Leading causes of blindness in developed countries include macular degeneration, glaucoma and diabetic retinopathy which are all manifested as dysfunction and degeneration of specific cell types in the retina. The molecular mechanisms and pathophysiology of these diseases are not well understood. Widely used techniques to study the pathophysiology of retinal diseases include In Vivo electroretinogram (ERG) and single cell electrophysiology. In Vivo ERG can assess the overall health state of the retina but is limited in providing quantitative information about the cellular and molecular origin of the functional deficits in a diseased retina. Single cell recordings, on the other hand, provide quantitative data from individual cells but are challenging, offer only limited recording time and are not easily scaled up to assess the function and signaling across the whole retina. The objective of this proposal is to advance techniques and methodology to dissect the function of photoreceptor, bipolar and Mller-glial cells in the intact mouse, primate, pig and ultimately human retinas. I wll first develop methodology to quantitatively assess the intrinsic functional state of these cells y using Ex Vivo ERG from isolated wild-type mouse retinas (K99). Ex Vivo ERG provides information about the average functional state of all cells across the lateral axis of the retina. However, some of the retinal diseases affect retina only locally (e.g. DR) or target primarily ganglion cells (e.g. glaucoma) that will not necessarily be observable in the Ex Vivo ERG signal. One objective of this proposal is to develop a novel device combining Ex Vivo ERG and multi-electrode array (MEA) methods. This device will be used to assess the local function of photoreceptors, bipolar, Mller-glial and ganglion cells across the whole wild-type mouse retinas (K99). Ex Vivo ERG (K99) will be applied to determine how the function of rod and cone bipolar, Mller-glial and ganglion cells are affected in the mouse models of retinitis pigmentosa (RP, P23H rhodopsin mutation) and diabetic retinopathy (DR, Streptozotocin-induced diabetes) and MEA-ERG device will be used to assess the local function of the retina that has been focally injured by laser (K99 and R00). These experiments will advance the understanding of pathophysiology of these diseases known to affect primarily outer and inner retina, respectively. The methodology developed for mouse retinas will be used to establish protocols to dissect the function of photoreceptors, bipolar and Mller-glial cells in primate, pig, and ultimately human donor retinas. I will first develop the recording protocols to obtain viable responses from primate retinas dissected from eye balls enucleated immediately following the euthanasia (K99). Then, in collaboration with Dr. Hanneken, we will determine the acceptable time frame between death and enucleation by using pig eyes (R00). Finally, based on the primate and pig experiments we will design and conduct recordings to assess function of photoreceptor, bipolar and Mller glia cells in a macula and peripheral regions of the human retina (R00).

Public Health Relevance

The most common causes of blindness in developed countries include age-related macular degeneration (AMD), diabetic retinopathy and glaucoma that are all manifested as dysfunction of specific cells in the retina. This proposal will develop novel techniques and methodology that can be used to assess the functional 'health state' of the retinal cells and effects of drugs on them in intact retinas from mouse to humans. The methodology will be used to advance our understanding of the cellular and molecular origins of diabetic retinopathy and retinitis pigmentosa as well as the functional similarities and difference between mouse, primate, pig and human retinas.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Career Transition Award (K99)
Project #
5K99EY026651-02
Application #
9249586
Study Section
Special Emphasis Panel (ZEY1-VSN (04))
Program Officer
Agarwal, Neeraj
Project Start
2016-04-01
Project End
2018-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
$94,826
Indirect Cost
$7,024
Name
Washington University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Sakurai, Keisuke; Vinberg, Frans; Wang, Tian et al. (2016) The Na(+)/Ca(2+), K(+) exchanger 2 modulates mammalian cone phototransduction. Sci Rep 6:32521