Chick Retina Research Articles

An Immunohistochemical Analysis of Hypoxia in Multi-layer Avascular Retina and Vascular Pecten Oculi of the Developing Chicken

The retina lines the internal surface of the back of the eye. In order to function, the retina requires a supply of oxygen. Retinal hypoxia often precedes proliferative diabetic retinopathy in human. In vertebrate eyes, there is a blood supply between retina and choroid, which supports the outer retinal layers. In humans, additional blood vessels enter the retina from the vitreal side and ramify in the plexiform layers. However, in chicks, such plexiform blood vessels are absent. The spatial oxidative state distribution in multi-layer avascular chick retina was studied using the oxygen-sensing reagent EF5. As development of the chick retina proceeds, different oxidation states in different layers were found. The hypoxic staining was almost evenly distributed throughout the tissues for E(mbryonic day) 5 chick retina. For E7 chick retina, the hypoxic stain was concentrated in a subset of ganglion cells. The inner portion of the inner nuclear layer and ganglion cell layer were hypotic for E11 chick retina,. For E14 chick retina, hypoxia was observed at the ganglion cell layer, but additional reaction was seen in the inner portion of the inner nuclear layer and the outer plexiform layer. There was also some reaction in the region of budding photoreceptors. The Ganglion cell layer, and the Inner Nuclear Layer were more hypoxic for E19 chick retina,. In most cases, the regions of hypoxia did not colocalize with the positions that have high mitochondrial density. Pecten oculi is a corrugated flap of tissue that extends along the back of the eye and is embedded in the vitreous body of the avian eye. It also contains a highly efficient glucose transporter. Hypoxia upregulates glucose transport activity. We found that pecten oculi is much less hypoxic than retina tissue nearby. There are more mitochondrial densities in pecten oculi than in retina.

NR2A contributes to genesis and propagation of cortical spreading depression in rats.

Cortical spreading depression (CSD) is a transient propagating excitation of synaptic activity followed by depression, which is implicated in migraine. Increasing evidence points to an essential role of NR2A-containing NMDA receptors in CSD propagation in vitro; however, whether these receptors mediate CSD genesis in vivo requires clarification and the role of NR2A on CSD propagation is still under debate. Using in vivo CSD in rats with electrophysiology and in vitro CSD in chick retina with intrinsic optical imaging, we addressed the role of NR2A in CSD. We demonstrated that NVP-AAM077, a potent antagonist for NR2A-containing receptors, perfused through microdialysis probes, markedly reduced cortex susceptibility to CSD, but also reduced magnitude of CSD genesis in rats. Additionally, NVP-AAM077 at 0.3 nmol perfused into the contralateral ventricle, considerably suppressed the magnitude of CSD propagation wave and propagation rate in rats. This reduction in CSD propagation was also observed with TCN-201, a negative allosteric modulator selective for NR2A, at 3 μM, in the chick retina. Our data provides strong evidence that NR2A subunit contributes to CSD genesis and propagation, suggesting drugs selectively antagonizing NR2A-containing receptors might constitute a highly specific strategy treating CSD associated migraine with a likely better safety profile.

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR).

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as "visual cycle" involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.

Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird.

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.

Differential gene expression of BMP2 and BMP receptors in chick retina & choroid induced by imposed optical defocus.

Recent studies have demonstrated the defocus sign-dependent, bidirectional gene expression regulation of bone morphogenetic proteins, BMP2, 4 and 7 in chick RPE. In this study, we examined the effects of imposed positive (+10 D) and negative (-10 D) lenses on the gene expression of these BMPs and BMP receptors (BMPR1A, BMPR1B, BMPR2) in chick retina and choroid after monocular lens treatment for 2 or 48 h, as indicators of the roles of retinal and choroidal BMPs and receptors in postnatal eye growth regulation. In retina, although all genes were expressed, neither +10 nor -10 D lenses, worn for either 2 or 48 h, significantly altered gene expression. In contrast, treatment-related differential gene expression was detected in the choroid for both BMPs and their receptors, although interestingly, with the +10 D lens, BMP2 was up-regulated by 156.7 ± 19.7% after 2 h, while BMPR1A was down-regulated to 82.3 ± 12.5% only after 48 h. With the -10 D lens, only the gene expression of BMPR1B was significantly altered, being up-regulated by 162.3 ± 21.2% after 48 h. Untreated birds showed no difference in expression between their two eyes, for any of the genes examined. The finding that retinal gene expression for BMP2, 4, 7 and their receptors are not affected by short-term optical defocus contrasts with previous observations of sign-dependent expression changes for the same genes in the RPE. The latter changes were also larger and more consistent in direction than the choroidal gene expression changes reported here. The interrelationship between these various changes and their biological significance for eye growth regulation are yet to be elucidated.

MTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Delayed neurogenesis with respect to eye growth shapes the pigeon retina for high visual acuity.

The macula and fovea located at the optical centre of the retina make primate visual perception unique among mammals. Our current understanding of retina ontogenesis is primarily based on animal models having no macula and no fovea. However, the pigeon retina and the human macula share a number of structural and functional properties that justify introducing the former as a new model system for retina development. Comparative transcriptome analysis of pigeon and chicken retinas at different embryonic stages reveals that the genetic programmes underlying cell differentiation are postponed in the pigeon until the end of the period of cell proliferation. We show that the late onset of neurogenesis has a profound effect on the developmental patterning of the pigeon retina, which is at odds with the current models of retina development. The uncoupling of tissue growth and neurogenesis is shown to result from the fact that the pigeon retinal epithelium is inhibitory to cell differentiation. The sum of these developmental features allows the pigeon to build a retina that displays the structural and functional traits typical of primate macula and fovea.

Wnt/β-catenin-signaling and the formation of Müller glia-derived progenitors in the chick retina.

Müller glia can be stimulated to de-differentiate, proliferate, and form Müller glia-derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de-differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt-pathway. The purpose of this study was to investigate how Wnt-signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA-damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt-signaling, including dkk1, dkk3, axin2, c-myc, tcf-1, and cd44. We find accumulations of nuclear β-catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA-treatment. Inhibition of Wnt-signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt-signaling, with GSK3β-inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt-signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK-signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β-catenin. In FGF2-treated retinas, inhibition of Wnt-signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt-signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt-signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt-signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983-1002, 2016.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

α-Melanocyte-stimulating hormone prevents glutamate excitotoxicity in developing chicken retina via MC4R-mediated down-regulation of microRNA-194.

Glutamate excitotoxicity is a common pathology to blinding ischemic retinopathies, such as diabetic retinopathy, glaucoma, and central retinal vein or artery occlusion. The development of an effective interventional modality to glutamate excitotoxicity is hence important to preventing blindness. Herein we showed that α-melanocyte-stimulating hormone (α-MSH) time-dependently protected against glutamate-induced cell death and tissue damage in an improved embryonic chicken retinal explant culture system. α-MSH down-regulated microRNA-194 (miR-194) expression during the glutamate excitotoxicity in the retinal explants. Furthermore, pharmacological antagonists to melanocortin 4 receptor (MC4R) and lentivirus-mediated overexpression of pre-miR-194 abrogated the suppressing effects of α-MSH on glutamate-induced activities of caspase 3 or 7, the ultimate enzymes for glutamate-induced cell death. These results suggest that the protective effects of α-MSH may be due to the MC4R mediated-down-regulation of miR-194 during the glutamate-induced excitotoxicity. Finally, α-MSH attenuated cell death and recovered visual functions in glutamate-stimulated post-hatch chick retinas. These results demonstrate the previously undescribed protective effects of α-MSH against glutamate-induced excitotoxic cell death in the cone-dominated retina both in vitro and in vivo, and indicate a novel molecular mechanism linking MC4R-mediated signaling to miR-194.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

Of ghrelin, cone cultivation, dinoflagellate eyes, and the cyanobacterial circadian clock.

Of ghrelin, cone cultivation, dinoflagellate eyes, and the cyanobacterial circadian clock.

Differential Expression of AQP1 and AQP4 in Avascular Chick Retina Exposed to Moderate Light of Variable Photoperiods.

Aquaporins (AQPs) are integral membrane proteins which maintain cellular water and ion homeostasis. Alterations in AQP expression have been reported in rod-dominated rodent retinas exposed to light. In rodents and also in birds, light of moderate intensities (700-2000lux) damages the retina, though detailed changes were not examined in birds. The aim of our study was to see if light affects cone dominated retinas, which would be reflected in expression levels of AQPs. We examined AQP1 and AQP4 expressions in chick retina exposed to 2000lux under 12h light:12h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod) and 24L:0D (constant light). Additionally, morphological changes, apoptosis (by TUNEL) and levels of glutamate and GFAP (a marker of injury) in the retina were examined to correlate these with AQP expressions. Constant light caused damage in outer and inner nuclear layer (ONL, INL) and ganglion cell layer (GCL). Also, there were associated increases in GFAP and glutamate levels in retinal extracts. In normal photoperiod, AQP1 was expressed in GCL, outer part of INL and photoreceptor inner segments of. AQP4 was additionally expressed in nerve fiber layer. Immunohistochemistry and Western blotting revealed over all decreased AQP1 and AQP4 expression in constant light condition compared to those in other two groups. The elevated GFAP and glutamate levels might be involved in the reduction of AQPs in constant light group. Such decreases in AQP expressions are perhaps linked with retinal cell damage seen in constant light condition, while their relatively enhanced expression in two other conditions may help in maintaining a normal retinal architecture, indicating their neuroprotective potential.

Activation of glucocorticoid receptors in Müller glia is protective to retinal neurons and suppresses microglial reactivity

Reactive microglia and macrophages are prevalent in damaged retinas. Glucocorticoid signaling is known to suppress inflammation and the reactivity of microglia and macrophages. In the vertebrate retina, the glucocorticoid receptor (GCR) is known to be activated and localized to the nuclei of Müller glia (Gallina et al., 2014). Accordingly, we investigated how signaling through GCR influences the survival of neurons using the chick retina in vivo as a model system. We applied intraocular injections of GCR agonist or antagonist, assessed microglial reactivity, and the survival of retinal neurons following different damage paradigms. Microglial reactivity was increased in retinas from eyes that were injected with vehicle, and this reactivity was decreased by GCR-agonist dexamethasone (Dex) and increased by GCR-antagonist RU486. We found that activation of GCR suppresses the reactivity of microglia and inhibited the loss of retinal neurons resulting from excitotoxicity. We provide evidence that the protection-promoting effects of Dex were maintained when the microglia were selectively ablated. Similarly, intraocular injections of Dex protected ganglion cells from colchicine-treatment and protected photoreceptors from damage caused by retinal detachment. We conclude that activation of GCR promotes the survival of ganglion cells in colchicine-damaged retinas, promotes the survival of amacrine and bipolar cells in excitotoxin-damaged retinas, and promotes the survival of photoreceptors in detached retinas. We propose that suppression of microglial reactivity is secondary to activation of GCR in Müller glia, and this mode of signaling is an effective means to lessen the damage and vision loss resulting from different types of retinal damage.

Resistivity profiles of wild-type, rd1, and rd10 mouse retina.

Electrical impedance of the retina is a critical factor in retinal prostheses, determining the intraretinal current flow and potential distribution of electrical stimulation. Previous resistivity measurements in retina were limited to healthy retina, and didn't include mouse models, a common and important animal model in retinal research. This experimental study measured the resistivity profiles of wild-type, rd1, and rd10 mice, providing basis for computational simulations and predictive modeling studies. The peak resistance frequency method has been utilized to measure the resistivity profiles of the retina cross section, and the results show agreement with previous studies in retina of normal rats and embryonic chicks. Retinal degeneration affects the width of the profile, which is in agreement with histological measurements. Degeneration also results in lower peak resistivity. The results indicate that, on the mesoscopic scale, resistivity is dominated by spatial factors, while influence of remodeling on the cellular level is not apparent under such scale.

GABAA receptor α2 subtype activation suppresses retinal spreading depression

Cortical spreading depression (SD) is a transient propagating neuronal excitation followed by depression, which is generally accepted as the underlying cause of migraine. The inhibitory γ-aminobutyric acid type A (GABAA) receptor activation not only reduces cortical SD frequency and propagation, but also relieves migraine headache. This study aims to determine the role of major α subtypes of GABAA receptor in mediating SD genesis and propagation using an efficient in vitro chick retinal model. We firstly demonstrated that abundant α2, and to a lesser extent, α5 of GABAA receptor expression in the chick retina, enabled the tissue useful for studying GABAA receptor pharmacology and SD. Marked suppression of SD by SL651498 and TPA023 was observed at 10μmolL−1 and 50μmolL−1, respectively, suggesting a critical role of GABAA receptor α subtypes, in particular α2, in modulating retinal SD elicitation and propagation. The negative data on NS11394 at 3μmolL−1 on SD and the little positive selectivity of TPA023 for α5 did not support that α5 subtype is involved in SD genesis and propagation. Our data provide strong evidence that α2, but not α5 is involved in the early stage of migraine, indicating that α2 subtype is a possible drug target related to migraine with aura.

Hedgehog-signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the retina

Müller glia can be stimulated to de-differentiate and become proliferating progenitor cells that regenerate neurons in the retina. The signaling pathways that regulate the formation of proliferating Müller glia-derived progenitor cells (MGPCs) are beginning to be revealed. The purpose of this study was to investigate whether Hedgehog (Hh) signaling influences the formation of MGPCs in the chick retina. We find that Hh signaling is increased in damaged retinas where MGPCs are known to form. Sonic Hedgehog (Shh) is normally present in the axons of ganglion cells, but becomes associated with Müller glia and MGPCs following retinal damage. Activation of Hh signaling with recombinant human SHH (rhShh) or smoothened agonist (SAG) increased levels of Ptch1, Gli1, Gli2, Gli3, Hes1 and Hes5, and stimulated the formation of proliferating MGPCs in damaged retinas. In undamaged retinas, SAG or rhShh had no apparent effect upon the Müller glia. However, SAG combined with FGF2 potentiated the formation of MGPCs, whereas SAG combined with IGF1 stimulated the nuclear migration of Müller glia, but not the formation of MGPCs. Conversely, inhibition of Hh signaling with KAAD-cyclopamine, Gli antagonists or antibody to Shh reduced numbers of proliferating MGPCs in damaged and FGF2-treated retinas. Hh signaling potentiates Pax6, Klf4 and cFos expression in Müller glia during the formation of MGPCs. We find that FGF2/MAPK signaling recruits Hh signaling into the signaling network that drives the formation of proliferating MGPCs. Our findings implicate Hh signaling as a key component of the network of signaling pathways that promote the de-differentiation of Müller glia and proliferation of MGPCs.

Synergistic Suppression of Cortical Spreading Depression under NR2A and NR2B Inhibition

Cortical spreading depression (CSD) is a transient synaptic excitation, followed by depression, which can propagate slowly across cortex, subcortex and retina. CSD is implicated in migraine with aura and may lead to migraine pain. CSD can be suppressed by inhibition of both NR2A and NR2B containing N-methyl-D-aspartic acid (NMDA) receptors. However, whether there is a synergistic effect of co-inhibition of NR2A and NR2B containing receptors on CSD remains unknown. In this study, an efficient in vitro model of migraine with intrinsic optical imaging approach was applied to address this role of co-activation of both NR2A and NR2B in CSD. The results showed that co-application of NVP-AAM077, a drug selectively targeting for NR2A and Ro 25-6981, a drug selectively targeting for NR2B containing receptors, showed synergistic inhibitory effects on propagation rate and the magnitude of CSD in a concentration dependent manner in chick retina. Inhibition of CSD propagation was also observed by a clinical acceptable drug that antagonizes both NR2A and NR2B containing receptors, memantine, at 10 μM. These data suggest that combinational use of antagonists selectively targeting NR2A and NR2B containing receptors could be a useful strategy for preventative treatment of migraine with improved efficacy with potential alleviated side effects.

Expression of Heat Shock Protein 27 and Alpha B Crystallin in the Retina and Optic Nerve of the Chick Embryo

Heat shock protein 27 (HSP27) and alpha B crystallin (aBC) belong to the small heat shock protein (sHSP) family and have similar amino acid sequences. However, no study has compared the distributional patterns of these two sHSPs in the retina and optic nerve. In this study, we compared the spatiotemporal distributions of the expressions of HSP27 and aBC in the developing chick retina and optic nerve. Both HSP27 and aBC were first expressed in the retina and optic nerve at embryonic day 16 (E16). At E20 the expressions of the two proteins were increased in the retina and optic nerve. Double immunofluorescence demonstrated that HSP27 and aBC were expressed in oligodendrocytes of the retina and optic nerve. In addition, HSP27 was also found to be expressed in ganglion cells in the retina. The findings of this study suggest that HSP27 and aBC act to protect ganglion cells and oligodendrocytes during late development of the chick retina and optic nerve.