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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Exp Eye Res. 2021 Feb 2;205:108480. doi: 10.1016/j.exer.2021.108480

Assessment of Inner Retinal Oxygen Metrics and Thickness in a Mouse Model of Inherited Retinal Degeneration

Mansour Rahimi a,1, Sophie Leahy a,1, Nathanael Matei a, Norman P Blair b, Shinwu Jeong a, Cheryl Mae Craft a,c, Mahnaz Shahidi a
PMCID: PMC8044016  NIHMSID: NIHMS1671345  PMID: 33539865

Abstract

The retinal degeneration 1 (rd1) mouse is a well-established model of inherited retinal degenerations, displaying photoreceptor degeneration and retinal vasculature damage. The purpose of the current study was to determine alterations in the rate of oxygen delivery from retinal circulation (DO2), the rate of oxygen extraction from the retinal circulation for metabolism (MO2), and oxygen extraction fraction (OEF) in rd1 mice. The study was performed in a total 18 WT mice and 10 rd1 mice at both 3-weeks and 12-weeks of age. Retinal arterial and venous oxygen contents (O2A and O2V) were measured using phosphorescence lifetime imaging. Total retinal blood flow (TRBF) was determined by fluorescence and red-free imaging. DO2 and MO2 were determined as TRBF × O2A and TRBF × (O2A-O2V), respectively. OEF was calculated as MO2/DO2. The thickness of individual retinal layers was measured from histology sections and inner retina (IR) and total retina (TR) thickness were calculated. TRBF, DO2 and MO2 were lower in rd1 mice compared to WT mice (P ≤ 0.001), whereas OEF was not significantly different between rd1 and WT mice (P = 0.4). TRBF and DO2 were lower at 3-weeks of age compared to 12-weeks of age (P ≤ 0.01), while MO2 was not significantly different between age groups (P = 0.4) and OEF was higher at 3-weeks of age compared to 12-weeks of age (P = 0.003). Additionally, the outer and inner retinal cell layer thicknesses were decreased in rd1 mice at 12-weeks of age compared to both age-matched WT mice and rd1 mice at 3-weeks of age (P ≤ 0.02). MO2 was directly correlated with both IR and TR thickness (R ≥ 0.50; P ≤ 0.03, N=20). The findings indicate that the rate oxygen is supplied by the retinal circulation is decreased and the reduction in oxygen extracted for metabolism is related to retinal cell layer thinning in rd1 mice.

Keywords: Retinal Degeneration, Retinal Blood Flow, Retinal Oxygen Delivery Rate, Retinal Oxygen Metabolism Rate

1. Introduction

Inherited retinal degenerations (IRD) are a group of diverse genetic disorders that together constitute the most common inherited form of retinal dystrophy with a worldwide prevalence of approximately 1:4000 (Pagon, 1988). IRD are characterized by the initial loss of rod photoreceptors, which typically results in night blindness and then the loss of cones and concentric visual field loss. In later stages of IRD, patients may experience profound vision loss because of extensive photoreceptor cell degeneration (Verbakel et al., 2018). Histologically, the earliest defect in IRD is degeneration of the cells in the outer nuclear layer, including their rod photoreceptors and connecting fibers (Gartner and Henkind, 1982).

Energy demand and oxygen consumption in retinal photoreceptors are high in order to generate neuronal signals needed to process light information (Braun et al., 1995). To facilitate these energy demands, copious and constant blood flow is necessary for adequate oxygen delivery to retinal mitochondria (Caprara and Grimm, 2012). In patients with advanced IRD, several studies have reported reduced retinal blood flow (Grunwald et al., 1996; Konieczka et al., 2012), blood velocity (Beutelspacher et al., 2011; Grunwald et al., 1996), and vascular diameter (Eysteinsson et al., 2014; Grunwald et al., 1996; Iacono et al., 2017; Ueda-Consolvo et al., 2015), while oxygen saturation was increased in retinal venules (Eysteinsson et al., 2014; Türksever et al., 2014; Ueda-Consolvo et al., 2015).

Retinal degeneration 1 (rd1) mice have been widely used for decades as a model for human IRD (Chang, 2013; Farber et al., 1994; Keeler, 1966). They have a spontaneous genetic point mutation that leads to early degeneration of rod photoreceptors (Farber and Lolley, 1974; Pittler and Baehr, 1991) at postnatal day 10 (P10) (Acosta et al., 2005; Farber et al., 1994; Gibson et al., 2013). Most rod and cone photoreceptors are lost by P25 and P100, respectively (Farber et al., 1994). Because humans with autosomal recessive IRD may carry this mutation, the rd1 mouse model may help identify functional and anatomical changes resulting from IRD (McLaughlin et al., 1993).

In addition to photoreceptor degeneration, retinal vasculature is also damaged in rd1 mice. Specifically, oxidative stress has been shown to retard retinal vascular development in deep layers (Fukuda et al., 2014). Recently, loss of small blood vessel branches in the deep retinal layers followed by larger vessels in intermediate and superficial layers have also been reported (Hanna et al., 2018). Recently, optical coherence tomography angiography has become available for visualizing perfusion in retinal vasculature. However, this technique does not measure blood flow or oxygen content. Although rd1 mice are well-characterized in terms of structure and electrophysiological function, limited information is available about retinal vascular physiology.

Previous studies have investigated changes in retinal oxygenation and oxygen consumption in animal models of IRD. Photoreceptor homeostasis and survival are highly sensitive to altered aerobic energy metabolism.(Kooragayala et al., 2015) In rd1 mice, photoreceptors were shown to have low mitochondrial reserve capacity based on ex vivo measurements of oxygen consumption rate, thus explaining the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations (Kooragayala et al., 2015). In Royal College of Surgeons rat (Vollrath et al., 2001) and P23H, an autosomal dominant transgenic IRD rat model with rhodopsin mutations, intra-retinal oxygen tension profiles generated in vivo showed increased oxygen tension and reduced oxygen uptake in the outer retina coupled with preservation of oxygen uptake in the inner retina (Yu and Cringle, 2001, 2005). Similar findings were reported in Abyssinian cats with rd, though inner retinal oxygen consumption was slightly increased at the latest stage of degeneration (Padnick-Silver et al., 2006).

Under normal physiological conditions, choroidal circulation is the main source of oxygen supply for the high oxygen consuming photoreceptors. It has been shown that in some normal rats, retinal circulation also supplies oxygen to the photoreceptors (Lau and Linsenmeier, 2012). With photoreceptor loss, unconsumed oxygen from the choroidal circulation becomes available that can affect oxygenation of the inner retinal neurons. However, there is a lack of knowledge of alterations in the rate of oxygen delivery from retinal circulation (DO2), the rate of oxygen extraction from the retinal circulation for metabolism (MO2), and oxygen extraction fraction (OEF) under retinal degenerative conditions. The purposes of the current study were to test the hypotheses that DO2, MO2, and OEF are altered in rd1 mice and impairments in MO2 are correlated with reduction in retinal thickness.

2. Materials and Methods

2.1. Animals

All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee. All experiments adhered to the guidelines of the statement of Use of Animals in Ophthalmic and Vision research by the Association for Research in Vision and Ophthalmology. The experiments have been reported following the Animal Research: Reporting in Vivo Experiments guidelines.

The study was performed in 18 WT mice and 10 rd1 mice, both groups evaluated at 3-weeks and 12-weeks of age. Thirteen WT mice (male) (age, 12.2 ± 0.4 weeks; weight, 26.0 ± 1.8 g) and five WT mice (2 male and 3 female) (age, 3.2 ± 0.3 weeks; weight, 9.2 ± 1.1 g) were used for the study. The WT mice were either purchased from Jackson Laboratory (Bar Harbor, ME, USA) (C57BL/6J; N=10) or Charles River Laboratories (Hollister, CA, USA) (C57BL; N=8). Using C57BL/6J mice, homozygous rdle (rd1, Pde6brd1) mice were generated in-house, as previously described (Carter-Dawson et al., 1978; Kim et al., 2018; LaVail and Sidman, 1974). These included six rd1 mice (5 male and 1 female) (age, 12.6 ± 1.0 weeks; weight, 26.5 ± 2.6 g) and 4 rd1 mice (male) (age, 3.1 ± 0.1 weeks; weight, 9.5 ± 1 g) were used for the study. There was no significant difference in mean age between WT and rd1 mice at 12-weeks (P= 0.33) and 3-weeks of age (P=0.48).

Mice were kept under environmentally controlled conditions with a 12-hour/12-hour light/dark cycle and had free access to food and water. Prior to imaging, mice were anesthetized with intraperitoneal injections of ketamine (90mg/kg) and xylazine (5mg/kg), with additional doses administered as needed. A catheter was placed in the femoral artery, and injections were given through the catheter for velocity, diameter, and oxygen tension measurements. Fluorescent microspheres (size, 2-μm) were injected for blood velocity imaging. Fluorescein angiography (FA) was performed with the injection of 10% fluorescein sodium (5 mg/kg, AK-FLUOR; Akorn, Decatur, IL, USA) for diameter imaging. Phosphorescence imaging was performed by injection of Pd-Porphine (Frontier Science, Boston, MA) at a dosage of 20 mg/kg for vascular oxygen tension imaging. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine. For imaging, mice were placed on an animal holder with circulating heated water. A glass coverslip with 2.5% hypromellose ophthalmic demulcent solution (HUB Pharmaceuticals, Plymouth, MI) was applied to the cornea to maintain hydration and eliminate its refractive power. Imaging was performed in one eye. Mice were euthanized following imaging, and eyes were then enucleated, fixed in Davidson’s fixative, and embedded in paraffin. Retinal sections (5-micron) were cut, and these included the pupil, optic nerve, and the retina temporal and nasal to the optic nerve head.

2.2. Retinal Oxygen Delivery, Metabolism, Extraction Fraction

Total retinal blood flow (TRBF) was calculated using our previously described imaging system (Blair et al., 2016). FA images were analyzed to detect vessel boundaries for diameter (D) measurements. D in individual vessels were averaged to obtain mean arterial and venous D (DA, Dv). For velocity (V) measurements, the displacement of intravascular fluorescent microspheres along each vein was determined over time using image sequences. Measurements in each vein were averaged to calculate a mean venous blood V (Vv). In each vein, blood flow (VπD2/4) was calculated and then summed over all the veins to derive TRBF. Retinal vascular oxygen tension (PO2) was measured using our established optical section phosphorescence lifetime imaging system (Blair et al., 2016; Wanek et al., 2013). Phosphorescence lifetimes of Pd-Porphine within all major retinal arteries and veins were determined using a frequency-domain approach and converted to PO2 measurements using the Stern-Volmer equation (Lakowicz et al., 1992; Shonat and Kight, 2003). Using the mouse hemoglobin dissociation curve (Gray and Steadman, 1964), PO2 values were converted to O2 content in individual vessels. The O2 content values were averaged to obtain mean arterial and venous O2 content (O2A, O2V). Arteriovenous O2 difference (O2A-V) was calculated as the difference between O2A and O2V. Inner retinal DO2 and MO2 were determined as the product of blood flow with O2A and O2A-V, respectively. OEF was calculated as the ratio of MO2 to DO2.

2.3. Retinal Thickness

Retinal tissues for histology were available from 10 of 13 WT mice at 12-weeks of age and all rd1 mice in both age groups. Retinal sections were stained with Hematoxylin and Eosin using established protocols (Ji et al., 2012). Images were acquired using an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) in the nasal and temporal regions next to the optic nerve head. As previously described (Matei et al., 2020), image analysis was performed using ImageJ software (ImageJ, U. S. National Institutes of Health, Bethesda, MD) by manually identifying retinal layer bounders at 45 μm intervals to provide ten measurements per region. Thicknesses were derived for the combined nerve fiber layer (NFL) and the retinal ganglion cell layer (RGCL) (NFL+RGCL) and measured for the inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor layer (PL), inner retina (IR) (from NFL to INL), and total retinal (TR) (from NFL to PL). The thickness of the combined NFL+RGCL was calculated as follows: IR - (IPL+INL). Measurements obtained in regions nasal and temporal to the optic nerve head were averaged.

2.4. Data Analysis

Statistical analyses were performed using SPSS software (version 25.0; IBM Corp., Armonk, NY). Normality of the data distribution was evaluated using Shapiro-Wilk tests and no outliers were identified. General linear model analysis was performed to determine the effects of condition (rd1, WT) and age (12-week, 3-week) on oxygen metrics. Retinal layer thicknesses were compared between conditions and age groups using unpaired t-tests. Pearson correlations were performed to assess the association of MO2 with IR and TR thickness. Statistical significance was accepted at P≤0.05. GraphPad Prism version 9.0.0 for Mac OS (La Jolla, CA, USA) was used for generating the figures.

3. Results

3.1. Retinal Vascular Metrics and Blood Flow

Figure 1 depicts the methodology for measurements of PO2V, DV, and Vv in representative WT and rd1 mice at 12-weeks of age. Reduced DV and Vv the rd1 mice compared to the WT mice are displayed, whereas PO2V appears similar.

Figure 1:

Figure 1:

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Illustration of the method for measurements of retinal vascular oxygen tension (PO2), vessel diameter, and blood velocity. Fluorescein angiograms are shown in wild type (WT) and retinal degeneration 1 (rd1) mice at 12-weeks of age. PO2 measurements in retinal veins are depicted by pseudo color. Boundaries of retinal veins, as detected automatically for measurement of diameter, are shown by red lines. Position of one microsphere at 2 time-points, showing the distance traveled over a given time period (blood velocity), is shown in the yellow boxes.

The mean and standard deviation of retinal vascular O2 content in rd1 and WT mice are displayed in Figure 2. There was a significant interaction between condition (rd1, WT) and age (12-weeks, 3-weeks) on the O2A (P=0.04). In rd1 mice, O2A was not different by age (P=0.2), but in WT mice, it was higher at 3-weeks of age compared to 12-weeks of age (P<0.001). At 3-weeks of age, O2A was lower in rd1 mice compared to WT mice (P=0.04), but not different by condition at 12-weeks of age (P=0.6). There was no significant interaction between condition and age on O2V and O2A-V (P≥0.2). There were no significant main effects of condition or age on O2V (P≥0.1). There were significant main effects of condition and age on O2A-V (P≤0.02), such that O2A-V was lower in rd1 mice compared to WT mice and higher at 3-weeks of age compared to 12-weeks of age.

Figure 2:

Figure 2:

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(A) arterial oxygen content (O2A), (B) venous oxygen content (O2V), and (C) arteriovenous oxygen content difference (O2A-V) in wild type (WT) and retinal degeneration 1 (rd1) mice at 3-weeks and 12-weeks of age. Error bars indicate standard deviations. Asterisks indicate significance at P≤0.05.

Mean and standard deviation of DA, DV, Vv, and TRBF in rd1 and WT mice are presented in Figure 3. There was no significant interaction between condition and age on DA, DV, Vv, and TRBF (P≥0.07). There was a significant main effect of condition on DA and DV (P≤0.001), but no significant effect of age (P≥0.3). DA and DV were lower in rd1 mice compared to WT mice. There was no significant main effect of condition on Vv (P=0.08), but Vv was lower at 3-weeks of age compared to 12-weeks of age (P≤0.001). There were significant main effects of condition and age on TRBF (P≤0.001). TRBF was lower in rd1 mice compared to WT mice and also lower at 3-weeks of age compared to 12-weeks of age.

Figure 3:

Figure 3:

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(A) arterial diameter (DA), (B) venous diameter (DV), (C) venous blood velocity (VV), and (D) total retinal blood flow (TRBF) in wild type (WT) and retinal degeneration 1 (rd1) mice at 3-weeks and 12-weeks of age. Error bars indicate standard deviations. Asterisks indicate significance at P≤0.05.

3.2. Inner Retinal Oxygen Delivery, Metabolism, and Extraction Fraction

The mean and standard deviation of DO2, MO2, and OEF in rd1 and WT mice are presented in Figure 4. There was no significant interaction between condition and age on DO2, MO2, and OEF (P≥0.3). There were significant main effects of condition and age on DO2 (P≤0.02). DO2 was lower in rd1 mice compared to WT mice and also lower at 3-weeks of age compared to 12-weeks of age. There was a significant main effect of condition on MO2 (P≤0.001), such that MO2 was lower in rd1 mice compared to WT mice. However, MO2 was not significantly different between age groups (P=0.4). OEF was not significantly different between rd1 and WT mice (P=0.4). However, there was a significant main effect of age on OEF (P=0.003), such that OEF was higher at 3-weeks of age compared to 12-weeks of age.

Figure 4:

Figure 4:

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(A) oxygen delivery (DO2), (B) oxygen metabolism (MO2), and (C) oxygen extraction fraction (OEF) in wild type (WT) and retinal degeneration 1 (rd1) mice at 3-weeks and 12-weeks of age. Error bars indicate standard deviations. Asterisks indicate significance at P≤0.05.

3.3. Retinal Layer Thickness

Figure 5 shows examples of retinal histology sections from the same retinal region in WT and rd1 mice at 12-weeks of age, displaying thinning of retinal layers in the rd1 mouse compared to the WT mouse. Mean and standard deviation of retinal layer thicknesses in rd1 and WT mice at 12-weeks of age and rd1 mice at 3-weeks and 12-weeks of age are displayed in Figure 6. At 12 weeks of age, thicknesses of NFL+RGCL, IPL, IR, and TR were decreased in rd1 mice compared to in WT mice (P≤0.02). OPL, ONL, and PL thicknesses were each 0 μm in rd1 mice and, therefore, significantly lower than those in WT mice (P≤0.0001). INL thickness did not significantly differ between the two groups (P=0.19). In rd1 mice, IPL, INL, IR, and TR thicknesses were significantly decreased at 12-weeks of age compared to 3-weeks of age, (P≤0.04), but thickness of NFL+RGCL was not significantly different (P= 0.84). OPL, ONL, and PL thicknesses were each 0 μm in rd1 mice in both age groups.

Figure 5:

Figure 5:

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Examples of hematoxylin and eosin-stained histology sections from the same retinal regions in wild type (WT) and retinal degeneration 1 (rd1) mice at 12-weeks of age, displaying individual retinal layers. Outer retinal layers were not visualized in the rd1 mouse. Scale bars represent 25 microns. NFL, nerve fiber layer; RGCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PL, photoreceptor layer; IR, inner retina.

Figure 6:

Figure 6:

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Comparison of nerve fiber layer and retinal ganglion cell layer (NFL+ RGCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor layer (PL), inner retina (IR), and total retina (TR) between (A) retinal degeneration 1 (rd1) and WT mice at 12-weeks of age and (B) rd1 mice at 3-weeks and 12-weeks of age. Error bars indicate standard deviations. Asterisks indicate significance at P≤0.05.

3.4. Correlation of MO2 with Retinal Thickness

Based on compiled data from WT and rd1 mice at 12-weeks and rd1 mice at 3-weeks of age, MO2 was linearly related to both IR and TR thickness. MO2 was correlated with IR thickness (R= 0.50; P = 0.03, N=20) and TR thickness (R= 0.74; P < 0.0001, N=20).

4. Discussion

We report for the first-time, to the best of our knowledge, measurements of DO2, MO2, and OEF in rd1 mice. The present study confirmed the hypotheses of reductions in DO2 and MO2 in rd1 mice and a correlation between reduction in MO2 and thinning of IR and TR. Additionally, DO2 was found to be lower at a younger age, whereas MO2 was not significantly affected by age, presumably due to the counteracting effects of reduced blood flow and increased oxygen extraction. Finally, retinal thickness was reduced in older rd1 mice compared to both same-age WT mice and younger rd1 mice. Our findings are of interest because they present experimental support for a degeneration-induced reduction in DO2 and MO2 and the development of retinal thinning in the setting of both photoreceptor and vascular loss.

In the current study, DA, DV, VV, and TRBF were significantly decreased in rd1 mice. Matthes and Bok (Matthes and Bok, 1984), and later Blanks and Johnson (Blanks and Johnson, 1986), demonstrated using light microscopy, a reduction in the number of retinal blood vessels of P14 rd1 mice. They also showed capillaries within the INL were most affected by degeneration, followed by a notable decrease in venules, arterioles, and capillaries within IPL. Using confocal scanning laser ophthalmoscopy, Hanna et al. showed a progressive loss in small branches of blood vessels and subsequent loss in larger main vessels in rd1 mice. They confirmed their findings by histology, demonstrating a pattern of blood vessel regression in the deep plexus, followed by intermediate and superficial retinal plexuses (Hanna et al., 2018). Here, we report, for the first time, narrowing of large retinal vessels coupled with decreased blood flow in rd1 mice. This finding is consistent with studies performed in human IRD that showed reduced retinal vessel diameter (Eysteinsson et al., 2014; Grunwald et al., 1996; Sodi et al., 2016; Ueda-Consolvo et al., 2015).

There was a reduction in DO2 in rd1 mice compared to WT mice which is predominantly due to the observed reductions in TRBF. Increased influx of oxygen from the choroidal circulation due to photoreceptor degeneration that can lead to inner retinal tissue hyperoxia is likely the predominant underlying factor for the observation of narrowing of vessels, reduced blood flow and DO2. Specifically, with photoreceptor cell death leading to degeneration, oxygen supply to the inner retina from the choroidal circulation increases due to reduced oxygen consumption by photoreceptors (Yu and Cringle, 2005), exacerbated by thinning of outer retina cell layers, and the consequently decreased oxygen diffusion distance. This increased oxygen availability in the inner retina, also shown during hyperoxia in cats (Linsenmeier and Yancey, 1989), likely results in narrowing of retinal vessels and decreased blood flow, as previously reported in human IRD (Eysteinsson et al., 2014; Grunwald et al., 1996). Moreover, in a transgenic mouse model of autosomal dominant IRD, Penn et al. demonstrated retinal vessel attenuation due to excess oxygen supply from the choroid and its reversal by ambient hypoxia (Penn et al., 2000). Additionally, with long durations of the disease, as shown in human IRD, extravascular leakage of serum proteins and subsequent progressive thickening of the extracellular matrix surrounding retinal vessels could lead to a decrease in luminal cross-section that may compromise the blood supply to the inner retina (Li et al., 1995). The finding of reduced DO2 at 3-weeks of age compared to 12-weeks of age is attributed to the observed decrease in blood velocity and TRBF. This finding may be attributed to lower body weight and smaller eyes with reduced perfused retinal volume in younger mice, consistent with a previous study that showed children with lower birth weight had narrower retinal arteries and reduced retinal vasculature complexity (Gopinath et al., 2010).

The current study demonstrated, for the first time, a reduction in MO2 of rd1 mice. This finding is based on the observed reductions in both TRBF and O2A-V. In contrast, there was no significant effect of age on MO2, presumably due to the counteracting effects of reduced TRBF and increased of O2A-V. MO2 (in units of volume of oxygen × time−1) represents the rate at which oxygen is extracted from the retinal circulation for metabolism by an uncertain volume of the inner retina. It can be expressed as the product of inner retinal tissue volume that is supplied by the retinal circulation and oxygen utilization (in units of volume of oxygen × time−1 × volume of tissue−1). With photoreceptor loss, there is increased oxygen supply to the inner retina coupled with inner retinal degeneration due to disease process, leading to a reduction in the volume of tissue supplied by the retinal circulation. Although the amount of increased oxygen from the choroidal circulation and the volume of consuming inner retinal tissue are not known, MO2 provides information about their combined effects. Therefore, the observed reduction in MO2 appears to be due to some combination of three factors: the increased oxygen supply, loss of inner retinal neurons, and decreased oxygen utilization of surviving neurons.

First, the loss of photoreceptors and the consequent reduction in outer retina oxygen consumption result in increased oxygen diffusion to the inner retina from the choroidal circulation. Therefore, a smaller volume of the inner retinal tissue is likely supplied by the retinal circulation, which may be reflected in a lower measurement of MO2. This notion is supported by previously reported oxygen profiles through the retinal depth in a cat model of retinal degeneration (Padnick-Silver et al., 2006). These studies showed increased inner retinal oxygen content concurrent with reduced outer retinal oxygen consumption. Furthermore, under systemic hyperoxia, part of inner retina was shown to be supplied by the choroidal circulation in healthy humans (Werkmeister et al., 2015) and cats (Linsenmeier and Yancey, 1989). Our current finding of narrowing of the retinal vessels in rd1 mice is also indicative of increased oxygenation to the inner retina from choroidal circulation. A similar finding of vasoconstriction and reduction in retinal blood flow in IRD (Eysteinsson et al., 2014; Grunwald et al., 1996; Konieczka et al., 2012; Ueda-Consolvo et al., 2015) may also be suggestive of the presence of an impairment in MO2.

Second, the demise of photoreceptors has a secondary effect on the survival of the inner retinal neurons, which can also cause a decrease in MO2. In a postmortem study on the sectioned macula in human IRD, the authors reported a reduction in the number of ganglion cells in the inner retina of IRD patients (Stone et al., 1992). Additionally, our current finding of thinning of the inner retinal cell layers, as well as previous similar reports in rd1 mice (Grafstein et al., 1972; Pennesi et al., 2012; Ward, 1982) and human IRD (Hartong et al., 2006; Oishi et al., 2013; Schuerch et al., 2016; Vamos et al., 2011) support the contribution of inner retinal neuron cell loss as a factor in the observed reduction in MO2.

Third, dysfunction of surviving inner retinal neurons may also contribute to the observed reduction in inner retinal MO2. Several investigations have been published reporting functional changes of inner retinal neurons following photoreceptor loss secondary to inherited retinal dystrophies (Barhoum et al., 2008; Cuenca et al., 2004; Marc et al., 2007; Varela et al., 2003). In the P23H rat model of retinal degeneration, progressive changes in rod relay pathway and neurotransmitter receptors function have been reported. These functional changes were reflected in variations of ERG response patterns, namely as reductions in a-wave amplitude, rod and cone-associated b-waves, and the amplitude of oscillatory potentials (Cuenca et al., 2004). In another study, after the degeneration of rod photoreceptors in rd1 mice, rod bipolar cells lost their glutamate (rod-neurotransmitters) input, and this change in neurotransmitter sensitivity affects bipolar cells (Varela et al., 2003). Furthermore, in both the rd1 mouse model of retinal degeneration and a sample of human IRD, a permanent loss of bipolar cell glutamate receptor expression was demonstrated (Marc et al., 2007).

OEF was not significantly different between rd1 and WT mice. OEF is the ratio of MO2 to DO2 and indicates the adequacy of tissue’s oxygen supply relative to its metabolic demand. Since TRBF is a factor in both MO2 and DO2, OEF can be alternatively expressed as the ratio of O2A-V and O2A, independent of TRBF (Felder et al., 2015). In the current study, O2A at 3-weeks of age and O2A-V at both age groups were reduced in rd1 mice, accounting for the maintained OEF. This finding is in contrast to previous reports in human IRD that showed an increase in retinal arterial and venous oxygen saturation (Eysteinsson et al., 2014; Todorova et al., 2016; Türksever et al., 2014; Ueda-Consolvo et al., 2015), potentially attributed to neurovascular remodeling (Türksever et al., 2014). Variations in the type of genetic defects and time course of phenotype presentation, as well as the small sample size, may have contributed to the observed difference. Nevertheless, the finding of maintained OEF in rd1 mice is due to a similar reduction in both MO2 and DO2. The finding of increased OEF at 3-weeks of age is attributed to the observed reduced DO2 and maintained MO2.

In the current study, IR and TR thicknesses were reduced in rd1 mice at 12-weeks of age, compared to both age-matched WT mice and 3-weeks of age rd1 mice. The reduction in IR thickness was due to corresponding thinning of NFL+RGCL+ IPL at 12-weeks of age. As expected, with increasing age and more advanced stage of degeneration, thickness of IPL+INL was further decreased in rd1 mice, while thinning of NFL+RGCL was maintained. A previous study showed that in rd1 mice, retinal thickness was markedly decreased by three weeks when the degeneration of photoreceptors became complete (Matthes and Bok, 1984). In the evaluation of retinal thickness in homozygous rd1 mice by optical coherence tomography, ONL thickness was reduced along with the obliteration of photoreceptors at two weeks (Hanna et al., 2018; Pennesi et al., 2012). This observation is consistent with our finding of negligible thickness of outer retinal layers in both age groups. Furthermore, reduced thickness of retinal layers has been demonstrated in human IRD (Oishi et al., 2013; Stone et al., 1992). Moreover, histopathologic examinations of human IRD retinal tissue showed that all retinal layers were affected, predominately in photoreceptor outer segments and ONL, and to a lesser degree in RGCL and INL (Milam et al., 1998; Santos et al., 1997). The finding of an association between decreased MO2 and IR thickness suggests inner retina cell loss likely contributed to the observed reduction in MO2. This finding is consistent with investigations on RGCL in rd1 mice (Grafstein et al., 1972) and human IRD (Grunwald et al., 1996; Stone et al., 1992) that showed a decrease in viable RGCL that can result in impaired inner retinal metabolism.

The current study had some limitations. First, the sample size was small, though we were able to find highly significant differences due to the stage of degeneration. Nevertheless, future studies with a larger sample size are needed to further characterize the findings. Second, since rd1 and WT mice used in the study were not littermates, genetic factors may have contributed to the observed results. Additionally, 8 of 18 WT mice that were purchased from Charles River Laboratories may have carried the rd8 mutation(Pak et al., 2015), which may have resulted in detection of a smaller difference between WT and rd1 mice. Third, findings of this pilot study were limited to a specific animal model, after a significant loss of photoreceptors, and thus may not be generalized to other models or an earlier stage of degeneration. Moreover, histology sections were not available in the 3-week old WT mice which limited comparison of retinal layer thickness between rd1 and WT mice at this younger age. However, retinal thinning has been previously reported in 3-week old rd1 mice (Farber et al., 1994). The current study reported changes in rd1 mice at the earliest age feasible for evaluation by our method. Nonetheless, future studies may be conducted in other mice models of IRD with slower rate of degeneration such as rd10. The findings would advance knowledge of retinal oxygen metabolic and anatomical changes due to progression of degeneration and potentially serve as biomarkers for testing therapeutic interventions. Fourth, retinal arterial O2 was low in both rd1 and WT mice, presumably due to anesthesia, and may have contributed differently to measurements of MO2. Specifically, in WT mice under systemic hypoxia, MO2 may be either maintained or reduced depending on the effectiveness of vascular autoregulation. In contrast, rd1 mice are expected to have a higher MO2 under systemic hypoxia since the retinal circulation can autoregulate, as opposed to the choroid, resulting in reduced choroidal oxygen content and reduced diffusion toward the inner retina. Therefore, the difference in MO2 between groups may have been underestimated, assuming effective autoregulation in WT mice. Overall, future studies investigating longitudinal changes in MO2 and retinal anatomy during the progression of degeneration in various models are needed to elucidate the time course of the changes observed in the current study.

5. Conclusion

In conclusion, our findings indicate that retinal degeneration has a significant effect on the rate of oxygen extracted from the retinal circulation for metabolism, hemodynamics, and anatomy of the inner retina of rd1 mice. Assessment of inner retinal oxygen delivery and metabolism may be particularly helpful in studying the pathophysiology of retinal degeneration and the evaluation of therapeutic modalities to prevent vision loss due to retinal degenerative diseases.

  • Alterations in the rates of oxygen delivery from retinal circulation and oxygen extraction from the retinal circulation for metabolism were reported for the first-time in retinal degeneration 1 mice.

  • The findings demonstrated reduction in the rates of oxygen supplied by and extracted from the retinal circulation.

  • Reduction in oxygen extraction from the retinal circulation for metabolism was correlated with thinning of retinal cell layers.

Acknowledgments

The authors thank James Burford for performing animal procedures.

Funding

This work was supported by the National Eye Institute, Bethesda, MD [EY017918 and EY029220]; the Mary D. Allen Foundation and an unrestricted departmental award from Research to Prevent Blindness, New York, NY.

Abbreviations:

DO2

rate of oxygen delivery from retinal circulation

FA

fluorescein angiography

INL

inner nuclear layer

IRD

inherited retinal degenerations

IPL

inner plexiform layer

IR

inner retina

MO2

rate of oxygen extraction from the retinal circulation for metabolism

NFL

nerve fiber layer

O2A

retinal arterial oxygen content

O2V

retinal venous oxygen content

OEF

oxygen extraction fraction

ONL

outer nuclear layer

OPL

outer plexiform layer

PL

photoreceptor layer

PO2

retinal vascular oxygen tension

rd1

retinal degeneration 1

RGCL

retinal ganglion cell layer

TR

total retina

TRBF

total retinal blood flow

WT

wild type

Footnotes

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Declaration of Conflicting Interest

MS holds a patent on the imaging system. All other authors have no conflicting interests.

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