ABSTRACT The development of non-invasive live ocular imaging and electrophysiological test systems for rodent eyes provides new tools for not only averaged analysis of the entire retina but also the ability to see, test, and compare different subregions of the same retina. These new capabilities provide the possibility for more detailed examinations of local structural and functional relationships within a single eye and the ability to also follow changes longitudinally over time. We have developed protocols based around the Micron-III/IV retinal imaging camera system for combining fluorescent imaging of the neural retinal micro-vasculature by FA (fluorescein angiography), imaging of all neural retinal layers by SD-OCT (Spectral-Domain Ocular Coherence Tomography), and focal “spot” light-targeted electroretinography (Focal-ERG) to relate the local neurovascular unit structure to the inner (photoreceptor) and outer-retinal electrical response to light stimulation. For demonstration purposes we have used the popular mouse oxygen induced retinopathy (OIR) model, which causes radial central patches of retinal neuron loss mostly in zones away from and between the primary retinal arteries and veins. In this model, the loss of central microvasculature is induced developmentally in mouse litters exposed to 75% oxygen from age P7 to P11. Return to room air on P12, causes several days of retinal ischemia during which neurons, mostly of the inner retina, perish. Bipolar and ganglion cell death ends as neovascular growth revascularizes the central retina. This model provides for non-uniform retinal damage as well as gradual progression and resolution over time. The OIR model was used to generate regions of inner retinal neuron loss in B6.Cg-Tg Thy1-YFP mice. Using image-guided focal-ERG, the dark-adapted mixed rod-cone light response was compared using stimulation of small circular (0.27 mm diameter) target areas located in the central retinas of the same eyes (OIR and control). The same areas of the same retinas were followed over three ages after revascularization (P21, P28 and P42). Conclusions Combined FA and SD-OCT imaging can provide local geographic specific information on retinal structural changes and be used to select different retinal areas within the same eye for testing of local light response. This analysis strategy can be employed for studies with rodent disease models that do not uniformly impact the entire retinal area. Combining these techniques would also be useful for testing gene and cell replacement therapies in retinal degeneration models where typically a small zone of the retina is treated. Both treated and untreated retinal zones within the eye can be followed non-invasively over many weeks. SUMMARY Mouse models utilized for retinal disease research including retinal vascular models can display nonuniform changes over the entire retina. Damage or loss of retinal layers and retinal neurons due to hypoxia can impact some retinal areas while leaving adjacent regions unaltered. Combining vascular imaging by fluoresceine angiography, vascular imaging and retinal layer imaging by SD-OCT, and focal-ERG provides us with new tools to examine retinal structure-function relationships within a single retina.

Mouse models of Human retinal diseases continue to be developed to support molecular investigations and therapeutic development. We tested the capability of the novel ERG / image-guided Micron-III System (Phoenix Research Labs) to obtain focal ERG data from discrete areas of the murine retina, using an oxygen induced retinopathy model, to monitor A-wave and B-wave changes in regions small enough to have uniform effects. These regions were monitored longitudinally in the same eye of the same animals over several weeks. Work began with a prototype and data for this paper were obtained with a final version of the Focal ERG module, with a gold corneal electrode integrated into the ocular lens mount. Dark-adapted B6.Cg-TgThy1-YFP mice, which express YFP in ganglion cells, were anesthetized after dilation of pupils. Continuous LED illumination in the red spectrum, and real time video imaging, was used to locate the disc and then target discrete retinal zones. LED illumination was switched to white light and pulse mode for control of ERG acquisition using the Micron-III ERG controller (Phoenix Research Labs). A robust dark-adapted ERG response with characteristic A-wave, B-wave, and oscillatory potentials was obtained with illumination zones as small as the murine disc. Focal ERG in the murine eye is efficient with the Micron-III system as tested. Using a temperature control pad, Focal-ERG and fluorescein angiography could be completed on the same eye before the formation of transient lens opacities. Targets are small enough to isolate the ERG response from control and treated regions of the same eye, or to test degenerations in different retinal zones. Live imaging capability makes targeting certain and provides a visual record of the ERG testing, which should be very useful for documentation during therapeutic testing. In the case of the OIR mouse model, the Focal-ERG system demonstrated clearly for the first time, that the B-wave is at first lost completely before it recovers to about 50% of normal after neovascularization. Photoreceptors remain responsive at all times, without loss of the Awave.

Increased levels of extracellular L-glutamate have been suggested to play a role in retinal damage in a number of blinding diseases such as glaucoma and diabetic retinopathy. Although glutamate can cause retinal damage in part by hyperstimulating its receptors ("excitotoxicity"), the downstream events that lead to retinal damage are poorly understood. In this study, we injected kainic acid (KA), a glutamate receptor agonist that specifically hyperstimulates non-NMDA- type receptors, into the vitreous humor of CD-1 mice and have investigated the role of plasminogen activators (PAs) [tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)] in excitotoxicity-induced retinal damage. Injection of KA into the vitreous humor led to an up-regulation in tPA and an induction in uPA activity in the retina and this was associated with activation of zymogen plasminogen to active plasmin. Immunocytochemical analysis indicated that retinal ganglion cells (RGCs), constitutively express tPA and release it into the extracellular space upon KA injection. Immunocytochemical analysis also indicated an increase in uPA in the nerve fiber layer after KA injection that was absent in the control retinas. These events were associated with apoptotic death of cells initially in the ganglion cell layer and subsequently in the inner and outer nuclear layer, associated with loss of RGCs and amacrine cells. These phenomena were inhibited when recombinant plasminogen activator inhibitor (rPAI-1) or tPA-STOP were injected into the vitreous humor with KA, whereas a plasmin inhibitor, alpha-2-antiplasmin, failed to attenuate KA-induced retinal damage. Taken together, these results suggest that inhibition of plasminogen activators might attenuate retinal damage in blinding retinal diseases in which hyperstimulation of glutamate receptors is implicated as a causative factor to retinal damage.

purpose. Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated. methods. Normal adult CD-1 mice were treated with KCl by intravitreal injection. MMP activity in retinal protein extracts was determined by gelatin zymography. Tissue localization of MMP-9 in the retina was determined by immunohistochemistry. MMP-9, MMP-2, tissue inhibitor of MMP (TIMP)-1, TIMP-2, Bax, and BCl-2 proteins in retinal extracts were determined by Western blot analysis. Apoptotic cell death in the retina was determined by TUNEL assays. Retinal damage was assessed by immunolocalization studies with antibodies against neurofilament-light (NF-L) and calretinin. results. Depolarizing concentrations of KCl induced a dose- and time-related upregulation in MMP-9 activity and protein in the retina. KCl-mediated MMP-9 upregulation was associated with an increase in proapoptotic protein Bax and apoptotic death of cells in the ganglion cell (GCL) and inner nuclear layer (INL), and subsequent loss of NF-L-positive ganglion cells and calretinin-positive amacrine cells. Intravitreal injection of KCl along with an N-methyl-d-aspartate (NMDA)-type glutamate receptor antagonist, MK-801, and a non-NMDA-type glutamate receptor antagonist, NBQX, resulted in a reduction in KCl-mediated MMP-9 upregulation in the retina. Furthermore, a synthetic MMP inhibitor inhibited KCl-mediated MMP-9 upregulation, which led to a significant attenuation of KCl-induced retinal damage. conclusions. These results suggest that upregulation of MMP-9, in part, plays a causative role in KCl-induced retinal damage.

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction.Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2(.)5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transcluction could be found after PIT In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells. (C) 2004 Elsevier Ltd. All rights reserved.