Name: 视频: Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System
Uploaded: 2024-08-16T14:20:00
Description: 44 次观看. Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System

measuring changes in mitochondrial metabolism in hypoxic rpe using resipher system

Novel OCR Monitoring System for Differentiated RPE Cultures

The retinal pigment epithelium (RPE) is a monolayer of functionally postmitotic, highly polarized epithelial cells that form a barrier between light-sensitive photoreceptors in the retina and their blood circulation, a capillary bed termed the choriocapillaris. Like the role of glia-supporting neurons, the RPE carries out myriad functions to support photoreceptors, including phagocytosis of photoreceptor outer segments, transport of nutrients and metabolic support for photoreceptors, and secretion of essential growth factors, all critical for maintaining visual function.
Degeneration of the RPE underlies several common degenerative disorders of vision. In age-related macular degeneration (AMD), one of the most common causes of incurable vision loss in the world, the RPE dies, and overlying photoreceptors therefore suffer a secondary degeneration. In proliferative vitreoretinopathy (PVR), the RPE instead exits its normally quiescent postmitotic state, proliferating and dedifferentiating into a mesenchymal state (a so-called epithelial-to-mesenchymal transition [EMT]) with alterations in its metabolism. RPE dedifferentiation causes a loss of RPE support to photoreceptors while also triggering a more fibrotic state. This results in both photoreceptor degeneration and RPE-induced scarring, both of which trigger vision loss1,2.
A major part of the RPE&#39;s support to photoreceptors is metabolic, and metabolic dysregulation is a critical factor in numerous retinal diseases, including AMD and PVR. The RPE serves as a regulatory barrier between photoreceptors and their source of oxygen and nutrients, the choriocapillaris. Thus, what the RPE chooses to metabolize versus what the RPE chooses to pass through from the choriocapillaris to the photoreceptors strongly governs photoreceptor metabolism and survival. Numerous studies have shown that the RPE is heavily dependent on mitochondrial metabolism for its normal health, and that photoreceptors instead rely heavily on glycolysis3. This has introduced the concept of complementary, intertwined metabolic states between photoreceptors and the RPE. Specifically, the RPE reduces its metabolism of preferred photoreceptor metabolic substrates and instead utilizes the byproducts of photoreceptor metabolism combined with the metabolites that photoreceptors do not consume. In diseases such as PVR and AMD, evidence strongly suggests that the RPE becomes more glycolytic and less dependent on mitochondrial metabolism; this shift towards RPE glycolysis may starve photoreceptors of metabolites it needs, triggering degeneration4,5,6. Given how interdependent RPE and photoreceptor metabolism are and how much altered metabolism underlies retinal disease, there is strong interest in modeling and manipulating RPE metabolism for therapeutic purposes.
While studying RPE mitochondrial metabolism in vivo is ideal, many aspects of RPE mitochondrial metabolism can only be practically probed in an in vitro culture system. Significant progress towards high-fidelity RPE cultures has been made in the past several decades, to the point that the most carefully groomed RPE cultures are now being used for cell replacement therapy in human clinical trials7. To maintain such high-fidelity cultures, the RPE needs to be grown on particular substrates in particular media for months prior to experimentation. With these conditions, RPE cultures are maximally differentiated and polarized, approximating in vivo RPE. Unfortunately, there is no equipment currently available that can measure mitochondrial metabolism specifically from the RPE in vivo. While oxygen monitoring of the retinal capillary network has been achieved in vivo using electron paramagnetic resonance (EPR) oximetry8, this is not possible for RPE analysis. Differences between RPE metabolism in vivo and in vitro are not well-described, but RPE cultures have been shown to have high mitochondrial activity, similar to RPE in vivo3,9, suggesting significant insight into RPE mitochondrial metabolism can be gained using high-fidelity RPE cultures.
As all mitochondrial metabolism leads to oxygen consumption, measuring RPE oxygen consumption rates (OCR) is a faithful proxy for mitochondrial metabolism. Measuring OCR in RPE cultures has been difficult, as the conditions that promote maximal RPE polarization and differentiation often preclude long-term accurate OCR measurements with currently available techniques, such as the Seahorse Analyzer. In this methods paper, a novel device, termed the Resipher (hereafter referred to as &#34;the system&#34;), is introduced, which allows continuous measurement of OCR over weeks in RPE grown under conditions that maximally promote polarization and differentiation. The ease with which OCR can be measured by this system in RPE culture conditions that maximally promote RPE differentiation and polarization is unique among existing OCR-measuring devices.
This paper provides tips and tricks for using the system with RPE cultures, followed by a demonstration of two particular applications. First, RPE EMT, mimicking PVR, is triggered by exposure to transforming growth factor-beta (TGF&#946;)1,10,11,12. The system is used to monitor how RPE metabolism evolves during the EMT process. Second, the role of hypoxia in RPE metabolism is explored using this system. Hypoxia is an important contributor to the pathogenesis of AMD, as the choriocapillaris thins with age13,14. Combining this system with hypoxia chambers allows one to model altered RPE mitochondrial metabolism with the subtle hypoxia that accompanies aging. Finally, an online calculator using Resipher data is introduced to allow one to determine whether RPE cultures are in hypoxic, normoxic, or hyperoxic conditions. Such information is critical for drawing any conclusions about RPE metabolism from in vitro RPE culture studies.

Author Spotlight: Extended Oxygen Consumption Measurement in Retinal Pigment Epithelium Using Resipher

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System

measuring-changes-mitochondrial-metabolism-hypoxic-rpe-using-resipher

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44 次观看. Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System