VO-CRO In Vivo Readouts
We offer a number of readouts for each of our animal models to evaluate the effects of experimental compounds. We are able to provide consultation on which readouts will most effectively assess the disease components of interest.
In order to measure the efficacious effects of putatively therapeutic compounds on retinal function, we use a combination of electroretinography, optical coherence tomography/morphometry, and optokinetic reflex/optokinetic tracking. In addition to their use in measuring the efficacy provided by therapeutic agents, we also utilize these methods to determine retinal toxicity caused by the test compounds.
We longitudinally assess visual function after drug treatment using electroretinography (ERG). ERG measures the electrical activity generated by the retinal cells of dark-adapted animals in response to light.
Optical Coherence Tomography/Morphometry
As a complementary approach, we also examine and measure changes in retinal structure and morphometry using optical coherence tomography (OCT) and conventional histological methods.
Optokinetic Reflex/Optokinetic Tracking
Visually evoked compensatory head movements or eye movements are extensively used in our experimental models to test both the efficacy and toxicity of therapeutic manipulations.
Neovascular area can be assessed for both the OIR and LCNV models. We carefully dissect the retina or choroid, stain for blood vessels, and flat-mount the tissue. We then measure neovascular growth using computer-assisted image analysis tools. Our methods allow for precise and highly reproducible data.
Increased vascular permeability is a damaging component of diabetic retinopathy. To test whether a drug has an effect on permeability, we use one or more of several methods. We measure the extravasation of Evan’s blue dye or fluorescein-conjugated dextrans from the vasculature into the retinal tissue, we employ quantitative fluorescein angiography or we evaluate retinal thickness via OCT.
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Diabetic retinopathy is characterized by a loss of retinal pericytes and endothelial cells. Trypsin digests can be used to carefully remove the retinal vasculature from the retina to assess its characteristics. Once removed and flat-mounted, the digest allows for determination of pericyte to endothelial cell ratio and quantification of pericyte and endothelial cell apoptosis. Digests can also serve as the source of information related to expression levels of targets of interest.
Leukostasis, the adherence of leukocytes to endothelium, is a major component of non-proliferative diabetic retinopathy and is a general indicator of tissue inflammation. We measure retinal leukostasis by infusing animals with fluorescein-conjugated concanavalin A, flushing the circulation with buffer perfusion, carefully dissecting and flat-mounting retinas, and counting the number of leukocytes remaining in the lumenal space of blood vessels per unit retina area.
Vasoregression refers to the progressive obliteration of capillaries that occurs early in some microvascular diseases. Using a specially-designed staining technique and computer-assisted image analysis, we can quantitatively assess capillary dropout under different stimuli.
As is the case for cells, RNA sequencing, quantitative RT PCR, and single-cell RNA sequencing are all methods used to determine expression levels in tissue samples derived from in vivo experiments.
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