2.1.

Animal Preparation

Adult Brown Norway rats (17 weeks old) were included in this study. Seven eyes were randomly chosen (four left eyes and three right eyes from seven animals). Animals were anesthetized with 5% isoflurane initially for 10 min and 2.5% isoflurane during the imaging session, mixed with 100% ${\mathrm{O}}_2$. Following stabilization in a custom-made imaging stage with multidimensional manipulation, the animal was positioned with the eye facing vertically (Fig. 1). The pupil was dilated with a 1% tropicamide ophthalmic solution. To keep the cornea moist, sterile irrigating salt solution (Alcon Laboratories Inc.) was applied to the eye every other minute. Body temperature was maintained with a 38.5°C water-warming blanket. Exhaust gas, including isoflurane, was removed by a vacuum pump to avoid carbon dioxide accumulation and was collected by an anesthesia gas filter (OMNICON F/air, Bickford) before releasing to the open air.

Fig. 1

Experimental setup of intraocular pressure elevation in rats. The rats were stabilized in a customized head holder with inhalation gas delivery. A vertically oriented light beam illuminated the eye.

To regulate IOP, the anterior chamber was cannulated with a 31-gauge needle and connected to a reservoir filled with balanced salt solution (BSS PLUS, Alcon Laboratories Inc.). The height of the reservoir was adjustable to increase and decrease IOP (Fig. 1), which was monitored by a calibrated pressure transducer (Harvard Apparatus, Model 724436). Mean arterial blood pressure was continuously monitored by noninvasive CODA tail-cuff tonometry (Kent Scientific, Torrington, Connecticut).

For each imaging session, IOP was increased, starting at 10 mmHg (baseline), in 10-mmHg increments up to 100 mmHg, which was empirically comparable to the measured mean arterial pressure.¹⁴ At each IOP level, 3 min were allowed for stabilization before acquiring images. Following 100 mmHg, IOP was returned to baseline, to ascertain recovery from the acute IOP elevation.

All experimental procedures complied with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Oregon Health & Science University (OHSU).

2.2.

Image Acquisition

A previously reported fiber-based 50-kHz vis-OCT prototype,³¹ custom-built by the Center for Ophthalmic Optics and Lasers at OHSU, was used in this study. To analyze ${\mathrm{sO}}_2$, the system wavelength spans from 510 to 610 nm, covering the high absorption contrast region of hemoglobin. The incident power on the cornea was 0.8 mw. A GPU-based real-time interface for visualizing structural and angiographic OCT was used to assist image acquisition.³³ The axial resolution was calibrated as $1.2\mu \mathrm{m}$ in tissue with a maximum sensitivity of 89 dB.

At each IOP level, a volumetric raster scan was acquired for angiography and ${\mathrm{sO}}_2$ analysis and three sets of dual-circular scans were acquired for total retinal blood flow measurement. The volumetric raster scan was taken in a $2.2\times 2.2\text{-}{\mathrm{mm}}^2$ field of view. In the fast transverse ($X$) scanning direction, 512 axial scans were sampled to form a B-scan. Three consecutive B-scans were captured before moving to the next slow axis ($Y$) position for motion contrast in OCTA. A total of 512 $Y$ positions were sampled. The dual circular scans ($r_1=0.6\mathrm{mm}$, $r_2=0.8\mathrm{mm}$) around the optic disc consisted of 4096 A-lines for each circle with 30 repetitions. The dual circular scan transected all retinal major arteries and veins. The whole experiment for full IOP regulation on a single rat was completed within $\sim 45\min$.

2.3.

Data Processing

Postprocessing was performed in the MATLAB platform. The recorded interferometric fringe was processed by DC subtraction, lambda-to-k resampling, and dispersion compensation successively. After that, the dataset was applied by fast Fourier transform to retrieve the depth-resolved structural information.³⁴ Retinal layers were segmented by searching the boundaries automatically and correcting with manual assistance.³⁵

The OCTAs were generated with the split-spectrum amplitude decorrelation algorithm (SSADA) on the volumetric raster scans.⁸ The vascular/capillary plexuses were then generated by projecting the flow signal within specific slabs. For the superficial vascular plexus (SVP), a slab containing the NFL and ganglion cell layer (GCL) was used. For the intermediate capillary plexus (ICP) and deep capillary plexus (DCP), slabs corresponding to the inner and outer boundaries, respectively, of the inner nuclear layer (INL) were used. The en face images of the vascular plexuses were enhanced by a Gabor filter, and then a Frangi multiscale vessel filter³⁶ was used to delineate the vessel binary mask. The vessel density was calculated as the percentage of area occupied by vascular pixels.

The procedure of measuring ${\mathrm{sO}}_2$ was detailed in our previous report.²⁵ Briefly, oxygen saturation is the concentration ratio of oxygenated hemoglobin to total hemoglobin and can be expressed as ${\mathrm{sO}}_2={\mathrm{C}}_{\mathrm{HbO}2}/({\mathrm{C}}_{\mathrm{HbO}2}+{\mathrm{C}}_{\mathrm{Hb}})$, where ${\mathrm{C}}_{\mathrm{HbO}2}$ and ${\mathrm{C}}_{\mathrm{Hb}}$ indicate the concentration of oxygenated and deoxygenated hemoglobin, respectively. To obtain ${\mathrm{sO}}_2$, we fit the obtained OCT spectrum at the posterior vascular pixels^25,37 to a model^15,38 [Eq. (1)] that contains the absorption features and concentrations of oxygenated and deoxygenated hemoglobin

The blood flow $F$ ($\mu \mathrm{L}/\min$) for each major vessel was determined from dual circular scans according to⁴⁰

Oxygen metabolic rate is defined as the oxygen consumption of the tissue over time, which can be deduced from the difference between oxygen content supplied by arteries and that carried away by veins per unit time.^27,32,40 In the inner retina, the oxygen demand is supplied by the retinal circulation, where, in the rat eye, all major arteries and veins enter the eye ventral to the optic disc.^13,42 Thus, the retina oxygen metabolic rate ${\mathrm{rMRO}}_2(\mathrm{g}/\min )$ can be quantified as

3.1.

Reflectivity

As shown in Fig. 2, the boundaries of the different retinal layers became vague and hard to distinguish at higher IOPs. In addition, the reflectivity of retinal layers was largely reduced at high IOP levels, especially for the NFL. In normal conditions, the NFL is highly reflective and has extraordinary reflectance amplitude shown by structural OCT. However, as shown in the volumetric visualization [Fig. 2(c)], the brightness of the NFL layer diminished as IOP increased. To remove the effect of potential defocus and aberrations due to tissue bending at $\mathrm{IOP}\ge 40\mathrm{mmHg}$ and to examine the dynamic changes of NFL more objectively, we calculated the relative reflectance (the mean reflectance of the NFL normalized by the mean reflectance of the entire retina). This decreased from $1.41\pm 0.22$ at $\mathrm{IOP}=10\mathrm{mmHg}$ to $0.99\pm 0.45$ and $0.72\pm 0.49$ at IOPs of 70 and 100 mmHg, respectively, and returned to $1.52\pm 0.19$ with IOP back to 10 mmHg.

Fig. 2

Reflectivity of the retinal NFL is reduced with acute IOP elevation, as demonstrated by sequential darkening in (a) B-scans, (b) en face images ($2.2\times 2.2{\mathrm{mm}}^2$), and (c) volumetric visualization. Each B-scan was averaged by three repeated frames. The white dashed line in (b) indicates the position of B-scans in (a). NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; EZ, ellipsoid zone; RPE, retinal pigment epithelium; CH, choroid; SVP, superficial vascular plexus; ICP, intermediate capillary plexus; and DCP, deep capillary plexus.

3.2.

Angiography

To visualize the responses of vascular perfusion clearly, retinal layers were segmented and en face images of SVP, ICP, and DCP were obtained by projecting the specific slabs described in Sec. 2.3. Figure 3 illustrates that capillary perfusion was well maintained for all three plexuses until IOP exceeded 70 mmHg. Above this, perfusion decreased dramatically, becoming invisible at 90 and 100 mmHg.

Fig. 3

En face images ($2.2\times 2.2{\mathrm{mm}}^2$) showing retinal vascular perfusion responses to acute IOP elevation. (a) SVP en face images were generated from the slabs containing NFL and GCL with first-15-maximum projection. (b) ICP en face images were generated from the slabs adjacent to the inner boundary of the INL with first-15-maximum projection. (c) DCP en face images were generated from the slabs adjacent to the outer boundary of the INL with first-15-maximum projection.

To quantitate the vascular perfusion during IOP elevation, vessel density was calculated as the percentage of the field of view covered by microvasculature. At $\mathrm{IOP}=10\mathrm{mmHg}$ baseline, the vessel densities were $21\%\pm 1\%$, $14\%\pm 2\%$, and $19\%\pm 2\%$ for SVP, ICP, and DCP, respectively. Overall, vessel densities were reduced at high IOPs and recovered when the IOP returned to 10 mmHg [Fig. 4(a)].

Fig. 4

The (a) actual and (b) normalized to baseline (10 mmHg) vessel densities of the three vascular/capillary plexuses SVP, ICP, and DCP at all IOP levels.

The vessel densities were normalized by baseline values to minimize population variation [Fig. 4(b)]. The normalized vessel densities remained at low IOPs and were significantly affected for IOPs greater than or equal to 70 mmHg ($p\text{-}\text{value}<0.05$ at $\mathrm{IOP}\ge 70\mathrm{mmHg}$), consistent with a previous report.¹⁴ From visual inspection of en face images in Fig. 3, it is clear that the vascular perfusion diminished quickly and the capillaries were hardly seen at IOP levels beyond 70 mmHg. Nonzero vessel densities calculated at these high IOP levels were likely due to the enhanced flow artifacts (background noise) by the vesselness filter³⁶ used before generating vascular binary images.

3.3.

Blood Flow

Arteries and veins were differentiated by their opposite flow directions in relation to the optic disc. Doppler OCT frames with reversal of arterial flow were found to appear at IOP of 50 mmHg occasionally and were more frequent at higher IOPs. The reversal could be appreciated by comparing the blood flow velocity distributions from a vein and an artery at $\mathrm{IOP}=10\mathrm{mmHg}$ and $\mathrm{IOP}=70\mathrm{mmHg}$ (Fig. 5). Venous blood flow velocities dropped evenly at all frames and remained negative at $\mathrm{IOP}=70\mathrm{mmHg}$. However, arterial blood flow velocities were positive at baseline in all frames, but decreased and reversed to negative at $\mathrm{IOP}=70\mathrm{mmHg}$ in 15 frames (black boxes) out of the total 30 frames.

Fig. 5

Arterial flow reversal at $\mathrm{IOP}=70\mathrm{mmHg}$ shown by a series of Doppler OCT (30 frames within 2.4 s) of a representative artery, compared to the constant flow direction in a vein. Doppler OCT images with arterial flow reversal are annotated with black boxes. Red: positive blood flow. Blue: negative blood flow.

The blood flow for all major retinal vessels was calculated from the dual circular Doppler scans, as described in Sec. 2.3. The total retinal blood flow (TRBF) for arteries and veins was summed up in all major arteries and veins, respectively. The TRBF at $\mathrm{IOP}=10\mathrm{mmHg}$ was measured at $8.37\pm 0.69\mu \mathrm{L}/\min$ for arteries and $-8.58\pm 0.86\mu \mathrm{L}/\min$ for veins [Fig. 6(a)], where positive and negative values indicate flow into and out of the retina, respectively. At high IOP levels, the blood flow was suspended as TRBF approached zero. Owing to the presence of flow reversal in most arteries at IOPs of 70 and 80 mmHg, current frame rate ($\sim 12\mathrm{Hz}$) was not considered able to fully sample this highly irregular dynamic pattern. Therefore, arterial TRBF for these two time points were not included. Furthermore, TRBF was not measurable at IOPs of 90 and 100 mmHg because absent blood flow prevented identification of vessel cross sections.

Fig. 6

Response of actual (a) and normalized (b) TRBF to the acute IOP elevation. Data points for arterial flow on IOPs of 70 and 80 mmHg were not recorded due to the impact of flow reversal. Data points on IOPs of 90 and 100 mmHg were not available due to severe perfusion loss. TRBF decreased as IOP was elevated and recovered as IOP was returned to 10 mmHg.

Retinal blood flow response to IOP elevation was also revealed by TRBF values normalized to their baselines. As shown in Fig. 6(b), normalized TRBF was set to 1.0 at baseline $\mathrm{IOP}=10\mathrm{mmHg}$ and remained unaffected at IOPs $<40\mathrm{mmHg}$ ($p\text{-}\mathrm{value}<0.05$ for arteries at $\mathrm{IOP}\ge 40\mathrm{mmHg}$; $p\text{-}\text{value}<0.05$ for veins at IOP $>40\mathrm{mmHg}$). At 60 mmHg, the normalized TRBF dropped to $<0.5$, even though capillary perfusion (Fig. 4) was unaffected at this pressure. When IOP returned to 10 mmHg, the normalized TRBF recovered to $0.98\pm 0.03$ and $1.00\pm 0.4$ for arteries and veins, respectively. This retinal blood flow response to elevated IOP was also consistent with a previous report.¹⁴

3.4.

Oxygen Saturation

The identification of arteries and veins by level of oxygen saturation agreed with that as determined by the direction of blood flow in all eyes. The ${\mathrm{sO}}_2$ in all arteries and veins within each eye was then averaged as the measured arterial and venous ${\mathrm{sO}}_2$ level in that eye. The ${\mathrm{sO}}_2$ was not measurable at IOPs of 90 and 100 mmHg due to insufficient vascular filling. As shown in Fig. 7, the arterial ${\mathrm{sO}}_2$ remained nearly 100% at all IOP levels, whereas venous ${\mathrm{sO}}_2$ decreased with higher IOPs ($p\text{-}\mathrm{value}<0.05$ at $\mathrm{IOP}\ge 40\mathrm{mmHg}$).

Fig. 7

The measured ${\mathrm{sO}}_2$ overlaid with en face images ($2.2\times 2.2{\mathrm{mm}}^2$) of retinal vessels at each IOP level. Note that the measurements at $\mathrm{IOP}=90$ and 100 mmHg were not available due to severe nonperfusion.

The oxygen extraction A-V ${\mathrm{sO}}_2$, calculated as the difference between arterial ${\mathrm{sO}}_2$ and venous ${\mathrm{sO}}_2$, increased from $13\%\pm 2\%$ at baseline to $34\%\pm 8\%$ at $\mathrm{IOP}=80\mathrm{mmHg}$ (Fig. 8). Moreover, this increase seemed to be nonlinear, with increments of 1%, 2%, 3%, 3%, 3%, 5%, and 5% as IOP levels increased from 20 to 80 mmHg. Restoration of A-V ${\mathrm{sO}}_2$ was also observed as IOP returned to baseline.

Fig. 8

Retinal response of ${\mathrm{sO}}_2$ in major vessels to acute IOP elevation. As IOP increases, the venous ${\mathrm{sO}}_2$ drops in nonlinear fashion while arterial ${\mathrm{sO}}_2$ is well maintained, resulting in an increased oxygen extraction (A-V ${\mathrm{sO}}_2$). Data points at IOPs of 90 and 100 mmHg were not obtainable due to severe nonperfusion.

3.5.

Oxygen Metabolism

Total retina oxygen metabolic rate was calculated as $276\pm 15\mathrm{ng}/\min$ at baseline and remained $\sim 250\mathrm{ng}/\min$ until IOP exceeded 40 mmHg (Fig. 9). At IOPs of 50 and 60 mmHg, the arterial blood flow reversal, mentioned above, began to appear sporadically in some frames in some vessels. These reversals, which contributed to the significantly decreased arterial blood supply in Fig. 6(b), produced a reduced calculated oxygen metabolism at these IOP levels, despite the increased oxygen extraction (Fig. 8). However, response to acute IOP elevation differed among subjects, which caused a large variation in calculated oxygen metabolism. The measurements at IOPs of 70 to 100 mmHg were not obtainable due to the lack of blood flow and oxygen saturation results.

Fig. 9

Calculated retinal response of oxygen metabolism rate to acute IOP elevation. The measurements at IOPs of 70 to 100 mmHg were not attainable due to the lack of blood flow or oxygen saturation results.