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

Abstract

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.

Preventing diabetic retinopathy by mitigating subretinal space oxidative stressin vivo

“…We found that the RPE-specific toxin sodium iodate produces less rod mitochondrial oxidative stress and rod death in 129S6/ev mice than in C57BL/6J mice (Berkowitz et al, 2017;Berkowitz et al, 2018b). Further, 129S6/ev mice retina have an 11% greater mitochondrial reserve than C57BL/6J mice from The Jackson Laboratory as measured by an ex vivo Seahorse assay (Berkowitz et al, 2018b;Berkowitz et al, 2020). When compared in vivo with those of dark-adapted C57BL/6J mice, rod mitochondria of dark-adapted 129S6/ev mice have a greater capacity to respond to a protonophore-evoked shrinkage of the ELM-RPE region as measured by OCT, consistent with the signaling pathway in Fig.…”

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

“…In this scenario (summarized in Fig 10 ), elevating cGMP levels in light-adapted mice causes sustained opening of rod photoreceptor outer segment cyclic nucleotide-gated channels, an energy-intensive event [ 64 ]. As a result of this increase in mitochondrial respiration, the subretinal space fills with acidified waste-water [ 14 , 17 – 19 , 24 , 28 , 65 ]. The lowering of the subretinal space pH upregulates water removal co-transporters in RPE with the end result of dehydrating / thinning of the ELM-RPE space ( Fig 10 ) [ 14 , 17 – 19 , 24 , 28 , 65 ].…”

“…As a result of this increase in mitochondrial respiration, the subretinal space fills with acidified waste-water [ 14 , 17 – 19 , 24 , 28 , 65 ]. The lowering of the subretinal space pH upregulates water removal co-transporters in RPE with the end result of dehydrating / thinning of the ELM-RPE space ( Fig 10 ) [ 14 , 17 – 19 , 24 , 28 , 65 ]. Evidence for subretinal space thinning following sildenafil is shown in Fig 3 [ 14 , 17 – 19 , 24 , 28 , 65 ].…”

Rod Photoreceptor Neuroprotection in Dark-RearedPde6brd10Mice

“…Previous quantitative studies performed in P40 or older WT C57BL/6J mice indicate a cGMP / mitochondria / pH / water removal signaling pathway that causes ELM-RPE to be measurably thinner in the dark than in the light; this phenotype is apparent in a representative P20 WT C57BL/6J mouse, which also demonstrates a clear dark less than light ELM-RPE thickness difference, as shown in Figure 1B. 40,41,[49][50][51][52][53][54][55][56] In contrast, male P24 Pde6brd10 mice lack a statistically significant dark-light ELM-RPE difference (inferior = -1.31; 95% confidence interval [CI] = -3.29 to 0.66; superior = -2.53, 95% CI = -5.56 to 0.49; P > 0.05 for both), consistent with a panretinal PDE6B mutation-induced increase in cGMP levels in the light (see Fig. 1C).…”

“…72 For comparison, we note that WT brown 129S6/ev mice, which do not have the Nnt mutation, have an 11% greater mitochondrial reserve than C57BL/6J mice from the Jackson Laboratory as measured by an ex vivo Seahorse assay and by our OCT studies. 52,53 Further, this mutation likely affects rescue potential because sodium iodate produces less rod mitochondrial oxidative stress and rod death in 129S6/ev mice than in C57BL/6J mice. 53,73 We anticipate future studies that address this potential problem by comparing Pde6brd10 mice on the C57BL/6J background with Pde6brd10 C57BL/6 mice from a different vendor without the Nnt mutation, or with Pde6brd10 mice on the 129S6/ev background.…”