Mitochondrial Retinal Imaging – What Do We Know So Far?

INTRODUCTION

The mitochondrion is considered to be the powerhouse of eukaryotic cells. Oxidative phosphorylation produces energy, leading to adenosine triphosphate production. In addition, mitochondria perform numerous other functions in cellular homeostases such as apoptosis regulation, steroid biosynthesis, and nucleotide metabolism. For this reason, mitochondrial dysfunction can severely compromise cellular homeostasis and has been highlighted as a crucial mechanism in the pathophysiology of normal aging and pathologic processes including retinal diseases.^[1,2]

Mitochondrial dysfunction is recognized to play a crucial role in a wide range of diseases and organs, with the most affected organs being those that have a high metabolic rate such as brain and heart.^[3] For example, previous studies have shown evidence of mitochondrial involvement in the pathophysiology of neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease, and in cardiac ischemia-reperfusion injury.

Recent research also highlighted the importance of mitochondria in another highly metabolic active tissue: The retina. Mitochondria are abundantly present in active retinal cells such as retinal pigmented epithelium (RPE) cells. The mitochondria play a central part in homeostasis. Reactive oxygen species (ROS) can typically alter mitochondria functions leading to respiratory chain impairment and loss of membrane potential; these changes precede apoptosis.^[4]

Thus, rising comprehension of mitochondria’s role in retinal functioning led to the development of a novel device to assess oxidative stress as a measure of retinal health. In contrast with other organs, the retina medium is transparent and allows for non-invasive detection of mitochondrial dysfunction.

To date, many papers have been published testing this device in different diseases. The purpose of this review is to provide a concise source about current understandings of mitochondrial non-invasive imaging in normal and retinal diseases.

LITERATURE SEARCH

A literature search of PubMed was conducted and the papers included were published between 2008 and 2018. Articles published in peer-reviewed journals on mitochondrial retinal imaging were included. The search included the following keywords: Non-invasive detection mitochondrial dysfunction and/or mitochondria dysfunction imaging and/or retina flavoprotein fluorescence (FPF). Publications cited in the references that were relevant to our subject were also selected. After reviewing the abstracts, studies not directly related to the topic were excluded from the study.

RETINAL DISEASES AND MITOCHONDRIAL DYSFUNCTION

Multiple studies have shown the importance of mitochondria in the pathophysiology of retinal disorders such as diabetic retinopathy (DR), age-related macular degeneration (AMD), and glaucoma.

DR

In diabetes mellitus (DM), hyperglycemia induces chronic overproduction of ROS that impair antioxidant defenses,^[5] damaging mitochondrial DNA (mtDNA), and disrupting mitochondrial membrane potential. This leads to cytochrome C release and initiation of apoptosis. In response, damaged mitochondria produce more ROS creating a vicious cycle of oxidative damage (Figure 1).^[6,7]

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Figure 1: Synoptic image of pathophysiological pathways involving mitochondrial dysfunction. Different factors like hyperglycemia, genetics, aging and intraocular pressure can trigger the alterations that lead to an increase in oxidative stress which causes mitochondrial dysfunction. In turn, mitochondrial dysfunction increases oxidative stress and leads to a vicious cycle of oxidative damage.

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OXIDIZED FLAVOPROTEINS AS A MEASURE OF RETINAL MITOCHONDRIAL DYSFUNCTION

The growing interest in understanding the mitochondrial role in human pathology established the need for measuring its activity or inactivity. There is a very broad spectrum of techniques that have been proposed, all of which are dependent on the precise definition authors give to the vague term “dysfunction.” In the pertinent literature, dysfunction is primarily defined as uncoupling of the respiratory chain that leads to a rise in oxidative stress and subsequently to apoptosis.^[1] Oxidative stress impairs mitochondrial equilibrium as increased ROS production and damage overshadows antioxidant defenses. Under these conditions, flavoproteins involved in the electron transport chain become oxidized.^[11] Mitochondrial dysfunction can be estimated assessing the presence of oxidized flavoproteins, which absorbs blue light and emits green autofluorescence.^[12] This measurement is called FPF and estimates oxidative stress by measuring average intensity (FPF score) and standard deviation (SD) of the autofluorescence emitted. In vitro studies have previously combined NAD(P)H fluorescence with FPF, but this cannot be replicated in humans because excitation wavelengths for NAD(P)H have phototoxic effects on retinal cells.^[13]

METABOLIC ANALYSIS OF RETINAL DISEASES

Eight peer-reviewed papers have been published regarding FPF as a biomarker in retinal diseases (Table 2).

DISCUSSION

Recent advances in a technology widely contributed to improve retinal imaging. However, the current standard of care devices focuses on the identification of structural changes rather than functional (metabolic) changes, despite the fact that functional changes often occur earlier in disease processes. The ability to analyze metabolic changes may allow for earlier anticipation of disease progression or even earlier diagnosis of previously unidentified disease as DR.^[27] Field et al. introduced the possibility that FPF can be a better index for progression of retinopathy than HBA1c.^[20] FPF analysis, first results have high potential in an area where an innovative diagnosis method is needed as demonstrated by the fact that none of the numerous functional techniques proposed for DR screening (e.g., electroretinography, microperimetry, color vision, and contrast sensitivity) have demonstrated a solid predictive value. In the same study, authors have shown a correlation between FPF and the presence of DR, but no analysis has been made to evaluate a relationship between DR severity and FPF score. Thus, FPF differences within DR severity needs to be further examined. This needs to be examined more thoroughly in not only DR but also additional retinal diseases with various stages of disease severity such as AMD. With long-term follow-up and larger sample sizes, future studies can explore FPF as a prognostic index and provide additional insight on manifestations at each disease stage that cannot be elucidated with current imaging technology.

Furthermore, preliminary results achieved in AMD, glaucoma, and CSR have shown that retinal functional alterations are present in many diseases, which pathophysiological pathways also indicate. Moreover, Ritch et al. demonstrated that optic disc FPF score decreases in glaucoma with antioxidant drug usage. Future studies can assess the efficacy of various treatments such as antioxidant drugs in different retinal diseases. In this scenario, mitochondrial retinal imaging represents a promising technology that transposes basic science knowledge to the clinical setting. This technology offers the possibility to perform a rapid, affordable, and non-invasive retinal functional study potentially applicable to all retinal diseases.

However, there are some divergences that merit further examination. According to the previous studies, FPF increases with aging and AMD.^[12,23] In contrast, Feher et al. have demonstrated that the number of mitochondria decreases with aging and it is even lower in patients with AMD.^[28] Based on Feher’s findings, it seems counterintuitive that FPF scores increase with greater mitochondrial cell death when considering Feher’s findings. However, one hypothesis is that the higher scores in more advanced stages of disease such as advance non-exudative and exudative AMD are actively indicating ongoing increases in mitochondrial dysfunction that will soon be followed by cell death. Henceforth, cell death and lower PFF scores may not be seen until a patient has experienced permanent vision loss. Additional studies are needed to clarify the correlation between a number of mitochondria and FPF. The points at which mitochondrial dysfunction level accelerates and later declines as cell death occurs for each retinal disease are subjected to uncertainty and exploration.

Moreover, a number of drawbacks are present. Two versions of the device have been used in these studies, and the field of view and FPF analysis software was different. Device model changes and the change in the field of view likely lead to differences in FPF score standardization. As a result, not all papers in the literature can be compared and examined together. Rigorous standardization of a single device that can measure FPF scores across all ophthalmic diseases is required to improve reliability and allow use in clinical settings. Present studies do not provide useful information to examine inter-individual variability. For example, FPF might be influenced by retinal mitochondrial density, lens status, lipofuscin concentration, and other factors. To define abnormal results, further studies to determine normality are needed, and any effect these factors have on FPF must be ascertained.

Finally, only eight investigations have been published, and all these studies rely on very limited sample size. Consequently, larger populations are needed to confirm the current findings.

CONCLUSION

FPF is a sensitive, albeit non-specific technology. This makes it suitable for a wide range of diseases, especially for screening and follow-up purposes. Future studies will eventually refine and solidify the present literature.