Biomarker evidence of early vision and rod energy-linked pathophysiology benefits from very low dose DMSO in 5xFAD mice.

Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.

Acetazolamide Challenge Changes Outer Retina Bioenergy-Linked and Anatomical OCT Biomarkers Depending on Mouse Strain.

Purpose: The purpose of this study was to test the hypothesis that optical coherence tomography (OCT) bioenergy-linked and anatomical biomarkers are responsive to an acetazolamide (ACZ) provocation. Methods: C57BL/6J mice (B6J, a strain with relatively inefficient mitochondria) and 129S6/ev mice (S6, a strain with relatively efficient mitochondria) were given a single IP injection of ACZ (carbonic anhydrase inhibitor) or vehicle. In each mouse, the Mitochondrial Configuration within Photoreceptors based on the profile shape Aspect Ratio (MCP/AR) index was determined from the hyper-reflective band immediately posterior to the external limiting membrane (ELM). In addition, we tested for ACZ-induced acidification by measuring contraction of the external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness; the hyporeflective band (HB) signal intensity at the photoreceptor tips was also examined. Finally, the nuclear layer thickness was measured. Results: In response to ACZ, MCP/AR was greater-than-vehicle in B6J mice and lower-than-vehicle in S6 mice. ACZ-treated B6J and S6 mice both showed ELM-RPE contraction compared to vehicle-treated mice, consistent with dehydration in response to subretinal space acidification. The HB intensity at the photoreceptor tips and the outer nuclear layer thickness (B6J and S6), as well as the inner nuclear layer thickness of B6J mice, were all lower than vehicle following ACZ. Conclusions: Photoreceptor respiratory efficacy can be evaluated in vivo based on distinct rod mitochondria responses to subretinal space acidification measured with OCT biomarkers and an ACZ challenge, supporting and extending our previous findings measured with light-dark conditions.

Do multiple physiological OCT biomarkers indicate age-related decline in rod mitochondrial function in C57BL/6J mice?

Purpose: To test the hypothesis that rod photoreceptor mitochondria function in vivo progressively declines over time. Methods: 2, 12, and 24 month-old dark- and light-adapted C57BL/6J (B6J) mice were examined by OCT. We measured (i) an index of mitochondrial configuration within photoreceptors measured from the profile shape aspect ratio (MCP/AR) of the hyperreflective band posterior to the external limiting membrane (ELM), (ii) a proxy for energy-dependent pH-triggered water removal, the thickness of the ELM-retinal pigment epithelium (ELM-RPE), and its correlate (iii) the hyporeflective band (HB) signal intensity at the photoreceptor tips. Visual performance was assessed by optokinetic tracking. Results: In 2 and 24 month-old mice, MCP/AR in both inferior and superior retina was smaller in light than in dark; no dark-light differences were noted in 12 month-old mice. Dark-adapted inferior and superior, and light-adapted superior, ELM-RPE thickness increased with age. The dark-light difference in ELM-RPE thickness remained constant across all ages. All ages showed a decreased HB signal intensity magnitude in dark relative to light. In 12 month-old mice, the dark-light difference in HB magnitude was greater than in younger and older mice. Anatomically, outer nuclear layer thickness decreased with age. Visual performance indices were reduced at 24 month-old compared to 2 month-old mice. Conclusion: While the working hypothesis was not supported herein, the results raise the possibility of a mid-life adaptation in rod mitochondrial function during healthy aging in B6J mice based on OCT biomarkers, a plasticity that occurred prior to declines in visual performance.

Transducin-Deficient Rod Photoreceptors Evaluated With Optical Coherence Tomography and Oxygen Consumption Rate Energy Biomarkers.

Purpose: To test the hypothesis that rod energy biomarkers in light and dark are similar in mice without functional rod transducin (Gnat1rd17). Methods: Gnat1rd17 and wildtype (WT) mice were studied in canonically low energy demand (light) and high energy demand (dark) conditions. We measured rod inner segment ellipsoid zone (ISez) profile shape, external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness, and magnitude of a hyporeflective band (HB) intensity dip located between photoreceptor tips and apical RPE; antioxidants were given in a subset of mice. Oxygen consumption rate (OCR) and visual performance indexes were also measured. Results: The lower energy demand expected in light-adapted wildtype retinas was associated with an elongated ISez, thicker ELM-RPE, and higher HB magnitude, and lower OCR compared to high energy demand conditions in the dark. Gnat1rd17 mice showed a wildtype-like ISez profile shape at 20 minutes of light that became rounder at 60 minutes; at both times, ELM-RPE was smaller than wildtype values, and the HB magnitude was unmeasurable. OCR was higher than in the dark. Light-adapted Gnat1rd17 mice biomarkers were unaffected by anti-oxidants. Gnat1rd17 mice showed modest outer nuclear layer thinning and no reduction in visual performance indexes. Conclusions: Light-stimulated changes in all biomarkers in WT mice are consistent with the established light-induced decrease in net energy demand. In contrast, biomarker changes in Gnat1rd17 mice raise the possibility that light increases net energy demand in the absence of rod phototransduction.

Functional Changes Within the Rod Inner Segment Ellipsoid in Wildtype Mice: An Optical Coherence Tomography and Electron Microscopy Study.

Purpose: To test the hypothesis that changing energy needs alter mitochondria distribution within the rod inner segment ellipsoid. Methods: In mice with relatively smaller (C57BL/6J [B6J]) or greater (129S6/ev [S6]) retina mitochondria maximum reserve capacity, the profile shape of the rod inner segment ellipsoid zone (ISez) was measured with optical coherence tomography (OCT) under higher (dark) or lower (light) energy demand conditions. ISez profile shape was characterized using an unbiased ellipse descriptor (minor/major aspect ratio). Other bioenergy indexes evaluated include the external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness and the magnitude of the signal intensity of a hyporeflective band located between the photoreceptor tips and apical RPE. The spatial distribution of rod ellipsoid mitochondria were also examined with electron microscopy. Results: In B6J mice, darkness produced a greater ISez aspect ratio, thinner ELM-RPE, and a smaller hyporeflective band intensity than in light. In S6 mice, dark and light ISez aspect ratio values were not different and were greater than in light-adapted B6J mice; dark-adapted S6 mice showed smaller ELM-RPE thinning versus light, and negligible hyporeflective band intensity in the light. In B6J mice, mitochondria number in light increased in the distal inner segment ellipsoid and decreased proximally. In S6 mice, mitochondria number in the inner segment ellipsoid were not different between light and dark, and were greater than in B6J mice. Conclusions: These data raise the possibility that rod mitochondria activity in mice can be noninvasively evaluated based on the ISez profile shape, a new OCT index that complements OCT energy biomarkers measured outside of the ISez region.

Correcting QUEST Magnetic Resonance Imaging-Sensitive Free Radical Production in the Outer Retina In Vivo Does Not Correct Reduced Visual Performance in 24-Month-Old C57BL/6J Mice.

Purpose: To test the hypothesis that acutely correcting a sustained presence of outer retina free radicals measured in vivo in 24-month-old mice corrects their reduced visual performance. Methods: Male C57BL/6J mice two and 24 months old were noninvasively evaluated for unremitted production of paramagnetic free radicals based on whether 1/T1 in retinal laminae are reduced after acute antioxidant administration (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]). Superoxide production was measured in freshly excised retina (lucigenin assay). Combining acute antioxidant administration with optical coherence tomography (i.e., QUEST OCT) tested for excessive free radical-induced shrinkage of the subretinal space volume. Combining antioxidant administration with optokinetic tracking tested for a contribution of uncontrolled free radical production to cone-based visual performance declines. Results: At two months, antioxidants had no effect on 1/T1 in vivo in any retinal layer. At 24 months, antioxidants reduced 1/T1 only in superior outer retina. No age-related change in retinal superoxide production was measured ex vivo, suggesting that free radical species other than superoxide contributed to the positive QUEST MRI signal at 24 months. Also, subretinal space volume did not show evidence for age-related shrinkage and was unresponsive to antioxidants. Finally, visual performance declined with age and was not restored by antioxidants that were effective per QUEST MRI. Conclusions: An ongoing uncontrolled production of outer retina free radicals as measured in vivo in 24 mo C57BL/6J mice appears to be insufficient to explain reductions in visual performance.

Functional regulation of an outer retina hyporeflective band on optical coherence tomography images.

Human and animal retinal optical coherence tomography (OCT) images show a hyporeflective band (HB) between the photoreceptor tip and retinal pigment epithelium layers whose mechanisms are unclear. In mice, HB magnitude and the external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness appear to be dependent on light exposure, which is known to alter photoreceptor mitochondria respiration. Here, we test the hypothesis that these two OCT biomarkers are linked to metabolic activity of the retina. Acetazolamide, which acidifies the subretinal space, had no significant impact on HB magnitude but produced ELM-RPE thinning. Mitochondrial stimulation with 2,4-dinitrophenol reduced both HB magnitude and ELM-RPE thickness in parallel, and also reduced F-actin expression in the same retinal region, but without altering ERG responses. For mice strains with relatively lower (C57BL/6J) or higher (129S6/ev) rod mitochondrial efficacy, light-induced changes in HB magnitude and ELM-RPE thickness were correlated. Humans, analyzed from published data captured with a different protocol, showed a similar light-dark change pattern in HB magnitude as in the mice. Our results indicate that mitochondrial respiration underlies changes in HB magnitude upstream of the pH-sensitive ELM-RPE thickness response. These two distinct OCT biomarkers could be useful indices for non-invasively evaluating photoreceptor mitochondrial metabolic activity.

Coronavirus Disease 2019 (COVID-19) Seropositivity and Asymptomatic Rates in Healthcare Workers Are Associated with Job Function and Masking.

BACKGROUND: Although the risk of exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is higher for frontline healthcare workers, not all personnel have similar risks. Determining infection rate is difficult due to the limits on testing and the high rate of asymptomatic individuals. Detection of antibodies against SARS-CoV-2 may be useful for determining prior exposure to the virus and assessing mitigation strategies, such as isolation, masks, and other protective equipment. METHODS: An online assessment that included demographic, clinical, and exposure information and a blood sample was collected from 20 614 participants out of ~43 000 total employees at Beaumont Health, which includes 8 hospitals distributed across the Detroit metropolitan area in southeast Michigan. The presence of anti-SARS-CoV-2 IgG was determined using the EUROIMMUN assay. RESULTS: A total of 1818 (8.8%) participants were seropositive between April 13 and May 28, 2020. Among the seropositive individuals, 44% reported that they were asymptomatic during the month prior to blood collection. Healthcare roles such as phlebotomy, respiratory therapy, and nursing/nursing support exhibited significantly higher seropositivity. Among participants reporting direct exposure to a Coronavirus Disease 2019 (COVID-19) positive individual, those wearing an N95/PAPR mask had a significantly lower seropositivity rate (10.2%) compared to surgical/other masks (13.1%) or no mask (17.5%). CONCLUSIONS: Direct contact with COVID-19 patients increased the likelihood of seropositivity among employees but study participants who wore a mask during COVID-19 exposures were less likely to be seropositive. Additionally, a large proportion of seropositive employees self-reported as asymptomatic. (Funded by Beaumont Health and by major donors through the Beaumont Health Foundation). CLINICALTRIALS.GOV NUMBER: NCT04349202.

Rod Photoreceptor Neuroprotection in Dark-Reared Pde6brd10 Mice.

Purpose: The purpose of this study was to test the hypothesis that anti-oxidant and / or anti-inflammation drugs that suppress rod death in cyclic light-reared Pde6brd10 mice are also effective in dark-reared Pde6brd10 mice. Methods: In untreated dark-reared Pde6brd10 mice at post-natal (P) days 23 to 24, we measured the outer nuclear layer (ONL) thickness (histology) and dark-light thickness difference in external limiting membrane-retinal pigment epithelium (ELM-RPE) (optical coherence tomography [OCT]), retina layer oxidative stress (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]); and microglia/macrophage-driven inflammation (immunohistology). In dark-reared P50 Pde6brd10 mice, ONL thickness was measured (OCT) in groups given normal chow or chow admixed with methylene blue (MB) + Norgestrel (anti-oxidant, anti-inflammatory), or MB or Norgestrel separately. Results: P24 Pde6brd10 mice showed no significant dark-light ELM-RPE response in superior and inferior retina consistent with high cGMP levels. Norgestrel did not significantly suppress the oxidative stress of Pde6brd10 mice that is only found in superior central outer retina of males at P23. Overt rod degeneration with microglia/macrophage activation was observed but only in the far peripheral superior retina in male and female P23 Pde6brd10 mice. Significant rod protection was measured in female P50 Pde6brd10 mice given 5 mg/kg/day MB + Norgestrel diet; no significant benefit was seen with MB chow or Norgestrel chow alone, nor in similarly treated male mice. Conclusions: In early rod degeneration in dark-reared Pde6brd10 mice, little evidence is found in central retina for spatial associations among biomarkers of the PDE6B mutation, oxidative stress, and rod death; neuroprotection at P50 was limited to a combination of anti-oxidant/anti-inflammation treatment in a sex-specific manner.

Age-related murine hippocampal CA1 laminae oxidative stress measured in vivo by QUEnch-assiSTed (QUEST) MRI: impact of isoflurane anesthesia.

Age-related impairments in spatial learning and memory often precede non-familial neurodegenerative disease. Ex vivo studies suggest that physiologic age-related oxidative stress in hippocampus area CA1 may contribute to prodromal spatial disorientation and to morbidity. Yet, conventional blood or cerebrospinal fluid assays appear insufficient for early detection or management of oxidative stress within CA1 sub-regions in vivo. Here, we address this biomarker problem using a non-invasive MRI index of CA1 laminae oxidative stress based on reduction in R1 (= 1/T1) after anti-oxidant administration. An R1 reduction reflects quenching of continuous and excessive production of endogenous paramagnetic free radicals. Careful motion-correction image acquisition, and avoiding repeated exposure to isoflurane, facilitates detection of hippocampus CA1 laminae oxidative stress with QUEnch-assiSTed (QUEST) MRI. Intriguingly, age- and isoflurane-related oxidative stress is localized to the stratum lacunosum of the CA1 region. Our data raise the possibility of using QUEST MRI and FDA-approved anti-oxidants to remediate spatial disorientation and later neurodegeneration with age in animals and humans.

Novel imaging biomarkers for mapping the impact of mild mitochondrial uncoupling in the outer retina in vivo.

PURPOSE: To test the hypothesis that imaging biomarkers are useful for evaluating in vivo rod photoreceptor cell responses to a mitochondrial protonophore. METHODS: Intraperitoneal injections of either the mitochondrial uncoupler 2,4 dinitrophenol (DNP) or saline were given to mice with either higher [129S6/eVTac (S6)] or lower [C57BL/6J (B6)] mitochondrial reserve capacities and were studied in dark or light. We measured: (i) the external limiting membrane-retinal pigment epithelium region thickness (ELM-RPE; OCT), which decreases substantially with upregulation of a pH-sensitive water removal co-transporter on the apical portion of the RPE, and (ii) the outer retina R1 (= 1/(spin lattice relaxation time (T1), an MRI parameter proportional to oxygen / free radical content. RESULTS: In darkness, baseline rod energy production and consumption are relatively high compared to that in light, and additional metabolic stimulation with DNP provoked thinning of the ELM-RPE region compared to saline injection in S6 mice; ELM-RPE thickness was unresponsive to DNP in B6 mice. Also, dark-adapted S6 mice given DNP showed a decrease in outer retina R1 values compared to saline injection in the inferior retina. In dark-adapted B6 mice, transretinal R1 values were unresponsive to DNP in superior and inferior regions. In light, with its relatively lower basal rod energy production and consumption, DNP caused ELM-RPE thinning in both S6 and B6 mice. CONCLUSIONS: The present results raise the possibility of non-invasively evaluating the mouse rod mitochondrial energy ecosystem using new DNP-assisted OCT and MRI in vivo assays.