retinal vasculature isolation from mouse eye and its mounting on a microscope slide

Novel Retina Protection Assay for Diabetic Retinopathy Pathogenesis Study

Diabetic retinopathy (DR) is one of the microvascular complications of diabetes mellitus (DM), and the leading cause of blindness in working-age individuals in developed countries1. Major risk factors for diabetic retinopathy are the duration and degree of hyperglycemia2,3,4. While DM causes dysfunction of both the vascular and neural components of the retina5, diagnosis of DR is based on morphological features of the retinal vasculature6.
Hyperglycemia-induced oxidative stress is one of the drivers of DR pathogenesis7. Increased oxidative stress causes widespread damage, which compromises the functionality of the mitochondria and thereby further increases the level of reactive oxygen species. These events are accompanied by leakage of retinal vessels, an increase in the level of inflammatory cytokines, and death of both neural and vascular cell types within the retina. Loss of vascular cells, and hence the functionality of the extensive capillary network of the retina, results in hypoxia, a potent stimulus for a variety of responses5. Such responses include increased expression of vascular endothelial growth factor (VEGF) that drives both permeability and angiogenesis, cardinal features of the advanced, sight-threatening stages of DR - diabetic macular edema, and proliferative diabetic retinopathy6.
Certain features of DR suggest that an organism (both patients and experimental animals) has an intrinsic ability to resist this indication. Patients experience several decades of DM before they develop sight-threatening DR8,9,10,11,12,13. While rodent models of DM do not develop the advanced, sight-threatening stages of DR14, the initial/mild form of DR that manifests does so only after a period of weeks or months of DM15,16. Furthermore, in both patients and experimental animals, DR is progressive, and retinal dysfunction/damage increases as the duration of DM is prolonged. Finally, some patients with DM never develop DR. In certain instances, this is because such individuals do not experience diabetes long enough for DR to develop. In other instances, it is because they display extraordinary resistance to DR; as is the case with participants of the Medalist study, who do not develop DR after 50 or more years of DM17. Despite such compelling support for the existence of protection from DR and its enormous translational relevance, the mechanism underlying protection has not been aggressively investigated.
The protection assay described herein was developed to facilitate investigation of why DR is delayed from the onset of DM in diabetic mice. The key steps of this assay, applied to DM and non-DM mice, include (1) delivering a sub-maximal death-inducing insult to the eye (ex vivo or in vivo), (2) isolating the retinal vasculature, (3) staining the vasculature with TUNEL and DAPI, (4) photographing the resulting images and quantifying the percentage of TUNEL/DAPI double-positive species.

Author Spotlight: Understanding Retinal Vessel Resilience and Disease Progression

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Our goal is to comprehend the reason behind the gradual onset of diabetic retinopathy, which occurs decades after the onset of diabetes. We're investigating the pathogenesis of diabetic retinopathy in two phases, loss of protection and damage accumulation. Our goal is to investigating the protection of retinal vessels against diabetes-induced insults through a protection assay.Our protection assay unveils the dual phases of protection loss, and accumulation of damage related to diabetic retinopathy. And so this innovative approach provide a valuable tool for understanding diabetic retinopathy pathogenesis. And the potential application could be extended beyond DR, it could apply to a broader physiological and pathological context.While existing literature predominantly focuses on the pathological alterations in diabetic retinopathy, there remains a notable gap in our knowledge concerning protective factors. This protection assay plays a crucial role in bridging the lacking of, and helps us understand the role of protective factors involved in the retinal vasculature resilience to damage induced by diabetes. This novel protection assay enhances our understanding of diabetic retinopathy progression, providing a targeted tool to investigate the delay between diabetes onset and DR manifestation in mice.It advances our research by dissecting the interplay between protection and damage, offering potential for targeted therapeutics and broader applications. Our future research will concentrate on delineating the specific cell types, endothelial cells, parasites, and neuronal cells in the mouse retina, and the molecular mechanisms underlying this protection. Additionally, we aim to identify the potential genes behind this protective mechanism.

Watch this Scientific Journal Video about Retinal Vasculature Isolation from Mouse Eye and its Mounting on a Microscope Slide at JoVE.com

Retinal Vasculature Isolation from Mouse Eye and its Mounting on a Microscope Slide

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Research

JoVE Journal

Medicine

298 visualizações.  University of Illinois at Chicago. Para começar, tome retinas isoladas em um prato de 24 poços cheio de água bidestilada. Em seguida, substitua a água bidestilada por 800 microlitros de solução de tripsina YL e incube a 37 graus Celsius com agitação suave ou nula por 4 horas e 15 minutos. Usando a pipeta de transferência de vidro mergulhada em solução de tripsina YL, transfira a retina para uma placa de Petri de 35 milímetros contendo água bidestilada sem fiapos.Para remover a camada nuclear externa, vire a...

Vídeo: Isolamento da Vasculatura Retiniana do Olho do Rato e sua Montagem em uma Lâmina de Microscópio

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...