A method for volumetric retinal tissue oxygen tension imaging

Abstract

Purpose: Inadequate retinal oxygenation occurs in many vision-threatening retinal diseases, including diabetic retinopathy, retinal vascular occlusions, and age-related macular degeneration. Therefore, techniques that assess retinal oxygenation are necessary to understand retinal physiology in health and disease. The purpose of the current study is to report a method for the three-dimensional (3D) imaging of retinal tissue oxygen tension (tPO₂) in rats.

Methods: Imaging was performed in Long Evans pigmented rats under systemic normoxia (N = 6) or hypoxia (N = 3). A vertical laser line was horizontally scanned on the retina and a series of optical section phase-delayed phosphorescence images were acquired. From these images, phosphorescence volumes at each phase delay were constructed and a 3D retinal tPO₂ volume was generated. Retinal tPO₂ volumes were quantitatively analyzed by generating retinal depth profiles of mean tPO₂ (M_tPO2) and the spatial variation of tPO₂ (SV_tPO2). The effects of systemic condition (normoxia/hypoxia) and retinal depth on M_tPO2 and SV_tPO2 were determined by mixed linear model.

Results: Each 3D retinal tPO₂ volume was approximately 500 × 750 × 200 μm (horizontal × vertical × depth) and consisted of 45 en face tPO₂ images through the retinal depth. M_tPO2 at the chorioretinal interface was significantly correlated with systemic arterial oxygen tension (P = 0.007; N = 9). There were significant effects of both systemic condition and retinal depth on M_tPO2 and SV_tPO2, such that both were lower under hypoxia than normoxia and higher in the outer retina than inner retina (P < 0.001).

Conclusion: For the first time, 3D imaging of retinal tPO₂ was demonstrated, with potential future application for assessment of physiological alterations in animal models of retinal diseases.

Keywords: Rat; phosphorescence lifetime imaging; retina; three-dimensional imaging; tissue oxygen tension.

Figure 1

A schematic diagram illustrating the steps for the generation of a retinal tissue oxygen tension (tPO₂) volume. (A) A vertical laser line is scanned laterally across the rat retina, as indicated by green lines, to acquire a series of phase-delayed optical section phosphorescence images. (B) The series of zero-phase delay optical section phosphorescence images are stacked along the x-axis. Vertical red lines on the rightmost optical section phosphorescence image indicate vitreoretinal (left) and chorioretinal (right) interfaces. (C) The zero-phase delay phosphorescence volume is shown. (D) Using the set of phase-delayed phosphorescence volumes, the phosphorescence lifetime was calculated at each voxel to generate a retinal tPO₂ volume, shown in pseudo color. Color bar indicates tPO₂ between 0 and 50 mmHg. (E) The tPO₂ volume contains 45 en face tPO₂ images in x–y planes across the retinal depth. Arrows (from left to right) point to en face tPO₂ images at 0%, 33%, 66% and 100% retinal depth.

Figure 2

(A) Representative retinal region imaged in a rat, indicated by green lines superimposed on the red-free image. Retinal tissue oxygen tension (tPO₂) volumes generated in the same retinal region under normoxia (B) and hypoxia (C). (D) From the tPO₂ volumes, en face tPO₂ images at four retinal depths under normoxia and hypoxia are displayed. Color bar indicates tPO₂ in mmHg.

Figure 3

Mean retinal tissue oxygen tension (M_tPO2) at the chorioretinal interface plotted as a function of systemic arterial oxygen tension (P_aO₂). Compiled data from nine rats.

Figure 4

(A) Mean retinal tissue oxygen tension (M_tPO2) depth profile under normoxia (red, N = 6) and hypoxia (blue, N = 3). (B) Retinal tissue oxygen tension spatial variation (SV_tPO2) depth profile under normoxia (red) and hypoxia (blue). Error bars indicate standard error of the means.