Inner Retinal Oxygen Extraction Fraction in Rat

Abstract

Purpose.: Oxygen extraction fraction (OEF), defined by the ratio of oxygen consumption to delivery, may be a useful parameter for assessing the retinal tissue status under impaired circulation. We report a method for measurement of inner retinal OEF in rats under normoxia and hypoxia based on vascular oxygen tension (PO₂) imaging.

Methods.: Retinal vascular PO₂ measurements were obtained in 10 rats, using our previously developed optical section phosphorescence lifetime imaging system. Inner retinal OEF was derived from retinal vascular PO₂ measurements based on Fick's principle. Measurements of inner retinal OEF obtained under normoxia were compared between nasal and temporal retinal sectors and repeatability was determined. Inner retinal OEF measurements obtained under normoxia and hypoxia were compared.

Results.: Retinal vascular PO₂ and inner retinal OEF measurements were repeatable (ICC ≥ 0.83). Inner retinal OEF measurements at nasal and temporal retinal sectors were correlated (R = 0.71; P = 0.02; n = 10). Under hypoxia, both retinal arterial and venous PO₂ decreased significantly as compared with normoxia (P < 0.001; n = 10). Inner retinal OEF was 0.46 ± 0.13 under normoxia and increased significantly to 0.67 ± 0.16 under hypoxia (mean ± SD; P < 0.001; n = 10).

Conclusions.: Inner retinal OEF is a promising quantitative biomarker for the adequacy of oxygen supply for metabolism under physiologically and pathologically altered conditions.

Introduction

The maintenance of retinal tissue requires adequate delivery of oxygen by the retinal and choroidal circulations. Indeed, under a severe hypoxic or ischemic insult, retinal function is compromised ^1–3 and irreversible functional loss can occur shortly following retinal vascular nonperfusion. ^2,4,5 Consequently, it would be advantageous to assess the status of the retina when impaired circulation is suspected. 

Oxygen extraction fraction (OEF) is a parameter defined by the ratio of oxygen consumption (MO₂) to oxygen delivery (DO₂). Cerebral OEF measured by positron emission tomography and magnetic resonance imaging has been widely accepted as a valuable parameter for assessing tissue ischemia. ^6–13 In fact, increased cerebral OEF, also termed “misery perfusion,” has been reported in subjects with acute ischemic stroke ^10–12 and carotid occlusion. ^7,8 Moreover, an increase in cerebral OEF has been shown to be an independent predictor of stroke. ^9,13  

Due to similarities between cerebral and retinal tissues, measurement of OEF may also be useful for assessment of ischemia that occurs in many retinal diseases; however, currently available brain imaging modalities lack adequate resolution to quantify OEF in the retinal tissue. Alternatively, OEF can be expressed as the ratio of the arteriovenous oxygen content difference to the arterial oxygen content based on Fick's principle, ¹⁴ as shown by the following equation.  

 

Therefore, inner retinal OEF can be derived without direct measurements of MO₂ and DO₂.  

In the current study, we report a method for quantitative measurement of inner retinal OEF based on retinal vascular oxygen tension (PO₂) imaging. ¹⁵ The repeatability, spatial variation, and hypoxia-induced alterations of inner retinal OEF measurements were determined. 

Methods

Animals

Ten Long Evans pigmented rats (weight 444 ± 99 g, mean ± SD) were used in this study. The rats were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Anesthesia was induced by intraperitoneal injections of ketamine (100 mg·kg⁻¹) and xylazine (5 mg·kg⁻¹), and maintained with supplemental injections of ketamine (20 mg·kg⁻¹) and xylazine (1 mg·kg⁻¹), as needed. The body temperatures of the rats were maintained at 37°C using an animal holder with a copper tubing water heater. A catheter was placed in a femoral artery and connected to a pressure transducer. Blood pressure and heart rate of the rats were monitored with a data acquisition system (Biopac Systems, Goleta, CA) linked to a pressure transducer connected to the catheter. 

The rats were mechanically ventilated with room air (21% O₂, normoxia) and then with 10% oxygen (hypoxia) through an endotracheal tube connected to a small animal ventilator (Harvard Apparatus, Inc., South Natick, MA). To verify the physiological condition, arterial blood was drawn from the femoral arterial catheter to measure systemic arterial oxygen tension (P_aO₂), carbon dioxide tension (P_aCO₂), and pH with a blood gas analyzer (Radiometer, Westlake, OH) and also hemoglobin concentration with a hematology system (Siemens, Tarrytown, NY). Animals were maintained normocapnic by adjusting the respiratory minute volume and performing blood gas analysis 5 minutes after each adjustment until P_aCO₂ was within the range of 35 to 45 mm Hg. An oxygen-sensitive molecular probe (Pd-porphine; Frontier Scientific, Logan, UT) was dissolved (12 mg·mL⁻¹) in bovine serum albumin solution (60 mg·mL⁻¹) and administered (20 mg·kg⁻¹) through the femoral arterial catheter, typically 10 minutes prior to imaging. The probe binds to albumin and does not permeate from the normal vasculature to the retinal tissue. The pupils were dilated with 2.5% phenylephrine and 1% tropicamide. Glass cover slips and 1% hydroxypropyl methylcellulose were applied to the corneas to eliminate the refractive power and to prevent dehydration. 

Retinal Vascular Oxygen Tension Imaging

Retinal vascular PO₂ was measured with our custom optical section phosphorescence lifetime imaging system. ¹⁵ In short, a laser beam was focused to a vertical line, and projected at an oblique angle on the retina. An optical section phosphorescence image of the retinal vasculature was acquired with an intensified charge-coupled device camera attached to a slit lamp biomicroscope. Since the incident laser beam was at an angle with respect to the imaging path, phosphorescence emissions of the Pd-porphine from the retinal vessels were depth-resolved. Phosphorescence lifetime was determined using a frequency-domain approach and converted to PO₂ measurements using the Stern-Volmer relationship defined by PO₂ = (1/κ _Q) · (1/τ − 1/τ ₀), where κ _Q (mm Hg⁻¹·μs⁻¹) is the quenching constant for the triplet-state of Pd-porphine, τ (μs) is the phosphorescence lifetime, and τ ₀ (μs) is the lifetime in a zero-oxygen environment. ^16,17 Measurements of PO₂ were obtained in a retinal sector, defined by a zone bounded by two major retinal arteries (PO_2A), with a major retinal vein (PO_2V) between the two arteries (Fig. 1). Three repeated measurements were obtained at nasal and temporal retinal sectors, within three optic disc diameters (∼600 μm) from the edge of the optic nerve head. A red-free retinal image was acquired for documenting the locations of the PO₂ measurements. Under both ventilation conditions, the laser power incident on the cornea was approximately 40 μW, which is safe for 1 hour of continuous viewing according to the American National Standard Institute for Safety Standards. ¹⁸  

Figure 1. 

 

Examples of cross-sectional vascular PO₂ maps (shaded rectangles) superimposed on red-free retinal images in a rat under normoxia (A) and hypoxia (B). Maps depict PO₂ in retinal arteries (a) and veins (v) in the temporal (left) and nasal (right) sides of the optic disc. Color bar displays PO₂ in mm Hg.

Quantification of OEF

Based on retinal vascular PO₂ measurements, the O₂ content of blood was estimated as follows:  

 

where O_2max is the maximum oxygen carrying capacity of hemoglobin (1.39 mLO₂·g⁻¹), ¹⁹ Hgb is the measured hemoglobin concentration of arterial blood (13.9 ± 0.5 g·dL⁻¹; n = 10), k is the oxygen solubility in blood (0.003 mLO₂·dL⁻¹·mm Hg⁻¹), ¹⁹ and SO₂ is the oxygen saturation. Based on vascular PO₂ and arterial blood pH measurements, SO₂ was calculated from the Hill equation, which models the oxygen dissociation curve and is defined as SO₂ = (PO₂/P₅₀)ⁿ/(1 + [PO₂/P₅₀]ⁿ), where n is an empirical constant taken to be 2.6 in rat, ²⁰ and P₅₀ is the PO₂ when SO₂ is 0.5 at a given pH, based on the Bohr effect. ²¹  

Inner retinal OEF was quantified in a retinal sector as follows,  

 

where mO_2A is the mean arterial O₂ content of the two major arteries, and O_2V is the venous O₂ content. The mean arterial PO₂ of the two major arteries (mPO_2A) was also calculated.  

Data Analysis

Repeatability of mPO_2A, PO_2V, and OEF was determined by calculating the intraclass correlation coefficients (ICCs) and the SDs of three repeated measurements obtained under normoxia at a retinal sector nasal to the optic disc. Repeated measurements of mPO_2A, PO_2V, and OEF at each retinal sector were averaged, and spatial variation was assessed by relating OEF measurements at nasal and temporal sectors using Pearson's correlation. In each rat, measurements of mPO_2A, PO_2V, and OEF at nasal and temporal retinal sectors were averaged, and the mean values under normoxia and hypoxia were compared with paired Student's t-test. Statistical significance was accepted at P less than 0.05. 

Results

Repeatability and Spatial Variation

The ICCs and mean SDs (averaged over data in 10 rats) obtained from three repeated mPO_2A, PO_2V, and OEF measurements in nasal retinal sectors under normoxia are shown in Table 1. Measurements of mPO_2A and PO_2V were repeatable, with ICCs greater than or equal to 0.86 and mean SDs less than or equal to 3 mm Hg. Similarly, inner retinal OEF measurements were also repeatable (ICC = 0.83 and mean SD = 0.08). As anticipated, measurements of OEF at nasal and temporal retinal sectors were correlated under normoxia (R = 0.71; P = 0.02; n = 10). 

Table 1. 

 

ICCs and mean SDs, Averaged over Data in 10 Rats, Based on Three Repeated mPO_2A, PO_2V, and OEF Measurements in the Nasal Sectors under Normoxia

	ICC	SD
mPO2A	0.86	3 mm Hg
PO2V	0.89	2 mm Hg
OEF	0.83	0.08

Systemic Physiological Status

The systemic physiological status of the rats under normoxia and hypoxia is presented in Table 2. P_aO₂ under hypoxia (34 ± 4 mm Hg) was significantly lower as compared with normoxia (93 ± 8 mm Hg) (P < 0.001; n = 10). Due to controlled ventilation, P_aCO₂ under normoxia and hypoxia were similar (P = 0.76). Arterial blood pH, blood pressure, and heart rate decreased significantly under hypoxia (P ≤ 0.001). 

Table 2. 

 

The Systemic Physiologic Status and Retinal Vascular PO₂ Measurements under Normoxia and Hypoxia

	Normoxia	Hypoxia	P Value
Systemic physiologic status
PaO2, mm Hg	93 ± 8	34 ± 4	<0.001*
PaCO2, mm Hg	40 ± 5	41 ± 5	0.76
pH	7.38 ± 0.05	7.31 ± 0.05	<0.001*
Blood pressure, mm Hg	113 ± 17	78 ± 22	0.001*
Heart rate, beats·min−1	214 ± 36	162 ± 45	0.002*
Retinal vascular PO2
mPO2A, mm Hg	44 ± 4	20 ± 4	<0.001*
PO2V, mm Hg	28 ± 5	11 ± 4	<0.001*

 Means and SDs of measurements in 10 rats are listed. Asterisks indicate statistically significant differences between normoxia and hypoxia.

Retinal Vascular PO₂ and OEF

Examples of cross-sectional vascular PO₂ maps obtained in the same rat under normoxia and hypoxia are shown overlaid on the corresponding red-free retinal images in Figure 1. As expected, in both nasal and temporal sectors, mPO_2A was higher than PO_2V under both normoxia and hypoxia. Both mPO_2A and PO_2V decreased under hypoxia as compared with normoxia. 

Measurements of mPO_2A and PO_2V averaged over all rats under normoxia and hypoxia are listed in Table 2. Under normoxia, mPO2_A and PO2_V were 44 ± 4 and 28 ± 5 mm Hg, respectively (n = 10). Under hypoxia, both mPO_2A (20 ± 4 mm Hg) and PO_2V (11 ± 4 mm Hg) decreased significantly as compared with normoxia (P < 0.001). Mean measurements of inner retinal OEF under normoxia and hypoxia are shown in Figure 2. Mean inner retinal OEF was 0.46 ± 0.13 under normoxia and increased significantly to 0.67 ± 0.16 under hypoxia (P < 0.001; n = 10). 

Figure 2. 

 

Inner retinal oxygen extraction fraction (OEF) under hypoxia (0.67 ± 0.16) was significantly higher than under normoxia (0.46 ± 0.13) (mean ± SD; P < 0.001; n = 10).

Discussion

Retinal ischemia is implicated in many retinal diseases that lead to visual impairment; however, methods that can quantitatively assess the retinal tissue status in vivo under ischemia are limited. One potential parameter is OEF, which indicates the adequacy of oxygen supply relative to the tissue's metabolic demand. In the current study, we report for the first time to our knowledge, quantitative measurements of inner retinal OEF in rats based on retinal vascular PO₂ imaging, and demonstrate a significant increase in inner retinal OEF under systemic hypoxia. 

Inner retinal OEF measurements in the current study were compared with calculated retinal and measured cerebral OEFs due to a lack of published retinal data. Based on oximetry studies in healthy subjects, ^22–24 we computed the inner retinal OEF to be 0.40, which is comparable to our measurements of 0.46 in rats under normoxia. Additionally, inner retinal OEF measured under normoxia in the current study was in agreement with published cerebral OEFs (0.37 to 0.57). ^25–30 Our finding of increased inner retinal OEF under systemic hypoxia was similar to elevated inner retinal OEF in chronic systemic hypoxia ³¹ and central retinal vein occlusion, ³² calculated from oximetry data in humans. Moreover, this finding was also consistent with the response of cerebral OEF under hypoxia. ^25,27,30  

An increase in inner retinal OEF can occur due to either an increase in retinal MO₂ or a decrease in DO₂. Under hypoxia, because MO₂ is unlikely to increase, the observed increase in inner retinal OEF would occur due to a decrease in DO₂ that can result from reductions in retinal blood flow and/or arterial blood oxygen content. Although retinal blood flow in principle could be affected by systemic blood pressure and/or blood pH, it was not likely influenced by these factors under our experimental conditions. Blood pressure remained within the autoregulatory range, ³³ even though it was reduced during hypoxia. Likewise, a change in blood pH was not expected to alter blood flow, according to findings in the brain. ^34,35 On the other hand, hypoxia has been reported to increase retinal blood flow, ^36–39 which would tend to maintain DO₂. Nevertheless, the compensatory increase in blood flow may not be adequate to maintain DO₂, if the hypoxia is severe enough. In that case, because of reductions in retinal arterial blood oxygen content, DO₂ decreases, thereby causing an increase in inner retinal OEF. 

One factor that likely affected our results was that oxygen contents in retinal veins were estimated from the oxygen dissociation curve using the arterial blood pH. Because blood pH is expected to be lower in veins than in arteries, ^40,41 the actual venous oxygen contents were likely lower due to a right shift of the oxygen dissociation curve. As a result, the actual inner retinal OEF should have been higher than the reported values under normoxia. The right shift of the oxygen dissociation curve would be expected to be even more prominent during hypoxia due to increased anaerobic metabolism. Because OEF was underestimated under both ventilation conditions, this limitation likely did not affect our finding of increased inner retinal OEF under hypoxia. 

Measurements of inner retinal OEF may provide information about the retinal tissue status during ischemic insult complementary to retinal blood flow measurements or conventional diagnostic tests, such as fundus photography and fluorescein angiography. An acute increase in retinal OEF indicates a state of misery perfusion, in which DO₂ declines, and thereby support for MO₂ becomes precarious. If DO₂ remains reduced chronically, irreversible anoxic damage may develop, leading to decreases in retinal MO₂ and OEF. Therefore, while OEF is elevated, retinal tissue may still remain viable, ⁴² thus potentially providing a window of opportunity for successful intervention, as has been demonstrated in cerebral tissue. ^43–45 In the current study, we evaluated inner retinal OEF under two ventilation conditions to establish a normal baseline and demonstrate a response to a severe hypoxic challenge. Additional studies that assess inner retinal OEF in response to varying levels of inspired oxygen may help establish a threshold at which OEF begins to increase due to maximized blood flow compensation. Furthermore, future studies are needed to provide knowledge of OEF response in experimental animal models of retinal vascular deficiency that may eventually be translated for clinical management of patients. 

In summary, we established a method for quantitative measurement of inner retinal OEF in rats and demonstrated an increase in OEF under acute systemic hypoxia. The method can be extended to provide a global assessment of inner retinal OEF by measuring O₂ contents in all major retinal blood vessels. Quantification of inner retinal OEF has potential value for providing information about the retinal energy metabolism under challenged physiological and pathological conditions. 

Footnotes

 Supported by the National Eye Institute, Bethesda, Maryland, EY17918 (MS) and EY01792 (University of Illinois at Chicago), and Research to Prevent Blindness, New York, New York, senior scientific investigator award (MS), and an unrestricted departmental award.

Footnotes

 Disclosure: P.-Y. Teng, None; J. Wanek, None; N.P. Blair, None; M. Shahidi, P