Rabbit Retina Research Articles

A p75 neurotrophin receptor-sparing nerve growth factor protects retinal ganglion cells from neurodegeneration by targeting microglia.

Retinal ganglion cells (RGCs) are the output stage of retinal information processing, via their axons forming the optic nerve (ON). ON damage leads to axonal degeneration and death of RGCs, and results in vision impairment. Nerve growth factor (NGF) signalling is crucial for RGC operations and visual functions. Here, we investigate a new neuroprotective mechanism of a novel therapeutic candidate, a p75-less, TrkA-biased NGF agonist (hNGFp) in rat RGC degeneration, in comparison with wild type human NGF (hNGFwt). Both neonate and adult rats, whether subjected or not to ON lesion, were treated with intravitreal injections or eye drops containing either hNGFp or hNGFwt. Different doses of the drugs were administered at days 1, 4 or 7 after injury for a maximum of 10days, when immunofluorescence, electrophysiology, cellular morphology, cytokine array and behaviour studies were carried out. Pharmacokinetic evaluation was performed on rabbits treated with hNGFp ocular drops. hNGFp exerted a potent RGC neuroprotection by acting on microglia cells, and outperformed hNGFwt in rescuing RGC degeneration and reducing inflammatory molecules. Delayed use of hNGFp after ON lesion resulted in better outcomes compared with treatment with hNGFwt. Moreover, hNGFp-based ocular drops were less algogenic than hNGFwt. Pharmacokinetic measurements revealed that biologically relevant quantities of hNGFp were found in the rabbit retina. Our data point to microglia as a new cell target through which NGF-induced TrkA signalling exerts neuroprotection of the RGC, emphasizing hNGFp as a powerful treatment to tackle retinal degeneration.

Fractal phototherapy: impact on the structure and function of the retina of rabbits with modelled retinal pigment epithelium atrophy

It is believed that in degenerative diseases of the retina, photostimulation by fractal dynamics signals activates neuroplasticity, thereby increasing the efficiency of visual rehabilitation. Previously, we showed a positive effect of fractal phototherapy (FF) on the electroretinogram (ERG) of healthy rabbits and demonstrated the safety of long-term photostimulation courses for the retina. The purpose of this work is to study the effect of FF on the functional activity and morphology of the retina in rabbits with a model of retinal pathology. Material and methods. We modelled an atrophy of retinal pigment epithelium (RPE) on both eyes of 50 rabbits. 30 days after the administration of bevacizumab, the animals were divided into two groups of 25 animals each. In the main group, photostimulation was performed using a device for FF, while in the control group incandescent lamps were used that create radiation of constant intensity. In both groups, 20-minute binocular light stimulation sessions were performed daily, five times a week. ERG and optical coherence tomography of the retina were performed before and after courses of treatment which lasted 1 week, 1 and 3 months. Results. Long-term courses of FF were shown to be safe for the morphology of the retina of animals with the RPE atrophy model. In all periods of observation, biochemical studies revealed no statistically significant changes in the content of norepinephrine and dopamine in the tear as compared with baseline values. In the main group, a slight positive effect of FF on rod and cone ERG was found after 5 FF sessions, while a significant increase in the amplitude of the transient and steady-state pattern-ERG (PERG), most pronounced after a 1-month FF course, was observed. Conclusions. A positive effect of FF on the functional activity of retinal ganglion cells (RGC) may suggest that prescribing a course of FF lasting up to 1 month (20 sessions) in diseases accompanied by a pathology of RGC is advisable, whereas for patients with a pathology of the macular region, such as AMD, an effective improvement in the activity of photoreceptors and bipolar cells could probably be achieved through a 1-week course of FF, conducted under the control of electroretinography.

In Vivo Ocular Pharmacokinetics and Toxicity of Siponimod in Albino Rabbits.

Siponimod is a promising agent for the inhibition of ocular neovascularization in diabetic retinopathy and age-related macular degeneration. Siponimod's development for ophthalmological application is hindered by the limited information available on the drug's solubility, stability, ocular pharmacokinetics (PK), and toxicity in vivo. In this study, we investigated the aqueous stability of siponimod under stress conditions (up to 60 °C) and its degradation behavior in solution. Additionally, siponimod's ocular PK and toxicity were investigated using intravitreal injection of two different doses (either 1300 or 6500 ng) in an albino rabbit model. Siponimod concentration was quantified in the extracted vitreous, and the PK parameters were calculated. The drug half-life after administration of the low and high doses was 2.8 and 3.9 h, respectively. The data obtained in vivo was used to test the ability of published in silico models to predict siponimod's PK accurately. Two models that correlated siponimod's molecular descriptors with its elimination from the vitreous closely predicted the half-life. Furthermore, 24 h and 7 days after intravitreal injections, the retinas showed no signs of toxicity. This study provides important information necessary for the formulation and development of siponimod for ophthalmologic applications. The short half-life of siponimod necessitates the development of a sustained drug delivery system to maintain therapeutic concentrations over an extended period, while the lack of short-term ocular toxicity observed in the retinas of siponimod-treated rabbits supports possible clinical use.

Topical Solution for Retinal Delivery: Bevacizumab and Ranibizumab Eye Drops in Anti-Aggregation Formula (AAF) in Rabbits

PurposeWet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF).MethodsBevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent.ResultsBevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies.ConclusionsBevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

No side effects on rabbit retina or vitreous microenvironment by nd:YAG laser vitreolysis.

To explore the safety of Neodymium:Yttrium-aluminum-garnet (Nd:YAG) laser vitreolysis based on the histological examination of the retina and the alteration of vitreous cytokines in the rabbits. Nine male New Zealand rabbits underwent Nd:YAG laser vitreolysis of 10mJ x 500 pulses in the left eyes, while the right eyes were used as controls. Intraocular pressure, color fundus photography, and ultrasound B scan were measured before, as well as 1day, 4 weeks, and 12 weeks after Nd:YAG laser vitreolysis. Three rabbits were euthanized 1day, 4 weeks, and 12 weeks after treatment, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and hematoxylin-eosin (H&E) staining were used to look for pathological changes in the retina. An enzyme-linked immunosorbent assay (ELISA) was utilized to detect the expression of vascular endothelial growth factor (VEGF) and some inflammatory cytokines, including interferon inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1) and interlenkin 6 (IL-6) in the vitreous humor. The ascorbic acid (AsA) and total reactive antioxidant potential (TRAP) in the vitreous humor were also measured. Following Nd:YAG laser vitreolysis, the levels of VEGF, IP-10, MCP-1, IL6, AsA, and TRAP in the vitreous humor did not change substantially (P > 0.05). There were no detectable pathological changes in the retinal tissues, and no apoptotic signal was found. Rabbits tolerate Nd:YAG laser vitreolysis without observable impact on retinal tissue or the microenvironment of the vitreous.

Effect of epiretinal electrical stimulation on the glial cells in a rabbit retinal eyecup model.

We examined how pulse train electrical stimulation of the inner surface of the rabbit retina effected the resident glial cells. We used a rabbit retinal eyecup preparation model, transparent stimulus electrodes, and optical coherence tomography (OCT). The endfeet of Müller glia processes line the inner limiting membrane (ILM). To examine how epiretinal electrode stimulation affected the Müller glia, we labeled them post stimulation using antibodies against soluble glutamine synthetase (GS). After 5 min 50 Hz pulse train stimulation 30 μm from the surface, the retina was fixed, immunostained for Müller glia, and examined using confocal microscopic reconstruction. Stimulus pulse charge densities between 133-749 μC/cm2/ph were examined. High charge density stimulation (442-749 μC/cm2/ph) caused significant losses in the GS immunofluorescence of the Müller glia endfeet under the electrode. This loss of immunofluorescence was correlated with stimuli causing ILM detachment when measured using OCT. Müller cells show potassium conductances at rest that are blocked by barium ions. Using 30 msec 20 μA stimulus current pulses across the eyecup, the change in transretinal resistance was examined by adding barium to the Ringer. Barium caused little change in the transretinal resistance, suggesting under low charge density stimulus pulse conditions, the Müller cell radial conductance pathway for these stimulus currents was small. To examine how epiretinal electrode stimulation affected the microglia, we used lectin staining 0-4 h post stimulation. After stimulation at high charge densities 749 μC/cm2/ph, the microglia under the electrode appeared rounded, while the local microglia outside the electrode responded to the stimulated retina by process orientation inwards in a ring by 30 min post stimulation. Our study of glial cells in a rabbit eyecup model using transparent electrode imaging suggests that epiretinal electrical stimulation at high pulse charge densities, can injure the Müller and microglia cells lining the inner retinal surface in addition to ganglion cells.

Temporospatial dynamics of the morphogenesis of the rabbit retina from prenatal to postnatal life: Light and electron microscopic study.

The retina consists of various cell types arranged in eight cell layers and two membranes that originate from the neuroectodermal cells. In this study, the timing of differentiation and distribution of the cellular components and the layers of the rabbit retina are investigated using light and electron microscopy and immunohistochemical techniques. There were 32 rabbit embryos and 12 rabbits used. The rabbit retina begins its prenatal development on the 10th day of gestation in the form of optic cup. The process of neuro- and gliogenesis occurs in several stages: In the first stage, the ganglionic cells are differentiated at the 15th day. The second stage includes the differentiation of Muller, amacrine, and cone cells on the 23rd day. The differentiation of bipolar, horizontal, and rod cells and formation of the inner segments of the photoreceptors consider the late stage that occurs by the 27th and 30th day of gestation. On the first week of age postnatally, the outer segments of the photoreceptors are developed. S100 protein is expressed by the Muller cells and its processes that traverse the retina from the outer to the inner limiting membranes. Calretinin is intensely labeled within the amacrine and displaced amacrine cells. Ganglionic cells exhibited moderate immunoreactivity for calretinin confined to their cytoplasm and dendrites. In conclusion, all stages of neuro- and gliogenesis of the rabbit retina occur during the embryonic period. Then, the retina continues its development postnatally by formation of the photoreceptor outer segments and all layers of the retina become established. RESEARCH HIGHLIGHTS: The aim of this study is to investigate the morphogenesis of the rabbit retina during pre- and postnatal life. The primordia of the retina could be observed in the form of the optic cup. The ganglionic cells are the first cells to differentiate, while the photoreceptor cells are the last. S100 protein is expressed by the Muller cells and its processes. Calretinin is intensely labeled in the amacrine and displaced amacrine cells and moderately expressed in the cytoplasm and dendrites of ganglionic cells.

Clinical and pathomorphological changes in the rabbit retina after an injection of various doses of the cytostatic melphalan

Background: In recent years, there have been individual reports on intracameral chemotherapy (ICC) for aqueous seeding in retinoblastoma. The effect of melphalan on the structures of the ocular anterior segment (including the cornea, iris and anterior lens capsule) is however, still unknown, since no relevant experimental studies have been carried out so far. Purpose: To experimentally assess the changes in the rabbit anterior segment induced by intracameral injection of various concentrations of the alkylating cytostatic melphalan. Material and Methods: Twelve adult Chinchilla rabbits (22 eyes; age, 5–6 months; weight, 2.5–3 kg) were involved in this experimental study and maintained in the vivarium of the Filatov institute in separate cages under standard conditions. Results: After a 5-µg melphalan injection, corneal and iris changes were reversible and the lens was still clear. With an increase in melphalan concentration in injection solution (to 10, 15 and 20 µg) and time point (to 1 month and 3 weeks) after injection, degenerative changes in some epithelial cells of the iris became irreversible, anterior capsular cataract developed, but the cornea and anterior chamber aqueous remained clear. After a single 20-µg intracameral injection of melphalan, there was depigmentation of the iris, posterior synechia and anterior capsular cataract. Conclusion: Clinical and ultrastructural responses of ocular anterior segment tissue to intracameral melphalan injection depended on the injected dose and time point after injection. Most cells of examined tissues showed the capability to restore their ultrastructure following ceasing of the toxic effect of the drug.

The effect of low-frequency ultrasound on the functional and morphological state of the retina of rabbits

Currently, three groups of scientists are developing the introduction and use of lowfrequency ultrasound in retinal and vitreous surgery. There is not enough data and work to study the effects of this type of energy on the functional activity and structural state of the retina. In this regard, we conducted experimental and diagnostic studies using modern and objective methods. The main purpose of this article is analyzing the parameters of the electroretinogram of the rabbit retina and compare it with histological data after removal of the vitreous body using low-frequency ultrasound and mechanical action. Experiments were conducted on Chinchilla rabbits (n=40). In the experimental group (n=20), vitreous removal was performed using low-frequency ultrasound, in the control group (n=20) using a guillotine mechanism. On the 1st, 7th, 14th and 14th days, the parameters of the a- and b-waves of the electroretinogram were recorded. The structural state of the retina was also evaluated on histological sections. On the 1st day after surgery in both study groups there was a decrease in all parameters of the electroretinogram, the evaluation of histological data showed the presence of edema in most layers of the retina. On days 7 and 14, the parameters of the a- and b-waves were restored, but did not return to normal in both groups, a decrease in edema in all layers of the retina was observed on histological preparations. On day 30, the a- and b-wave indicators returned to normal, which indicates the restoration of the functional properties of photoreceptor cells, bipolar and Muller cells in both groups, the retinal layers were clearly visualized on histological sections without signs of edema. Analysis of electroretinogram parameters and histological data showed that the use of low-frequency ultrasound for vitreous removal can be considered safe and promising for further development.

TARPγ2 Is Required for Normal AMPA Receptor Expression and Function in Direction-Selective Circuits of the Mammalian Retina.

AMPA receptors (AMPARs) are the major mediators of fast excitatory neurotransmission in the retina as in other parts of the brain. In most neurons, the synaptic targeting, pharmacology, and function of AMPARs are influenced by auxiliary subunits including the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is unclear which TARP subunits are present at retinal synapses and how they influence receptor localization and function. Here, we show that TARPɣ2 (stargazin) is associated with AMPARs in the synaptic layers of the mouse, rabbit, macaque, and human retina. In most species, TARPɣ2 expression was high where starburst amacrine cells (SACs) ramify and transcriptomic analyses suggest correspondingly high gene expression in mouse and human SACs. Synaptic expression of GluA2, GluA3, and GluA4 was significantly reduced in a mouse mutant lacking TARPɣ2 expression (stargazer mouse; stg), whereas GluA1 levels were unaffected. AMPAR-mediated light-evoked EPSCs in ON-SACs from stg mice were ∼30% smaller compared with heterozygous littermates. There was also loss of a transient ON pathway-driven GABAergic input to ON-SACs in stg mutants. Direction-selective ganglion cells in the stg mouse showed normal directional tuning, but their surround inhibition and thus spatial tuning was reduced. Our results indicate that TARPɣ2 is required for normal synaptic expression of GluA2, GluA3, and GluA4 in the inner retina. The presence of residual AMPAR expression in the stargazer mutant suggests that other TARP subunits may compensate in the absence of TARPɣ2.

Congenital cataracts affect the retinal visual cycle and mitochondrial function: A multi-omics study of GJA8 knockout rabbits

Congenital cataracts are a threat to visual development in children, and the visual impairment persists after surgical treatment; however, the mechanisms involved remain unclear. Previous clinical studies have identified the effect of congenital cataracts on retinal morphology and function. To further understand the molecular mechanisms by which congenital cataracts affect retinal development, we analyzed retina samples from 7-week-old GJA8-knockout rabbits with congenital cataracts and controls by four-dimensional label-free quantification proteomics and untargeted metabolomics. Bioinformatics analysis of proteomic data showed that retinol metabolism, oxidative phosphorylation, and fatty acid degradation pathways were downregulated in the retinas of rabbits with congenital cataracts, indicating that their visual cycle and mitochondrial function were affected. Additional validation of differentially abundant proteins related to the visual cycle and mitochondrial function was performed using Parallel reaction monitoring and western blot experiments. Untargeted metabolome analysis showed significant upregulation of the antioxidant glutathione and ascorbic acid in the retinas of rabbits with congenital cataracts, indicating that their oxidative stress balance was not dysregulated. SignificanceCongenital cataracts in children can alter retinal structure and function, yet the mechanisms are uncertain. Here is the first study to use proteomics and metabolomics approaches to investigate the effects of congenital cataracts on retinal development in the early postnatal period. Our findings suggest that congenital cataracts have an impact on the retinal visual cycle and mitochondrial function. These findings give insight on the molecular pathways behind congenital cataract-induced visual function impairment in the early postnatal period.

Aflibercept Suppression of angiopoietin-2 in a Rabbit Retinal Vascular Hyperpermeability Model.

Anti-vascular endothelial growth factor (anti-VEGF) therapies, which attenuate the capacity of VEGF to bind to VEGF receptors, are standard-of-care options for various retinal disorders that are characterized by pathologic retinal angiogenesis and vascular permeability. Multiple receptors and ligands have also been reported as being involved in these pathways, including angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). Electrochemiluminescence immunoassays were used to detect human VEGF (hVEGF), as well as rabbit ANG2 and basic fibroblast growth factor protein levels in vitreous samples derived from a study evaluating the efficacy of the anti-VEGF agents ranibizumab, aflibercept, and brolucizumab in an hVEGF165-induced rabbit retinal vascular hyperpermeability model. hVEGF was completely suppressed in rabbit vitreous after anti-VEGF treatment for 28 days. ANG2 protein in vitreous and ANGPT2 mRNA in retina tissue were similarly suppressed, although the anti-VEGF agents do not directly bind to ANG2. Aflibercept demonstrated the greatest inhibitory effect in ANG2 levels in vitreous, which correlated with strong, durable suppression of intraocular hVEGF levels. This study explored the effects of anti-VEGF therapies beyond direct binding of VEGF by evaluating protein levels and the expression of target genes involved in angiogenesis and associated molecular mechanisms in the rabbit retina and choroid. In vivo data suggest that anti-VEGF agents currently used for the treatment of retinal diseases could provide beneficial effects beyond direct binding of VEGF, including suppression of ANG2 protein and ANGPT2 mRNA.

Электронно-микроскопическое исследование зрительного нерва с токсическим невритом в эксперименте

Рurpose. To identify ultrastructural changes of the optic nerve in the area of the trellis plate in rabbits after exposure to methyl alcohol. Material and methods. A model of experimental optic neuritis with the introduction of methyl alcohol into the retrobulbar space was reproduced on 8 rabbits. The animals were removed from the experiment on the 14th, 30th, 90th and 180th days after the administration of methanol. Pieces of the optic nerve (1–2 mm) were isolated from the enucleated eyeballs in the area of the lattice plate and examined using a transmission electron microscope. Results. On the 14th day after the administration of methanol, signs of pronounced cytoplasmic edema and destruction of intracellular organelles were detected in the ganglion neurons of the rabbit retina in the area of the lattice plate. In individual nerve fibers of the optic nerve, myelin sheaths were stratified and unfolded, axons swelled. Structural changes were detected in astrocytes, especially in the processes around blood vessels. Macrophages of nervous tissue — microgliocytes — became active. Morphological changes indicated a violation of microcirculation and permeability of the hematoneural barrier. The basal membranes around the walls of blood vessels were compacted. Intracellular organelles were destroyed in the cytoplasm of endothelial cells, hemostasis was determined inside the vessels. Subsequently, on the 30–90th day of the experiment, dystrophic and destructive changes in the cytoplasm of ganglion cells and their processes forming the optic nerve, as well as surrounding gliocytes progressed. There were signs of gliofibrosis. Destructive processes and gliofibrosis of the retina and nerve fibers of the optic nerve in the area of the lattice plate increased in degree of expression up to 180th days of the experiment. Conclusion. The results of electron microscopic studies of the intrabulbar part of the optic nerve in experimental animals in dynamics after the introduction of methyl alcohol indicate that the main trigger for the development of visual neuritis may be a violation of microcirculation processes in the retina and optic nerve, which leads to an increase in the permeability of the hematoneural barrier. These events entail dystrophic and destructive changes in the nervous tissue, the progression of which naturally leads, with the assistance of glial cells to gliofibrosis of the optic nerve. Keywords: toxic optic neuritis, methanol, hematoneural barrier, axon demyelination, gliofibrosis

Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.

The light pathways are segregated into rod and cone pathways in which rods synapse with rod bipolar cells (RBCs), while cones contact cone bipolar cells (CBCs). However, previous studies found that cones can make synapse with RBCs (cone-RBC synapses) and rods can contact OFF CBCin primate and rabbit retinas. Recently, such cone-RBC synapses have been reported physiologically and morphologically in the mouse retina. Nevertheless, the precise subcellular evidence to determine whether it is the invaginating synapse or the flat contact remains absent. This is due to a lack of immunochemically verified ultrastructural data. Here, we investigated the precise expression of protein kinase C alpha (PKCα) using pre-embedding immunoelectron microscopy (immuno-EM) with a monoclonal antibody against PKCα, a biomarker for the RBCs. We determined the nanoscale localization of PKCα in the outer plexiform layer of the mouse and guinea pig retinas. Our results demonstrate the existence of both the direct invaginating synapse and the basal/flat contact of the cone-RBCs, providing for the first time immunochemically verified ultrastructural evidence for the cone-RBC synapse in the mouse and guinea pig retinas. These results suggest that the cross talk between cone and rod pathways is much more extensive than previously assumed.

LC/MS Assessment of Glycoform Clearance of A Biotherapeutic MAb in Rabbit Ocular Tissues

Many biotherapeutics such as monoclonal antibodies (mAbs) consist of various glycoforms, which can have different PK properties upon administration to animals and human. As a result, it is necessary to monitor the abundance of glycoforms and limit lot-to-lot variability during the manufacturing process. However, limited information is known about the clearance of mAb glycoforms from ocular space upon intravitreal injection. We present here an assessment of glycoform clearance of a biotherapeutic mAb (IgG1) from rabbit vitreous humor, aqueous humor and retina tissue using LC/MS. The results show that G0, G0F and G1F have similar T1/2, while mannose-5 has a longer T1/2 and is cleared slower in rabbit ocular space, which contradicted with what has been reported in the literature in which Mann5 was cleared faster systematically.

A novel recombinant human microplasminogen induced complete posterior vitreous detachment without morphological change of retina in juvenile rabbits

Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhμPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhμPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh μPlg could be activated by t-PA to generate microplasmin (μPlm). Moreover, rhμPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 μg rhμPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhμPlg has a potential value in the treatment of vitreoretinopathy.

Vitreoretinal Surgery with Temperature Management: A Preliminary Study in Rabbits.

The present study aimed to evaluate the structure of the rabbit retina after vitreoretinal surgery using prolonged irrigation with solutions of different temperatures. Thirty-six rabbits (72 eyes) were included in this study and randomly divided into 3 equal groups according to the temperature of the intraocular irrigating fluid they received during vitrectomy. Vitreoretinal surgery was performed with a 5°C irrigation solution in group 1 (12 rabbits, 24 eyes), a 22°C irrigation solution in group 2 (12 rabbits, 24 eyes), and a 36°C irrigation solution in group 3 (12 rabbits, 24 eyes). In each group of animals, the mean irrigation/aspiration time was 30 minutes for left eyes and 60 minutes for right eyes. Histological examination of the retina was performed 1, 7, and 30 days after surgery. During surgery, the temperature in the vitreous cavity of the eyes of rabbits of groups 1, 2, and 3 dropped by 26.0°C, 11.2°C (deep hypothermia), and 1.0°C (mild hypothermia), respectively. The highest rewarming rate was detected in group 1 (0.9°C/min) compared with group 2 (0.7°C/min) and group 3 (0.2°C/min). After 60 minutes of irrigation, retinal structural changes were detected in the animals of groups 1 and 2 (in contrast to the animals of group 3). After surgery with irrigation lasting 30 minutes, no retinal structural changes were observed. This study showed that temperature management, avoidance of intraoperative deep hypothermia, and prevention of rapid uncontrolled rewarming may protect the retinal morphology and increase the safety of prolonged vitreoretinal surgery.

Impact of Retinal Degeneration on Response of ON and OFF Cone Bipolar Cells to Electrical Stimulation.

In retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), the photoreceptors become stressed and start to degenerate in the early stages of the disease. Retinal prosthetic devices have been developed to restore vision in patients by applying electrical stimulation to the surviving retinal cells. However, these devices provide limited visual perception as the therapeutic interventions are generally considered in the later stages of the disease when only inner retinal layer cells are left. A potential treatment option for retinal degenerative diseases in the early stages can be stimulating bipolar cells, which receive presynaptic signals from photoreceptors. In this work, we constructed computational models of healthy and degenerated (both ON and OFF-type) cone bipolar cells (CBCs) with realistic morphologies extracted from connectomes of the healthy and early-stage degenerated rabbit retina. We examined these cells' membrane potential and axon terminal calcium current differences when subjected to electrical stimulation. In addition, we investigated how differently healthy and degenerated cells behave with respect to various stimulation parameters, including pulse duration and cells' distance from the stimulating electrode. The results suggested that regardless of the position of the OFF CBCs in the retina model, there is not a significant difference between the membrane potential of healthy and degenerate cells when electrically stimulated. However, the healthy ON CBC axon terminal membrane potential rising time-constant is shorter (0.29 ± 0.03 ms) than the degenerated cells (0.8 ± 0.07 ms). Moreover, the ionic calcium channels at the axon terminals of the cells have a higher concentration and higher current in degenerated cells (32.24 ± 6.12 pA) than the healthy cells (13.64 ± 2.88 pA) independently of the cell's position.

Impact of fractal visual stimulation on healthy rabbit retina: functional, morphometric and biochemical studies

Purpose: to investigate the changes of electrophysiological activity of healthy rabbit retina occurring in courses of fractal stimulation (FS) of varied duration in order to obtain new scientific data on how fractal visual signals of low intensity, self-similar with respect to time, affect the retina.Material and methods. 12 healthy Chinchilla rabbits (24 eyes), were examined before and after FS courses that lasted 1, 4 or 12 weeks, using electroretinographic (ERG), morphometric (optical coherence tomography) and biochemical methods (detection of dopamine in the tear). For FS of rabbits, a device with an LED emitter was developed, which generates nonlinear brightness fluctuation based on the Weierstrass — Mandelbrot fractal functions. The choice of fractal signal parameters used in the work was substantiated. Pattern ERG and ganzfeld ERG were registered according to ISCEV standards; also, photopic flicker ERG was recorded at 8.3, 10, 12, and 24 Hz.Results. No negative effects of a 12-month FS course on the activity and morphology of the retina, or on dopaminergic processes in the eye of a healthy animal were found, which confirms the safety of using low-intensity FS in the clinic. A statistically significant increase in the amplitude of low-frequency flicker ERGs, a shortening of peak latency, and an increase in the amplitude of the b-wave of the scotopic and photopic ERGs was noted.Conclusion. To assess possible therapeutic effects of FS, we need to continue the investigation on animal models and human patients with retinal pathology. Considering the changes of retinal activity as revealed in the present paper, we recommend the duration of FS courses from 1 week to 1 month for future studies.