Measuring mitochondrial respiration in vivo: From mouse to human

Introduction: The mitochondrial energy ecosystem can be non-invasively interrogated in photoreceptors by combing a clinical tool, optical coherence tomography (OCT), with a mitochondrial protonophore (2,4 dinitrophenol, DNP). It remains unclear if only supra-clinical doses of DNP will be useful for mouse studies or if lower but clinically relevant doses of DNP would facilitate translation from mice to humans. Methods: The experiment was a paired longitudinal design that took place over 2 days. On day 1, C57BL/6J mice were overnight dark adapted, then light-adapted for 5 h before OCT examination before regaining consciousness; a similar procedure was followed on day 2 but mice were injected IP with either saline or an acceptable human dose of DNP (0.5 mg/kg) 1 hour before OCT examination. Pre- and post-injection retinal laminae thickness were compared for evidence of toxicity. In particular, we measured the external limiting membrane – retinal pigment epithelium to look for thinning between day 1 and 2, which has a confirmed basis in DNP modulation of rod photoreceptor mitochondrial respiration upstream based on pH-triggering of RPE co-transporter-based water efflux. Results: Compared to uninjected controls, saline evoked no changes in retina layer thickness between days 1 and 2. On the other hand, a clinical dose of DNP caused a reduction in ELM-RPE thickness in the absence of any other changes in retinal layer thickness, a finding consistent with increased mitochondrial respiration. Conclusions: The promising results herein raise the possibility that combining a low dose of DNP with a standard clinical imaging tool will facilitate first-in-kind measurements of neuronal mitochondria in patients suffering from a range of diseases and morbidities

Impact of COVID-19 on a Free Clinic Patient Population

The Robert R. Frank Student Run Free Clinic at WSUSOM, surveyed patients to determine the impact of the COVID-19 pandemic on its patient population. The study examined any challenges faced in food, financial status, transportation, and healthcare. A Qualtrics survey was administered with ethnicity, age, and gender as controls, while testing patient responses to social factors using the Likert scale. The survey was targeted to a portion of the clinic’s patient pool (n=33) over a span of three months and responses were analyzed using SPSS 26 regression analysis, ANOVA, and paired sample T-tests. Significant responses were across demographics, categories of impact, and pre/post COVID-19. Results show that access to fresh foods was severely impacted by COVID-19 (t = -3.19, p\u3c 0.05). Linear regression models show a correlation between difficulty accessing healthcare and medications, before and after COVID-19, indicating that the pandemic may have exacerbated pre-existing barriers to treatment (correlation = 0.810). Financial status has been the most affected with many patients indicating changes in employment or income status. 55% of the participants noted a greater use of savings or retirement money to make ends meet. A moderate correlation (0.471) was found between the use of public transportation prior to the pandemic and transportation barriers during the pandemic. We have decided to compile resources to address the needs based on the study results. Future research includes a longitudinal, follow-up survey to gauge any changes. Limitations include the study sample size and participation bias among the patient population

Visual Performance Declines in 24 mo C57BL/6J Mice are Unrelated to Outer Retina Oxidative Stress in Vivo

Purpose : Age-related declines in visual performance increase the risk of morbidity and mortality from falls in humans and are so far untreatable; rats and mice also show reduced vision with age. The cause of age-related declines in visual performance is unclear but evidence ex vivo suggests a link to photoreceptor / retinal pigment epithelium oxidative stress. Methods : 2 and 24 mo male C57BL/6J mice were non-invasively evaluated for excessive production of paramagnetic free radicals based on whether R1 (= 1/T1) in retinal laminae are reduced after acute anti-oxidant (AO) administration [QUEnch-assiSTed (QUEST) magnetic resonance imaging (MRI)]. Superoxide production was measured in excised retina (lucigenin assay). Acute AO administration was also used to test for age-related oxidative stress-induced thinning of subretinal space (QUEST optical coherence tomography [OCT]) and cone-based visual performance declines (QUEST optokinetic tracking [OKT]). Results : At 2 mo, no evidence was found in vivo for oxidative stress in any retinal layer. At 24 mo, oxidative stress was localized only to superior outer retina. Yet, no age-related change in retinal superoxide production was noted suggesting that free radical species other than superoxide contributed to the positive QUEST MRI signal at 24 mo. Subretinal space did not show age-related thinning and was unresponsive to AO’s. Finally, visual performance declined with age and was not restored by AO’s that were effective in QUEST MRI. Conclusions : Outer retinal oxidative stress appears to be insufficient to explain the reduction in visual performance in 24 mo C57BL/6J mice

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

PurposeThe phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision.MethodsIn dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking.ResultsIn light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil.ConclusionsThe sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice