Rabbit Retina Research Articles

Connexin50 couples axon terminals of mouse horizontal cells by homotypic gap junctions.

Horizontal cells in the mouse retina are of the axon-bearing B-type and contribute to the gain control of photoreceptors and to the center-surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro-dendritic and axo-axonal gap junctions composed of connexin57 (Cx57). In Cx57-deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A-type horizontal cells of the rabbit retina. In the mouse retina, Cx50-immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50-immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57-deficient mice. However, Cx50 expression was unaffected in Cx57-deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina.

Chlorophyllin-M: A new substance for photodynamic therapy in the retina and choroid.

Chlorophyllin-M is a new chlorophyll-based derivative photosensitive compound developed by our research group with easy laboratorial synthesis and ideal properties for photodynamic therapy (PDT). It is intended for clinical treatments with simple and low cost techniques and reagents. The objective of this study is to evaluate if intravenous chlorophyllin-M is able to deliver a photosensitizer to rabbit retina and rabbit choroid and promote PDT after ocular irradiation with a 660 nm LASER. This is a pre-clinical study. Ten eyes of five pigmented Californian rabbits were included in the study. The right eyes served as the treatment group, and the left eyes served as the control group. All eyes had been ophthalmologically evaluated and were considered normal. Ophthalmic exam with anterior biomicroscopy, dilated fundus examination, and fluorescein angiography after the LASER procedure revealed normal anterior segment, retinal and choroid vessels occlusion, lumen narrowing, and capillary non-perfusion in the treated areas, indicating that PDT was successful in the treatment eyes group. The results of this pre-clinical study encourage future studies with this new compound. Chlorophyllin-M may become a new cost-effective agent in the retinal therapeutic arsenal.

Spike history neural response model

There is a potential for improved efficacy of neural stimulation if stimulation levels can be modified dynamically based on the responses of neural tissue in real time. A neural model is developed that describes the response of neurons to electrical stimulation and that is suitable for feedback control neuroprosthetic stimulation. Experimental data from NZ white rabbit retinae is used with a data-driven technique to model neural dynamics. The linear-nonlinear approach is adapted to incorporate spike history and to predict the neural response of ganglion cells to electrical stimulation. To validate the fitness of the model, the penalty term is calculated based on the time difference between each simulated spike and the closest spike in time in the experimentally recorded train. The proposed model is able to robustly predict experimentally observed spike trains.

High osmolarity effect of intravitreal plasmin enzyme on rabbit retina.

We conducted a study of the effect of intravitreal plasmin on retinal thickness in rabbits. Forty rabbit eyes were injected with 0.4, 1, 2, and 5 IU of plasmin (8 eyes/group). The same volume of BSS-plus(®) was injected in control eyes. Four eyes in each group underwent vitrectomy 60 minutes after the injections. The retinal thickness was measured in optical coherence tomographic (OCT) images before, 30 minutes, and 1 week after the injection. To study the effect of hyperosmolarity, 4 eyes received an injection of mannitol solution whose osmolarity was the same as the plasmin solutions. Thirty minutes after the plasmin injection, 4 eyes developed a serous retinal detachment (SRD). The mean retinal thickness including SRD was increased at 30 minutes in a dose-dependent way. The increase in eyes with 5 IU of plasmin was significantly greater than that in eyes with BSS-Plus(®) or 0.4 IU of plasmin (P = 0.0266, P = 0.0371, respectively). One week after the injection, SRD disappeared, and the mean retinal thickness decreased. The eyes injected with mannitol, the same osmolarity of 1, 2, 5 IU of plasmin, developed SRD at 30 minutes, and it disappeared after 1 week in all eyes. The results of this study demonstrated that an intravitreal injection of plasmin increases the retinal thickness in a dose-dependent way in rabbit eyes. The results with mannitol suggest that the increase in retinal thickness following plasmin is most likely caused by the hyperosmolarity of plasmin solution.

Effect of sunlight transformed by luminophore-containing materials on cell functions in vitro and in vivo.

Effect of sunlight transformed by luminophore-containing materials on cell viability and functional state of the retina was assessed using the photodamage model. Exposure to the luminescent component of light improved viability of NIH 3T3 cells and promoted recovery of electric activity in rabbit retina after photodamage.

Reliability of intravitreal nepafenac in rabbits.

The purpose of this experiment was to investigate the possible toxic effects of Nepafenac, a nonsteroidal anti-inflammatory molecule, after its intravitreal application in various concentrations. Forty pigmented rabbits were randomly divided into 4 groups, each including 10 rabbits. The active ingredient Nepafenac was prepared to be applied in different doses, for intravitreal use. Under topical anesthesia, following pupil dilatation, 0.3, 0.5, 0.75, and 1.5 mg doses of Nepafenac was applied intravitreally into the right eye. In each rabbit, the right eye was considered to be the study group. Saline was injected intravitreally into the left eye of each rabbit, and these eyes were considered to be the control group. Immediately after the injection and at the 1st, 4th, and 8th weeks, fundus examination by indirect ophthalmoscopy and intraocular pressure measurement were conducted. Furthermore, electroretinographic (ERG) recordings were taken at the 4th and 8th weeks. At the end of the 8th week, eyes of the surviving 26 rabbits were enucleated, and then animals were sacrificed. Following necessary fixation procedures, histopathological investigations were conducted by using a light and electron microscope. In the histological cross sections, differences between the eyes with injection and the control group were evaluated, and total retinal thickness, inner nuclear layer thickness, and outer nuclear layer thickness were measured. No pathology was found by clinical examination of either group. In the photopic and scotopic full-field ERG, conducted before the injection and in the 4th and 8th weeks after the injection, no statistically significant difference was determined between the study group and the control group. In the histological evaluation of the preparations, there were no statistically significant differences in the retina thickness of control and study groups. In the electron microscopic examinations, there were no toxicity findings in the eyes with injection. Our data show that intravitreal application of 0.3, 0.5, 0.75, and 1.5 mg doses of Nepafenac active substance is nontoxic to the rabbit retina.

Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α1c L-type Ca(2+) Channel.

Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca2+ influx through L-type Ca2+ channels. The purpose of this study was to determine whether the α1c subunit of L-type voltage-gated Ca2+ channel (α1c L-type Ca2+ channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α1c L-type Ca2+ channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α1c L-type Ca2+ channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α1c L-type Ca2+ channel indicates that the α1c L-type Ca2+ channel has a specific role in rod bipolar cells, and the antibody against the α1c L-type Ca2+ channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas.

Влияние тридцатидневной тампонады витреальной полости перфторорганическими соединениями на биоэлектрическую активность сетчатки глаза кролика

<p style="text-align: justify;"><span style="color: #993300;"><span style="line-height: 1.538em;"><span style="color: #993300;">https://doi.org/10.31288/oftalmolzh2014695100</span></span></span> <p style="text-align: justify;"><span style="color: #993300;"><strong><span style="line-height: 1.538em;">Influence of thirty day tamponade of vitreal cavity with perfluorineorganic compounds on bioelectrical activity of the rabbit retina</span></strong></span> <p style="text-align: justify;"><strong>Zhmurik D. V., Khramenko N. I,,Slobodyanik S. B., Milienko M. V.</strong>

Overexpression p21WAF1/CIP1 in suppressing retinal pigment epithelial cells and progression of proliferative vitreoretinopathy via inhibition CDK2 and cyclin E.

BackgroundP21 is one kind of cyclin-dependent kinase inhibitor that can prevent cells from going through the G1/S phase checkpoint and inhibit cell proliferation. Proliferative vitreoretinopathy (PVR) is a proliferative response in the eye. The aim of this study was to determine whether p21Waf1/Cip1 (p21) suppresses the proliferation and migration of retinal pigment epithelial (RPE) cells in vitro and controls PVR development in vivo.MethodsCell cycle analyses and transwell assays were conducted to assess cell proliferation characteristics and the migration ability of RPE cells after transfection with p21. Western blot and reverse-transcription polymerase chain reaction technologies were used to detect the expression of p21, CDK2 and cyclinE in RPE cells and rabbit retinal tissues. The impact of increasing p21 expression on PVR development was conducted by implantation of an adenovirus vector containing rabbit p21 (rAd-p21) in a PVR rabbit model. The prevalence of PVR and retinal detachment was determined by indirect ophthalmoscopy on days 3, 7, 14, and 21 after the injection of rAd-p21 into the vitreous. B scans and hematoxylin-eosin staining were employed to check rabbit retinas on day 21.ResultsCell cycle analyses and transwell assays showed that p21 inhibited the proliferation and migration of RPE cells. Increased expression of p21 was detected in cultured RPE cells and rabbit retinas after transfection with the p21 gene, whereas levels of CDK2 and cyclinE were decreased. The increase in p21 expression effectively suppressed the development of PVR in a rabbit model.ConclusionsThe increase in p21 expression in RPE cells not only inhibits the proliferation and migration of RPE cells in vitro, but also suppresses the development of PVR in vivo, which indicates its therapeutic potential in treating PVR.

Preclinical investigations of intravitreal ziv-aflibercept.

To investigate the retinal safety of intravitreal (IVT) ziv-aflibercept in rabbits. Eighteen rabbits were given an IVT injection of ziv-aflibercept (25 mg/mL) or aflibercept (40 mg/mL) and examined by funduscopy, electroretinography (ERG), optical coherence tomography (OCT), light microscopy, and transmission electron microscopy (TEM). Serum, aqueous, and vitreous were obtained afterward for osmolarity analysis. The effect of ziv-aflibercept on human retinal cultured cells (ARPE-19) was assessed by the MTT cell viability assay. All eyes showed normal funduscopy, OCT, and ERG findings at baseline and 24 hours or 7 days after the procedure. Median baseline serum, vitreous, and aqueous osmolarity remained unchanged. Histology and TEM showed no major anatomic signs of toxicity. No cytotoxic effect was observed in ARPE-19 cells exposed to ziv-aflibercept. IVT injection ziv-aflibercept at a concentration of 25 mg/mL proved to be safe for the rabbit retina.

Histopathologic examination of the retina following intra-vitreal propranolol administration in rabbits

Propranolol is a nonselective, beta-adrenergic receptor blocker with anti-angiogenic and anti-inflammatory effects that could be used in the treatment and control of many vascular disorder-related retinopathies. In this pilot study, the effects and complications of an intra-vitreal injection of propranolol HCl on the rabbit retina were investigated by clinical and histopathological means as the first step to determination the viability of intra-vitreal propranolol administration. Group 1 (n = 8) had 15 μg of propranolol HCL given, via intra-vitreal injection, into the right eye. Group 2 (n = 8) had a matching volume of balanced saline solution also via intra-vitreal injection into the right eye. After 4 weeks, all rabbits were killed humanely and the right eyes from both groups enucleated for histopathological study. Clinical observations and histopathological findings suggest no difference between propranolol and control groups (P ≥ 0.05). As a result of handling, in some cases, the eyes had light inflammation, hyperaemia and oedema with acute degeneration changes. This study shows that 15 μg of propranolol does not cause any significant abnormalities and is not a toxic dose for intra-vitreal injection in rabbits.

Effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits.

The aim of the present study was to evaluate the effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits. New Zealand male albino rabbits (n 21) were divided into two groups: group 1 (G1; n 11), fed a hypercholesterolaemic diet, and group 2 (G2; n 10), fed a hypercholesterolaemic diet enriched with flaxseed flour. The serum concentrations of total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, TAG and fasting blood glucose were determined at the start of the experiment and on the day of killing (8th week). Choroid and sclera samples were subjected to haematoxylin-eosin (HE) staining and histomorphometric and immunohistochemical analyses with the anti-oxidised LDL antibody. Sensory retina samples were subjected to an immunohistochemical analysis with the primary monoclonal nitrotyrosine antibody. At the end of the experiment, a significant increase was observed in TC and LDL-C concentrations in G1 rabbits when compared with G2 rabbits (P=0·008 and P=0·02, respectively). HE staining revealed a significant increase in choroid-sclera complex thickness in G1 rabbits when compared with G2 rabbits (P<0·001). Immunohistochemical analysis of choroid and sclera samples with the anti-oxidised LDL marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P<0·001). Immunohistochemical analysis of sensory retina samples with the anti-nitrotyrosine marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P=0·002). Flaxseed reduced the choroid-sclera complex thickness of diet-induced hypercholesterolaemic rabbits and the expression of oxidised LDL in the choroid-sclera complex as well as the expression of nitrotyrosine in the sensory retina.

Density and types of calretinin-containing retinal ganglion cells in rabbit

Single-cell injection with lipophilic dyes following immunocytochemistry is extremely valuable for revealing the morphology of a cell expressing a protein of interest, and is a more reliable technique for cell type classification than standard morphological techniques. This study focuses on calretinin (CR), which is used as a selective marker for distinct subpopulations of neurons in the rabbit retina. The present study used single-cell injection after immunocytochemistry to describe the density and types of CR-containing retinal ganglion cells (RGCs) in rabbit. The density of CR-immunoreactive cells in the rabbit RGC cell layer was 426cells/mm2. CR-containing RGCs were identified by immunocytochemistry and were then iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscope was used to characterize the morphology of CR-immunoreactive RGCs based on their dendritic field size, branching pattern, and stratification of the inner plexiform layer. Our results show that 10 morphologically different types of rabbit RGCs expressed CR. CR-containing RGCs were heterogeneous in their morphology. This approach to integrate the selective expression of a particular protein with spatial patterns of dendritic arborization will lead to a better understanding of RGCs.

Spike history model for neural control

There is increased interest from the research community and clinicians to implement closed-loop stimulation strategies in neurobionic devices. That is, to adjust stimulation levels dynamically based on the responses of neural tissue in real time. To adjust electrical stimulation in a closed-loop bionic device, a model-based controller design can be implemented. Here, we collect experimental data from retina slices and use data-driven technique to model neural dynamics. Our motivation comes from visual prostheses. In vitro experiments were conducted on NZ white rabbit retina tissue. Electrical stimulation of the retinal ganglion cells consisted of a train of pulses whose amplitudes had a white Gaussian distribution with zero mean and standard deviation of 1uA. In this pulse train, there were a total of 5000 biphasic pulses with 100µs/phase duration, equal phase amplitude, and no interphase delay. Frequency pulse trains of 25-1000Hz were used. Experimental data were used to characterize the response properties of neurons to the electrical stimulation. The reverse-correlation approach, widely used to predict neural response to light stimulation, was adapted to predict neural response of ganglion cells to electrical stimulation. In contrast to the traditional reverse-correlation schemes, the model proposed here incorporated the history of the response of a neuron. To emphasize this, we call it the “spike history” model. To estimate the responses of cells to electrical stimulation, we used a novel pseudo-random stimulation. To validate the fitness of the model, we performed statistical analysis of the simulated spike trains. In particular, we compared the values of the coefficient of variation of the interspike interval for the experimentally recorded spike train, for the simulated spike train with the history model, and for the simulated spike train using the model without the response kernel. To compare how well the simulated spike train approximated experimentally recorded spikes, we compared the penalty terms for the spike history model and for the model without the response kernel. The penalty term was calculated based on the time difference between each simulated spike and the closest spike in time in the experimentally recorded train. Cells responded to all stimulation frequencies. A phenomena of clusters of spikes followed by periods of suppression was observed in raw spike trains above 200 Hz stimulation. The phenomenon of cluster-suppression with white-noise stimulation may be indicative of memory in the system; i.e., the response depends not only on the current stimulus but also on the responses within a time window preceding the current time. To confirm this, we calculated the auto-correlation function of the recorded spike trains for different frequencies of stimulation. This showed that there was memory in the system for stimulation at frequencies higher than 100Hz. Robustness of the model parameters was confirmed by repeated stimulation of the same cell at the same frequency (up to 20 repetitions). Statistical comparison of the model with history and the model without history confirmed that the model with history approximates experimental data better. The proposed model can be used to design a stimulation strategy to control neural excitation.

Retinal Function and Morphology in the Rabbit Eye after Intravitreal Injection of the TNF Alpha Inhibitor Adalimumab

Aim: To study the effects of the tumor necrosis factor alpha inhibitor adalimumab on rabbit retina after injection into the vitreous body.Methods: Forty-eight rabbits of mixed strain (9–12 months old, weighing ≈ 3.5 kg) were randomized into four groups. Adalimumab was injected at one of two concentrations (1.25 mg or 2.5 mg) into the eyes of two groups, and balanced salt solution into the eyes of the third group. The fourth group acted as controls. Full-field electroretinography (ffERG) was performed before injection and 1 and 6 weeks post-injection. At 6 weeks post-injection the rabbits were euthanized and the sectioned retinas were studied. Retinal histology was studied with hematoxylin–eosin staining. Immunohistochemical analysis was performed on rods, cones, rod bipolar cells, horizontal cells, amacrine cells and Müller cells.Results: No significant difference in ffERG amplitudes or implicit times was observed between the four groups at any time point. Histological and immunohistochemical findings were similar in all groups.Conclusions: Injection of adalimumab into the vitreous body of healthy rabbits, at doses up to 2.5 mg, does not appear to be toxic to the rabbit retina.

Directional selective neurons in the awake LGN: response properties and modulation by brain state.

Directionally selective (DS) neurons are found in the retina and lateral geniculate nucleus (LGN) of rabbits and rodents, and in rabbits, LGN DS cells project to primary visual cortex. Here, we compare visual response properties of LGN DS neurons with those of layer 4 simple cells, most of which show strong direction/orientation selectivity. These populations differed dramatically, suggesting that DS cells may not contribute significantly to the synthesis of simple receptive fields: 1) whereas the first harmonic component (F1)-to-mean firing rate (F0) ratios of LGN DS cells are strongly nonlinear, those of simple cells are strongly linear; 2) whereas LGN DS cells have overlapped ON/OFF subfields, simple cells have either a single ON or OFF subfield or two spatially separate subfields; and 3) whereas the preferred directions of LGN DS cells are closely tied to the four cardinal directions, the directional preferences of simple cells are more evenly distributed. We further show that directional selectivity in LGN DS neurons is strongly enhanced by alertness via two mechanisms, 1) an increase in responses to stimulation in the preferred direction, and 2) an enhanced suppression of responses to stimuli moving in the null direction. Finally, our simulations show that these two consequences of alertness could each serve, in a vector-based population code, to hasten the computation of stimulus direction when rabbits become alert.

Compartment-specific tyrosine hydroxylase-positive innervation to AII amacrine cells in the rabbit retina

Tyrosine-hydroxylase-positive (TH+) amacrine cells release dopamine in a paracrine manner and also form GABA-ergic contact sites with inner retinal neurons. The best known sites are formed by TH+ fibrous rings and AII amacrine cell somata in stratum 1 of the inner plexiform layer (IPL). An AII amacrine cell is a highly compartmentalized neuron with relatively large soma, a stout dendritic stalk and two sets of processes, one showing lobular appearance and extending horizontally in stratum 1 and a second transversally elongated group of fibers in strata 4 and 5. Although, all of these compartments have been reported as tic sites, it is uncertain if TH+ amacrine cell inputs are homogeneously distributed or they rather target specific AII cell compartments. In this study we investigated the TH+/AII cell system by immunohistochemistry to map the potential synaptic contacts in the rabbit retina. We found numerous intimate contacts between the two amacrine cell populations throughout the IPL. However, TH+ fibers favored the soma/main stalk region of AII amacrine cells and only contacted lobular appendages and transversal processes sporadically. In addition to the well-studied contacts between AII cell somata and TH+ rings in stratum 1 we found that the main stalk region in stratum 3 serves as a secondary major target for TH+ axons. These data thus clearly show that TH+ contacts to AII amacrine cells are highly compartment specific.

NEV2lkit: A NEW OPEN SOURCE TOOL FOR HANDLING NEURONAL EVENT FILES FROM MULTI-ELECTRODE RECORDINGS

The analysis and discrimination of action potentials, or "spikes", is a central issue to systems neuroscience research. Here we introduce a free open source software for the analysis and discrimination of neural spikes based on principal component analysis and different clustering algorithms. The main objective is to supply a friendly user interface that links the experimental data to a basic set of routines for analysis, visualization and classification of spikes in a consistent framework. The tool has been tested on artificial data sets, on multi-electrode extracellular recordings from ganglion cell populations in isolated superfused mouse, rabbit and turtle retinas, and on electrophysiological recordings from mouse visual cortex. Our results show that NEV2lkit is very reliable and able to satisfy the experimental demands in terms of accuracy, efficiency and consistency across experiments. It performs fast unit sorting in single or multiple experiments and allows the extraction of spikes from over large time intervals in continuously recorded data streams. The tool is implemented in C++ and runs cross-platform on Linux, OS X and Windows systems. To facilitate the adaptation and extension as well as the addition of new routines, tools and algorithms for data analysis, the source code, binary distributions for different operating systems and documentation are all freely available at http://nev2lkit.sourceforge.net .

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity-purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at 〜24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS-immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1)-immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a-, SMI-32-, and melanopsin-immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs.

Topical Delivery of Avastin to the Posterior Segment of the Eye In Vivo Using Annexin A5‐associated Liposomes

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.