Large Retinal Ganglion Cells Research Articles

Dynamic changes in cell size and corresponding cell fate after optic nerve injury

Identifying disease-specific patterns of retinal cell loss in pathological conditions has been highlighted by the emergence of techniques such as Detection of Apoptotic Retinal Cells and Adaptive Optics confocal Scanning Laser Ophthalmoscopy which have enabled single-cell visualisation in vivo. Cell size has previously been used to stratify Retinal Ganglion Cell (RGC) populations in histological samples of optic neuropathies, and early work in this field suggested that larger RGCs are more susceptible to early loss than smaller RGCs. More recently, however, it has been proposed that RGC soma and axon size may be dynamic and change in response to injury. To address this unresolved controversy, we applied recent advances in maximising information extraction from RGC populations in retinal whole mounts to evaluate the changes in RGC size distribution over time, using three well-established rodent models of optic nerve injury. In contrast to previous studies based on sampling approaches, we examined the whole Brn3a-positive RGC population at multiple time points over the natural history of these models. The morphology of over 4 million RGCs was thus assessed to glean novel insights from this dataset. RGC subpopulations were found to both increase and decrease in size over time, supporting the notion that RGC cell size is dynamic in response to injury. However, this study presents compelling evidence that smaller RGCs are lost more rapidly than larger RGCs despite the dynamism. Finally, using a bootstrap approach, the data strongly suggests that disease-associated changes in RGC spatial distribution and morphology could have potential as novel diagnostic indicators.

The expression and function of TRPV4 channels in primate retinal ganglion cells and bipolar cells

The transient receptor potential vanilloid 4 (TRPV4) channel may be opened by mechanical stimuli to mediate Ca2+ and Na+ influxes, and it has been suggested to mediate glaucoma retinopathy. However, it has been mostly unclear how TRPV4 activities affect the function of primate retinal ganglion cells (RGCs). We studied RGCs and bipolar cells (BCs) in the peripheral retina of the old-world primate using whole-cell current-clamp and voltage-clamp recordings, immunomarkers and confocal microscopy. RGCs were distinguished from displaced amacrine cells (ACs) by the absence of GABA and glycine immunoreactivity and possession of an axon and a large soma in the RGC layer. Strong TRPV4 signal was concentrated in medium to large somas of RGCs, and some TRPV4 signal was found in BCs (including PKCα-positive rod BCs), as well as the end feet, soma and outer processes of Mȕller cells. TRPV4 immunoreactivity quantified by the pixel intensity histogram revealed a high-intensity component for the plexiform layers, a low-intensity component for the soma layers of ACs and Mȕller cells, and both components in the soma layers of RGCs and BCs. In large RGCs, TRPV4 agonists 4α-phorbol 12,13 didecanoate (4αPDD) and GSK1016790A reversibly enhanced the spontaneous firing and shortened the delay of voltage-gated Na+ (Nav) currents under current-clamp conditions, and under voltage-clamp conditions, 4αPDD largely reversibly increased the amplitude and frequency of spontaneous excitatory postsynaptic currents. In BCs, changes in the membrane tension induced by either applying pressure or releasing the pressure both activated a transient cation current, which reversed at ~ −10 mV and was enhanced by heating from 24 °C to 30 °C. The pressure for the half-maximal effect was ~18 mmHg. These data indicate that functional TRPV4 channels are variably expressed in primate RGCs and BCs, possibly contributing to pressure-related changes in RGCs in glaucoma.

Overexpression of Brain-Derived Neurotrophic Factor Protects Large Retinal Ganglion Cells After Optic Nerve Crush in Mice.

Brain-derived neurotrophic factor (BDNF), a neurotrophin essential for neuron survival and function, plays an important role in neuroprotection during neurodegenerative diseases. In this study, we examined whether a modest increase of retinal BDNF promotes retinal ganglion cell (RGC) survival after acute injury of the optic nerve in mice. We adopted an inducible Cre-recombinase transgenic system to up-regulate BDNF in the mouse retina and then examined RGC survival after optic nerve crush by in vivo imaging. We focused on one subtype of RGC with large soma expressing yellow fluorescent protein transgene that accounts for ∼11% of the total SMI-32–positive RGCs. The median survival time of this subgroup of SMI-32 cells was 1 week after nerve injury in control mice but 2 weeks when BDNF was up-regulated. Interestingly, we found that the survival time for RGCs taken as a whole was 2 weeks, suggesting that these large-soma RGCs are especially vulnerable to optic nerve crush injury. We also studied changes in axon number using confocal imaging, confirming first the progressive loss reported previously for wild-type mice and demonstrating that BDNF up-regulation extended axon survival. Together, our results demonstrate that the time course of RGC loss induced by optic nerve injury is type specific and that overexpression of BDNF prolongs the survival of one subgroup of SMI-32–positive RGCs.

Rank Order Coding: a Retinal Information Decoding Strategy Revealed by Large-Scale Multielectrode Array Retinal Recordings.

How a population of retinal ganglion cells (RGCs) encodes the visual scene remains an open question. Going beyond individual RGC coding strategies, results in salamander suggest that the relative latencies of a RGC pair encode spatial information. Thus, a population code based on this concerted spiking could be a powerful mechanism to transmit visual information rapidly and efficiently. Here, we tested this hypothesis in mouse by recording simultaneous light-evoked responses from hundreds of RGCs, at pan-retinal level, using a new generation of large-scale, high-density multielectrode array consisting of 4096 electrodes. Interestingly, we did not find any RGCs exhibiting a clear latency tuning to the stimuli, suggesting that in mouse, individual RGC pairs may not provide sufficient information. We show that a significant amount of information is encoded synergistically in the concerted spiking of large RGC populations. Thus, the RGC population response described with relative activities, or ranks, provides more relevant information than classical independent spike count- or latency- based codes. In particular, we report for the first time that when considering the relative activities across the whole population, the wave of first stimulus-evoked spikes is an accurate indicator of stimulus content. We show that this coding strategy coexists with classical neural codes, and that it is more efficient and faster. Overall, these novel observations suggest that already at the level of the retina, concerted spiking provides a reliable and fast strategy to rapidly transmit new visual scenes.

Non-linear retinal processing supports invariance during fixational eye movements

Fixational eye movements can rapidly shift the retinal image, but typically remain unnoticed. We identify and simulate a model mechanism for the suppression of erroneous motion signals under fixational eye movements. This mechanism exploits the non-linearities common to many classes of large retinal ganglion cells in the mammalian retina, and negates the need for extra-retinal signals or explicit gaze information. When tested using natural images undergoing simulated fixational eye movements, our model successfully distinguishes “real world” motion from retinal motion induced by eye movements. In addition, this model suggests a possible explanation for several fixational eye movement related visual illusions such as the Ouchi–Spillmann and “Out-of-focus” illusions.

Programmed cell death-1 is expressed in large retinal ganglion cells and is upregulated after optic nerve crush

Programmed cell death-1 (PD-1) is a key negative receptor inducibly expressed on T cells, B cells and dendritic cells. It was discovered on T cells undergoing classical programmed cell death. Studies showed that PD-1 ligation promotes retinal ganglion cell (RGC) death during retinal development. The purpose of this present study is to characterize PD-1 regulation in the retina after optic nerve crush (ONC). C57BL/6 mice were subjected to ONC and RGC loss was monitored by immunolabelling with RNA-binding protein with multiple splicing (Rbpms). Time course of PD-1 mRNA expression was determined by real-time PCR. PD-1 expression was detected with anti-PD-1 antibody on whole mount retinas. PD-1 staining intensity was quantitated. Colocalization of PD-1 and cleaved-caspase-3 after ONC was analyzed. Real-time PCR results demonstrated that PD-1 gene expression was significantly upregulated at day 1, 3, 7, 10 and 14 after ONC. Immunofluorescent staining revealed a dramatic increase of PD-1 expression following ONC. In both control and injured retinas, PD-1 tended to be up-expressed in a subtype of RGCs, whose somata size were significantly larger than others. Compared to control, PD-1 intensity in large RGCs was increased by 82% in the injured retina. None of the large RGCs expressed cleaved-caspase-3 at day 5 after ONC. Our work presents the first evidence of PD-1 induction in RGCs after ONC. This observation supports further investigation into the role of PD-1 expression during RGC death or survival following injury.

Intravitreal delivery of human NgR-Fc decoy protein regenerates axons after optic nerve crush and protects ganglion cells in glaucoma models.

Glaucoma is a major cause of vision loss due to retinal ganglion cell (RGC) degeneration. Therapeutic intervention controls increased IOP, but neuroprotection is unavailable. NogoReceptor1 (NgR1) limits adult central nervous system (CNS) axonal sprouting and regeneration. We examined NgR1 blocking decoy as a potential therapy by defining the pharmacokinetics of intravitreal NgR(310)-Fc, its promotion of RGC axonal regeneration following nerve crush, and its neuroprotective effect in a microbead glaucoma model. Human NgR1(310)-Fc was administered intravitreally, and levels were monitored in rat vitreal humor and retina. Axonal regeneration after optic nerve crush was assessed by cholera toxin β anterograde labeling. In a microbead model of glaucoma with increased IOP, the number of surviving and actively transporting RGCs was determined after 4 weeks by retrograde tracing with Fluro-Gold (FG) from the superior colliculus. After intravitreal bolus administration, the terminal half-life of NgR1(310)-Fc between 1 and 7 days was approximately 24 hours. Injection of 5 μg protein once per week after optic nerve crush injury significantly increased RGCs with regenerating axons. Microbeads delivered to the anterior chamber increased pressure, and caused 15% reduction in FG-labeled RGCs of control rats, with a 40% reduction in large diameter RGCs. Intravitreal treatment with NgR1(310)-Fc did not reduce IOP, but maintained large diameter RGC density at control levels. Human NgR1(310)-Fc has favorable pharmacokinetics in the vitreal space and rescues large diameter RGC counts from increased IOP. Thus, the NgR1 blocking decoy protein may have efficacy as a disease-modifying therapy for glaucoma.

Estimation of Retinal Ganglion Cell Loss in Glaucomatous Eyes With a Relative Afferent Pupillary Defect

To estimate retinal ganglion cell (RGC) losses associated with a relative afferent pupillary defect (RAPD) in glaucoma. A cross-sectional study was conducted including both eyes of 103 participants from the Diagnostic Innovations in Glaucoma Study. A total of 77 subjects had glaucoma in at least one eye and 26 were healthy. Pupil responses were assessed using an automated pupillometer that records the magnitude of RAPD as an "RAPD score." Standard automated perimetry (SAP) and optical coherence tomography (OCT) also were performed. Retinal ganglion cell counts were estimated using empirical formulas that combine estimates from SAP and OCT. The estimated percentage RGC loss was calculated using the combined structure function index (CSFI). There was good correlation between RAPD magnitude and intereye differences in estimated RGCs (R(2) = 0.492, P < 0.001), mean deviation (R(2) = 0.546, P < 0.001), retinal nerve fiber layer thickness (R(2) = 0.362, P < 0.001), and CSFI (R(2) = 0.484, P < 0.001). Therefore, a high RAPD score is likely to indicate large asymmetric RGC losses. The relationship between intereye difference in RGC counts and RAPD score was described best by the formula; RGC difference = 21,896 + 353,272 * RAPD score. No healthy subjects had an absolute RAPD score > 0.3, which was associated with asymmetry of 105,982 cells (or 12%). Good correlation between the magnitude of RAPD and intereye differences in mean deviation and estimated RGC counts suggests pupillometry may be useful for quantifying asymmetric damage in glaucoma. (ClinicalTrials.gov number, NCT00221897.).

The Relationship Between Cup-to-Disc Ratio and Estimated Number of Retinal Ganglion Cells

To investigate the relationship between cup-to-disc ratio (CDR) and estimates of retinal ganglion cell (RGC) number. This cross-sectional study included 156 healthy eyes, 53 glaucoma suspects, and 127 eyes with glaucoma. All eyes had standard automated perimetry (SAP), Cirrus SD-OCT, and stereoscopic optic disc photography within 6 months. CDR was determined from stereoscopic photographs by two or more masked graders. The number of RGCs in each eye was estimated using a published model that combines estimates of RGC number from SAP sensitivity thresholds and SD-OCT retinal nerve fiber layer measurements. The mean estimated RGC count was 1,063,809 in healthy eyes; 828,522 in eyes with suspected glaucoma; and 774,200 in early, 468,568 in moderate, and 218,471 in advanced glaucoma. Healthy eyes had a mean vertical CDR of 0.45 ± 0.15 vs. 0.80 ± 0.16 in glaucomatous eyes. There was good correlation between stereophotographic vertical CDR and SD-OCT vertical CDR (R(2) = 0.825; P < 0.001). The relationship between estimated RGCs and vertical CDR was best represented using a third degree polynomial regression model, including age and optic disc area, which accounted for 83.3% of the variation in estimated RGC counts. The nonlinear relationship between RGC estimates and CDRs indicated that eyes with a large CDR would require loss of large RGC numbers for a small increase in CDR. The relationship between estimated RGC counts and CDR suggests that assessment of change in CDR is an insensitive method for evaluation of progressive neural losses in glaucoma. Even relatively small changes in CDR may be associated with large losses of RGCs, especially in eyes with large CDRs. (ClinicalTrials.gov numbers, NCT00221923, NCT00221897.).

Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10μM ACh, 100μM nicotine or 100nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

Erythropoietin Protects Adult Retinal Ganglion Cells against NMDA-, Trophic Factor Withdrawal-, and TNF-α-Induced Damage

PurposeThis study aimed to evaluate the neuroprotective effect of EPO in the presence of N-methyl-d-aspartate (NMDA)-, trophic factor withdrawal (TFW)-, and tumor necrosis factor-alpha (TNF-α)-induced toxicity on total, small, and large retinal ganglion cells (RGCs).MethodsRetinal cells from adult rats were cultured in a medium containing brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), and forskolin. Expression of RGC markers and EPOR was examined using immunocytochemistry. RGCs were classified according to their morphological properties. Cytotoxicity was induced by NMDA, TFW, or TNF-α. RGC survival was assessed by counting thy-1 and neurofilament-l double-positive cells.ResultsEPO offered dose-dependent (EC50 = 5.7 ng/mL) protection against NMDA toxicity for small RGCs; protection was not significant for large RGCs. Time-course analysis showed that the presence of EPO either before or after NMDA exposure gave effective protection. For both small and large RGCs undergoing trophic factor withdrawal, EPO at concentrations of 1, 10, or 100 ng/mL improved survival. However, EPO had to be administered soon after the onset of injury to provide effective protection. For TNF-α-induced toxicity, survival of small RGCs was seen only for the highest examined concentration (100 ng/mL) of EPO, whereas large RGCs were protected at concentrations of 1, 10, or 100 ng/mL of EPO. Time-course analysis showed that pretreatment with EPO provided protection only for large RGCs; early post-treatment with EPO protected both small and large RGCs. Inhibitors of signal transduction and activators of transcription such as (STAT)-5, mitogen-activated protein kinases (MAPK)/extracellular-regulated kinase (ERK), and phosphatidyl inositol-3 kinase (PI3K)/Akt impaired the protective effect of EPO on RGCs exposed to different insults.ConclusionEPO provided neuroprotection to cultured adult rat RGCs; however, the degree of protection varied with the type of toxic insult, RGC subtype, and timing of EPO treatment.

Lack of early pattern stimulation prevents normal development of the alpha (Y) retinal ganglion cell population in the cat

Binocular deprivation of pattern vision (BD) early in life permanently impairs global motion perception. With the SMI-32 antibody against neurofilament protein (NFP) as a marker of the motion-sensitive Y-cell pathway (Van der Gucht et al. [2001] Cereb. Cortex 17:2805-2819), we analyzed the impact of early BD on the retinal circuitry in adult, perceptually characterized cats (Burnat et al. [2005] Neuroreport 16:751-754). In controls, large retinal ganglion cells exhibited a strong NFP signal in the soma and in the proximal parts of the dendritic arbors. The NFP-immunoreactive dendrites typically branched into sublamina a of the inner plexiform layer (IPL), i.e., the OFF inner plexiform sublamina. In the retina of adult BD cats, however, most of the NFP-immunoreactive ganglion cell dendrites branched throughout the entire IPL. The NFP-immunoreactive cell bodies were less regularly distributed, often appeared in pairs, and had a significantly larger diameter compared with NFP-expressing cells in control retinas. These remarkable differences in the immunoreactivity pattern were typically observed in temporal retina. In conclusion, we show that the anatomical organization typical of premature Y-type retinal ganglion cells persists into adulthood even if normal visual experience follows for years upon an initial 6-month period of BD. Binocular pattern deprivation possibly induces a lifelong OFF functional domination, normally apparent only during development, putting early high-quality vision forward as a premise for proper ON-OFF pathway segregation. These new observations for pattern-deprived animals provide an anatomical basis for the well-described motion perception deficits in congenital cataract patients.

A Novel High-Content Flow Cytometric Method for Assessing the Viability and Damage of Rat Retinal Ganglion Cells

PurposeThe aim of the study was to develop a high-content flow cytometric method for assessing the viability and damage of small, medium, and large retinal ganglion cells (RGCs) in N-methyl-D-aspartic acid (NMDA)-injury model.Methods/ResultsRetinal toxicity was induced in rats by intravitreal injection of NMDA and RGCs were retrogradely labeled with Fluoro-Gold (FG). Seven days post-NMDA injection, flatmount and flow cytometric methods were used to evaluate RGCs. In addition, the RGC area diameter (D(a)) obtained from retinal flatmount imaging were plotted versus apparent volume diameter (D(v)) obtained from flow cytometry for the same cumulative cell number (sequentially from small to large RGCs) percentile (Q) to establish their relationship for accurately determining RGC sizes. Good correlation (r = 0.9718) was found between D(a) and apparent D(v). Both flatmount and flow cytometric analyses of RGCs showed that 40 mM NMDA significantly reduced the numbers of small and medium RGCs but not large RGCs. Additionally, flow cytometry showed that the geometric means of FG and thy-1 intensities in three types of RGCs decreased to 90.96±2.24% (P<0.05) and 91.78±1.89% (P>0.05) for small, 69.62±2.11% (P<0.01) and 69.07±2.98% (P<0.01) for medium, and 69.68±6.48% (P<0.05) and 69.91±6.23% (P<0.05) for large as compared with the normal RGCs.ConclusionThe established flow cytometric method provides high-content analysis for differential evaluation of RGC number and status and should be useful for the evaluation of various models of optic nerve injury and the effects of potential neuroprotective agents.

The protective effects of ginkgo biloba extract on cultured human retinal ganglion cells

This study was designed to investigate the protective effects of ginkgo biloba extract (EGb) 761 on human retinal ganglion cells (RGC). It was an experimental study. Cultured human retinal cells were separated for four groups: control group, glutamate group, EGb761 group and EGb761+glutamate group. RGC survival rate was determined by immune Flow cytometry using Thy-1 as a special fluorescent antibody of RGC. RGC survival rate in Multi groups were analysed by repetitive measure analysis of variance (ANOVA), and the differences between groups were analysed by LSD t test. The effects of EGb761 on cultured human RGC were different under different interfere factors. The RGC survival rate was (61.94±7.75)% in control group, (44.59±4.19)% in glutamate group, (75.05±3.90)% in EGb761 group, and (63.19±9.44)% in EGb761+glutamate group. There were significant difference in different groups on survival rates of RGC (F=13.329, P<0.01). Compared with the control group, the survival rate of RGC in glutamate group was significantly decreased (P=0.010), while the EGb761 group was significantly increased (P=0.019), and the EGb761+glutamate group had no significant difference (P=0.801). Compared with the glutamate group, the survival rate of RGC in the EGb761 group and the EGb761+glutamate group was significantly increased (P=0.000, 0.020). There was no significant difference in the percentage of the large RGC in all groups. But compared with the control group (36.69±2.92)%, the percentage of large RGC in dead RGC in the EGb761 group (24.63±7.21)% and the EGb761+glutamate group (25.99±5.05)% was significantly decreased (P=0.001, 0.002). Compared with the glutamate group (40.78±3.34)%, the death rate of the large RGC in the EGb761 group and the EGb761+glutamate group was also significantly decreased (P=0.000, 0.000). EGb761 can protect RGC against glutamate induced neurotoxicity, and it has significant protective effect on cultured human RGC.

SEROTONERGIC SYNAPSES MODULATE GENERATION OF SPIKES FROM RETINAL GANGLION CELLS OF TELEOSTS

Serotonin [5-hydroxytryptamine (5-HT)] is a common neurotransmitter/neuromodulator found widely in the nervous system. Cellular morphology and retinal distribution of serotonergic amacrine cells in the channel catfish (Ictalurus punctatus) retina are identified using monoclonal anti-5HT antibody. These cells receive ribbon synapses from OFF-center (hyperpolarizing) bipolar cells as well as conventional synapses with other non-serotonergic amacrine cells. Output synapses from the serotonergic cells are mainly channel onto retinal ganglion cells. Output synapses from the serotonergic cells occur as "the branched synapses" onto the ganglion cell dendrites at the dyads of the ribbon synaptic sites, and are made onto the ganglion cells, apart from the ribbon synapses. Application of serotonin receptor agonist: 5HT(1A) serotonin receptor agonist, (+)-8-hydroxy-dipropylaminotetralin [8-OH-DPAT; 1-10 muM] is also known to activate 5HT(7) serotonin receptor, coupled with activation of adenylate cyclase, generated continuous repetitive spikes from large retinal ganglion cells of the adult goldfish (Carassius auratus) in flat-mounted preparations, using amphotericin-B-perforated patch-clamp. Under control conditions of bleached retina with continuous light illumination, goldfish large retinal ganglion cells had generated only few spikes. This is the first observation of positive neuromodulation promoting retinal ganglion cell excitation in the retina. The results confirm previous reports of a serotonergic system in the mammalian retina. These results support the presence of developed postsynaptic serotonin receptors in cyprinid fish retina together with other physiological and anatomical studies, and suggest that the action of serotonin in the retina may be more important than previously believed.

Functional and Structural Changes in a Canine Model of Hereditary Primary Angle-Closure Glaucoma

To characterize functional and structural changes in a canine model of hereditary primary angle-closure glaucoma. Intraocular pressure (IOP) was evaluated with tonometry in a colony of glaucomatous dogs at 8, 15, 18, 20, and 30 months of age. Retinal function was evaluated using electroretinography (scotopic, photopic, and pattern). Examination of anterior segment structures was performed using gonioscopy and high-frequency ultrasonography (HFU). A gradual rise in IOP was observed with an increase in age: 8 months, 14 mm Hg (median value); 15 months, 15.5 mm Hg; 18 months, 17.5 mm Hg; 20 months, 24 mm Hg; 30 months, 36 mm Hg. Provocative testing with mydriatic agents (tropicamide and atropine 1%) caused significant increases in IOP (35% and 50%, respectively). HFU analysis showed complete collapse of iridocorneal angles by 20 months of age. Scotopic and photopic ERG analysis did not reveal significant deficits, but pattern ERG analysis showed significantly reduced amplitudes in glaucomatous dogs (glaucoma, 3.5 +/- 0.4 muV; control, 6.2 +/- 0.3 muV; P = 0.004; Student's t-test). Histologic analysis revealed collapse of the iridocorneal angle, posterior bowing of the lamina cribrosa, swelling and loss of large retinal ganglion cells, increased glial reactivity, and increased thickening of the lamina cribrosa. Canine hereditary angle-closure glaucoma is characterized by a progressive increase in intraocular pressure, loss of optic nerve function, and retinal ganglion cell loss.

The protective effects of ginkgo biloba extract 761 on cultured large retinal ganglion cells in infantile rats

Objective To investigate the protective effects of ginkgo biloba extract (EGb) 761 on retinal ganglion cells (RGC) in rats,and to establish a method to define the rat RGC using fluorogold as a fluorescence dye.Methods RGC of 12 20-day-old Sprague-Dawley rats were labeled by injecting fluorogold into superior colliculus.The eyeball enucleation was performed 6 days later.Retinal stretched preparation was obtained from one eye to observe the label result under fluorescence microscope,and the retina from the other eye was detached to make the cell suspension to observe the configuration of stained RGC under the contrast fluorescence microscope.The cell suspension was divided into the control group and Egb761 groups with the concentration of 0.03%,0.10%,0.30%,1.00%,and 3.00%.Trypan blue dye was used to evaluate cells viability and the survival rate of the large retinal ganglion cells was calculated.Results The sign of the RGC was clear after labeled by fluorogold.The characteristics of large RGC were obvious.After detachment,large RGC died quickly in the cell suspension and the fluorescence disappeared.The result of Trypan blue staining indicated that large RGC died rapidly in the cell suspension.Large RGC in EGb761 group showed significantly better survival rates than that in control group at different time sites (P<0.01) in a dose-dependent manner (P<0.01).Conclusions EGb761 has a significant protective effect on large RGC cultivated in vitro,and retrolable method to identify RGC is feasible. Key words: Ginkgetin/therapeutic use; Ginkgolides/therapeutic use; Retinal ganglion cells/drug effects! Animal experimentation

Differential gene expression profiling of large and small retinal ganglion cells

Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.

Morphology and spatial arrangement of large retinal ganglion cells projecting to the optic tectum in the perciform fish Pholidapus dybowskii

Using retrograde HRP labeling from the optic nerve (ON) or optic tectum (OT), we have visualized large ganglion cells (LGCs) in wholemounted retinas of the teleost Pholidapus dybowskii and studied their morphology and spatial properties. In all, three LGC types were distinguished. In a previous paper, detailed data were provided on one type, biplexiform cells [Pushchin, I. I., & Kondrashev, S. L. (2003). Biplexiform ganglion cells in the retina of the perciform fish Pholidapus dybowskii revealed by HRP labeling from the optic nerve and optic tectum. Vision Research, 43, 1117–1133]. Here, we present data on the other two confirmed types, α a and α ab cells. The types differed in the level of dendrite stratification, dendrite arborization pattern, dendritic field size, and other features, and formed in the retina significantly non-random, spatially independent mosaics. Both types were labeled from the OT, indicating their participation in OT-mediated visual reactions. The comparison of spatial properties of α a and α ab mosaics labeled from the ON and OT suggests that the OT is the major or one of the major projection areas of both types. We also describe the morphology of cells resembling α c cells of other fishes, which were only labeled from the ON. The LGC types presently revealed were similar in their morphology to LGCs found in other teleosts supporting the hypothesis of LGC homology across the teleost lineage.

Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina

Transforming growth factor beta (TGF-β) is an extracellular signaling molecule known to mediate programmed cell death (PCD) in the developing retina. In the present study, we investigated the expression profiles and activity levels of TGF-β ligand and TGF-β receptors (TβR) during the successive physiological PCD periods of the developing postnatal mouse retina. The peak of TβR expression levels – revealed by Western Blots and MLEC assays – coincided with the main periods of postnatal (P) retinal murine PCD at P2, P9, and P15. Immunocytochemical studies showed that the localization of the TβRs is restricted to the ganglion cell layer. Application of a neutralizing anti-TGF-β antibody to E15 and P9 retinal cultures resulted in a significant decrease in the number of TUNEL-positive neurons specifically in the ganglion cell or prospective ganglion cell layer. Treatment of P2 and P15 organotypic murine retinal wholemount cultures with exogenous recombinant TGF-β significantly increased cell death levels. In the P15 retina, where PCD affects ganglion cells and photoreceptors, TGF-β induced cell death of large retinal ganglion cells, whereas small ganglion cells and photoreceptor neurons remained unaffected. Our data indicate that TGF-β mediated apoptosis during all postnatal retinal PCD phases specifically affects the fate of retinal ganglion cells.