The Role of Mitochondria, Oxidative Stress, and the Radical-binding Protein A1M in Cultured Porcine Retina

Abstract

Purpose: The purpose of this study was to explore the relationship between oxidative stress, antioxidant defense, mitochondrial structure and biomechanical tissue support in the isolated porcine retina. Methods:Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 2 or 48 hours using 1) a previously established low-support explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or 2) a high-support procedure developed by our group, apposing the Müller cell endfeet and inner limiting membrane (ILM) against the membrane. The grafts were analyzed by quantitative PCR, immunohistochemistry, and transmission electron microscopy (TEM), and culture medium was assayed for the cell damage and oxidative stress markers lactate dehydrogenase and protein carbonyls. Results:In explants cultured with physical support to the inner border, cone photoreceptors were preserved and lactate dehydrogenase levels were reduced, although an initial (2h), transient, increased oxidative stress was observed. Elevated expression of the antioxidants A1M (α1-microglobulin) and heme oxygenase-1 were seen in the mitochondria-rich inner segments after 48h compared to low-support counterparts.Housekeeping gene expression suggested a higher degree of structural integrity of mitochondria in high-support explants, and TEM of inner segments confirmed preservation of a normal mitochondrial morphology. Conclusion:Providing retinal explants with inner retinal support leads to mobilization of antioxidant proteins, preservation of mitochondrial function and increased cell viability.Consequently, the failure of low-support retinal cultures to mobilize an adequate response to 2 the oxidative environment may play an key role in their rapid demise. These findings shed new light on pathological reactions in biomechanically related conditions in vivo.

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“…The protective effects of rA1M treatment regarding both elevated BP and oxidative and nitrative stress supports its role as an endogenous radical-scavenging and tissue repair protein 27 . It has been shown that A1M has a protective role against oxidative stress and binds to mitochondria to help maintain morphological structure and ATP production 34,35 . The reduction in kidney expression levels of HO-1 , CAT and SOD2 in the rA1M-treated females supports this protective role, since these are genes involved in responses towards oxidative stress or apoptosis.…”

Update on the human and mouse lipocalin (LCN) gene family, including evidence the mouse Mup cluster is result of an “evolutionary bloom”

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Organ Cultures for Retinal Diseases

“…To establish a functional retinal model, several challenges need to be overcome. A major problem is the limited viability of most retinal organ culture systems due to axotomy or lack of blood flow and biomechanical tissue support leading to metabolic disorders and cell death (Åkerström et al, 2017;Schnichels et al, 2019). This mainly affects RGCs, but also other cell types such as macroglia, displaying gliosis (Tanihara et al, 1997;Wurm et al, 2011;Joachim et al, 2012;Hurst et al, 2017a) and reducing the time frame for experiments with these cultures.…”