a week. The ocular tissues are taken at the 22nd day and evaluated by Hematoxylin&Eosin staining. TGF‐beta and TNF‐alfa stains were planned.ResultsPreliminary results of this study revealed conjunctival epithelial degeneration, corneal neovascularization, mild‐severe retinal nerve fibre layer degeneration, inflammation and cystic dilatation of lacrimal gland, increased capillary vessel density and vacuolar degeneration of optic nerve. The findings were demonstrated in Fig 1,2 and 3.ConclusionsDTX cause serious ocular disorders and this is the first experimental study investigating these effects histologically. Increasing number of cancer cases and frequent use of Docetaxel, urges ophthalmologist to be aware of its ocular side effects.

Effects of Brimonidine on Microglia Morphology in the Transient Retinal Ischemia Model

Background: This study aimed to investigate the effects of brimonidine on microglia cell morphology by creating a transient retinal ischemia model in rats. Methods: In the right eyes of male Wistar rats (n = 12), a transient retinal ischemia model was created. The rats were divided into three groups: (1) eyes treated with topical brimonidine in the transient retinal ischemia model, (2) shamtreated eyes, and (3) control eyes. Four main phenotypes (ramified, primed, reactive, and amoeboid-phagocytic) of Iba-1 positive microglia cells were examined in the retinal layers. Results: In the transient retinal ischemia model, the number of Iba-1 positive microglia cells was 100.67 ± 7.50 cells in the sham group and 57.67 ± 14.64 cells in the topical brimonidine group. The decrease in the total number of Iba-1 positive microglial cells was statistically significant (p < 0.05). When we compared ramified and primed microglia cells, there was no statistically significant difference between the groups. The number of amoeboidphagocytic microglial cells was 9.5 ± 1.29 cells in the sham treatment group and 2.25 ± 0.50 cells in the topical brimonidine group (p < 0.05). There was a statistically significant decrease in the number of reactive and amoeboid cells. Conclusion: Studies in the central nervous system in rats demonstrated that cell metabolism and functions were changed after acute injury and that microglia transformed from a ramified form to an amoeboid-phagocytic form. In this study, the decrease in the transition of cells from ramified to reactive and amoeboid forms showed us that topical brimonidine treatment could have neuroinflammation suppressor features.

Analysis of vitreous opacities in chronic glaucoma by vitreo‐optical coherence tomography imaging

AbstractPurposeTo analyze the vitreous opacities of rat eyes by optical coherence tomography (OCT) in two models of chronic glaucoma. Hyperreflective opacities at the vitreoretinal interface, their dynamics over time (changes in number, size, intensity, eccentricity, and orientation) and as a function of intraocular pressure were analyzed in two different glaucoma models and compared to healthy controls.MethodsImages of the vitreoretinal interface were obtained by OCT and exported as videos (composed of cross‐sectional images acquired by means of 61 b‐scans measuring around 3 mm in length and centred on the optic nerve). These videos were analysed using a custom program implemented in Matlab. The vitreous opacities, closely related to the immune cells, were analysed.ResultsHyperreflective opacities, extrapolated as soma areas, were classified according to their size: isolated cells (<10 μm2), non‐activated cells (10–50 μm2), activated cells (50–250 μm2) and cell complexes (>250 μm2). In glaucoma models, right eyes showed significant increase of total area compared with left non‐induced eyes, and left eyes showed significant increase compared to healthy controls. The physiological number of opacities was 10–20. Glaucoma‐induced right eyes showed a mean of 70 opacities, a specular response between activated cells and non‐activated cells, and a greater variation in intensity in the smallest cells, that showed an early change in orientation compared with physiological eyes.ConclusionsOur method of analyzing vitreous opacities by OCT could serve as a reliable neuroimaging biomarker to detect disease onset and early progression applicable to glaucoma and potentially other neurodegenerative diseases impacting the retina and optic nerve.

A modified high-yield method for primary culture of rat retinal microglial cells

Microglial cells are the main immune cells of the retina. The primary culture of the retinal microglia is critically important in investigating the cells' properties and behaviors in neurodegenerative and inflammatory retinal disease. Here, we described a modified protocol of a microglial cell culture from the neonatal rat retina. In our culture protocol, the retina was isolated from the neonatal rat eye from postnatal day 1 to day 3 and trypsinized into a single-cell suspension. The cells were seeded into a T75 flask, which was pre-coated with poly-D-lysine (PDL) and cultured with dulbecco's modified eagle medium-F12 (DMEM/F12) that contained 10% fetal bovine serum (FBS) with different concentrations. Small bright rounded cells were observed on the top of mixed glial cells on the seventh day, and attained the maximum cell number on the 14th day. Then, the isolation was performed by a shaking method and isolated cells were identified with microglia markers ionized calcium-binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), cluster of differentiation 11b (CD11b), as well as astrocyte marker glial fibrillary acidic protein (GFAP) by immunofluorescence staining. Additionally, the initial plating ratio of the mixed glial cell, culture period of isolation, procedures of the isolation, as well as the purification procedure, were optimized for our primary microglial cell culture. The morphological changes and phagocytic function were performed after lipopolysaccharide (LPS) stimulation. Moreover, the release of pro-inflammatory cytokines at different time points of LPS activation were measured. In the present study, we found that the concentration of one retina/T75 flask could harvest the largest number of microglial cells. Besides, we continuously cultured the mixed glial cells as long as one month and isolated the mixed glial cells as much as three times. In our study, we used an isolation-shaking rate of 200 rpm for 2h, which guaranteed the steady rate and resulted in high purification of the primary retinal-microglial cells, with no need of an additional purification procedure. In conclusion, we provided a high-producing protocol for the primary culture of purified rat retinal-microglial cells.

Pharmacokinetics of Pullulan-Dexamethasone Conjugates in Retinal Drug Delivery.

The treatment of retinal diseases by intravitreal injections requires frequent administration unless drug delivery systems with long retention and controlled release are used. In this work, we focused on pullulan (≈67 kDa) conjugates of dexamethasone as therapeutic systems for intravitreal administration. The pullulan–dexamethasone conjugates self-assemble into negatively charged nanoparticles (average size 326 ± 29 nm). Intravitreal injections of pullulan and pullulan–dexamethasone were safe in mouse, rat and rabbit eyes. Fluorescently labeled pullulan particles showed prolonged retention in the vitreous and they were almost completely eliminated via aqueous humor outflow. Pullulan conjugates also distributed to the retina via Müller glial cells when tested in ex vivo retina explants and in vivo. Pharmacokinetic simulations showed that pullulan–dexamethasone conjugates may release free and active dexamethasone in the vitreous humor for over 16 days, even though a large fraction of dexamethasone may be eliminated from the eye as bound pullulan–dexamethasone. We conclude that pullulan based drug conjugates are promising intravitreal drug delivery systems as they may reduce injection frequency and deliver drugs into the retinal cells.