2.1.

Animal Preparation

Two female Lewis rats weighing 125 to 149 g were purchased from Harlan Laboratories and maintained with standard chow and water ad libitum under pathogen-free conditions. All animals were treated in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research under the animal protocol approved by the Animal Care and Use Committee of University of Washington.

2.2.

OCT-Based Microangiography

An anterior OCT system was used to implement OMAG, which was the system previously described in Ref. 31. In brief, it was a custom-built spectral-domain OCT system operated at a central wavelength of 1340 nm with a scan speed of 92,000 A lines per second. The spatial ($\text{axial}\times \text{lateral}$) resolution of the system was $7\mu \mathrm{m}\times 7\mu \mathrm{m}$ in air, respectively. The system sensitivity was measured at 100 dB at the focal spot of the sample beam, corresponding to 0.5 mm below the zero delay line.

The rat was anesthetized with inhalational isoflurane (1.5%) mixed with 20% oxygen. Each animal was placed in the right lateral decubitus position and secured in a custom-made stereotactic stage equipped with a heating pad and a nose cone for continuous inhaled isoflurane anesthesia. Complete exposure of the anterior segment was provided by traction sutures placed through the lower and upper eyelids, and cornea protection was provided with a topical balanced salt solution (BSS, Alcon, Johns Creek, GA) throughout the imaging session. The stereotactic stage was then positioned with the apex of the cornea centered beneath a scan lens in the sample arm of the OCT system.

A repetitive B-mode step scanning protocol³² was adopted to implement OMAG, in which five repeated B-scans (fast scan) at each C-scan (slow scan) position were performed on the anterior segment of the rat eye at a speed of 140 frames per second. This scanning protocol generated a total of 2000 B-frames (512 A lines per each B-frame) in a single 3-D OCT data volume. During the image acquisition, anesthesia was maintained using a breathing anesthesia machine (M3000, Supera Anesthesia Innovations, Clackamas, OR) to restrain bulk motions from its body except for breathing and pulsation.

B-mode blood flow images were computed from the acquired 3-D OCT data volume by applying an intensity-based OCT angiography algorithm.³¹ In this method, the intensity changes in time caused by randomly backscattered lights resulting from moving red blood cells in the vessel lumens can be decoupled from the surrounding static tissue regions by subtraction of adjacent two B-frames (a time interval of 7 ms) obtained at the same location, representing blood perfusion in the functional vessels. This strategy to contrast blood flow is quite similar to ultrahigh-sensitive optical microangiography,^25,26 an OMAG algorithm that utilizes the analytical forms of the OCT signals rather than the OCT amplitude signals. Using five repetition B-scans, one blood flow image was obtained through ensemble averaging of the calculated four B-scans of blood flow at a given C-scan position. Eventually, a total of 400 cross-sectional blood flow images together with the corresponding 400 structural images were reconstructed within the scanned tissue volume. To facilitate the visualization and analyses, the 3-D blood flow data were collapsed into an en face (XY) projection image (an OCT angiogram).

2.3.

Induction of Acute Anterior Uveitis

The experimental protocol for the rat model of AAU is illustrated in Fig. 1. To induce AAU, we modified an established experimental model of uveitis previously reported in rabbits to the rat.³³ Anterior uveitis was initiated in the right eye of each rat with an intravitreal injection of $10\mu \mathrm{g}$ of killed mycobacterium tuberculosis H37Ra antigen (DifcoLaboratories, Detroit, MI) in a $5\mu \mathrm{l}$ volume of phosphate buffered saline at day 0. This protocol induces intraocular inflammation that peaks at two days post-injection. To test the therapeutic effect of steroid treatment on AAU, the other rat was also treated with an additional periocular injection of steroids, 4 mg triamcinolone in 0.1 ml at day 0. The steroid is a potent anti-inflammatory agent. Anterior OCT imaging was performed with the right eyes of all rats at day 0 (preinjection) and at day 2 (peak-inflamed or steroid treated).

Fig. 1

Illustration of the experimental protocol for the rat model of acute anterior uveitis.

2.4.

Histology

After OCT imaging at day 2, the animals were euthanized and eyes were collected for histology. The collected eyes were fixed in 10% neutral buffered formalin (Triangle Biosciences, Durham, NC) and embedded in paraffin. $4\text{-}\mu \mathrm{m}$ sections were obtained and stained with hematoxylin and eosin for microscopic evaluation.

3.1.

Identification of Inflammation in the Rat Anterior Segment

The signs of intraocular inflammation in vivo include the infiltration of white blood cells (WBCs) into the AC and posterior chamber (PC), corneal edema, iris vascular dilation, and an increase in the protein concentration in the intraocular fluids.^18,34 When inflammation is severe, the protein concentration can form an inflammatory membrane on the anterior surface of the iris and across the pupil.¹⁸ To determine if OCT imaging could identify these features in a rodent model of anterior uveitis, we obtained OCT images at baseline and during peak inflammation and compared these to histology. Figure 2 shows the representative cross-sectional OCT images obtained at the central AC [Figs. 2(a) and 2(c)] and at the limbus [Figs. 2(b) and 2(d)]. At day 0, the OCT images [Figs. 2(a) and 2(b)] reveal typical anatomical features of the anterior segment of normal murine, including the hyper-reflective cornea, iris and CB, the optically empty AC, and the lens.³⁵ At day 2, the OCT images [Figs. 2(c) and 2(d)] reveal the inflammatory cell infiltration into both the anterior and posterior chambers and along the anterior lens capsule [arrows in Figs. 2(c) and 2(d)], the increase of corneal thickness [oblique bars in Figs. 2(a) and 2(c)], and fibrinous membranes on the surface of the iris and extending across the pupil [asterisks in Fig. 2(c)]. An unexpected finding was the increase in OCT signal intensity of the zonules during inflammation [dotted circle in Fig. 2(d)]. The zonules are a series of fibrous strands connecting the CB with the lens. At day 0, they can be seen as individual thin hyper-reflective bands, but at day 2, the individual fibers are obscured probably because of the surrounding inflammatory cells. Post mortem histology [Fig. 2(e)] confirms the inflammatory changes noted by the OCT images. An additional note regarding the OCT imaging is that the sclera in Fig. 2(d) looks down-curved, which is caused by the refractive index difference between the hydration medium (BSS) on the sclera and the air.

Fig. 2

In vivo optical coherence tomography (OCT) imaging of the anterior segment in the rat model of acute anterior uveitis. (a) and (b) Representative cross-sectional OCT structural images obtained at central anterior chamber (AC) and at limbal region at day 0 (baseline), respectively. (c) and (d) Representative cross-sectional OCT structural images obtained at the location similar to (a) and (b) at day 2 (peak inflammation), respectively, showing structural changes, such as infiltrating cells (arrows) in the AC and the posterior chamber (PC), thickened cornea, and fibrinous membranes (asterisks) on the surface of iris and across the pupil. The inflammation features in the OCT images [(c) and (d)] are confirmed with a histologic section through the anterior segment of the same rat eye (e). Sale bars: $500\mu \mathrm{m}$.

3.2.

Identification of Steroid Treatment Effect on Inflammation of the Rat Anterior Segment

Histology is the gold standard for the detection of intraocular inflammation in animal models of uveitis. To compare the ability of OCT with histology in the assessment of the effect of steroid treatment on inflammation, the rat induced with AAU was treated with the periocular injection of triamcinolone. OCT images of the eye were obtained at day 0 and day 2 as shown in Fig. 3. In contrast to the rat without treatment (see Sec. 3.1), at day 2, no inflammatory changes are detected by OCT images [Figs. 3(c) and 3(d)] when compared to the baseline [Figs. 3(a) and 3(b)] in the steroid-treated animal. This finding is confirmed with the histologic assessments [Fig. 3(e)] by the absence of infiltrating WBCs in the AC, PC, or surrounding the CB and the zonules. These data are consistent with clinical results in humans that triamcinolone effectively inhibits ocular inflammatory activity in patients with uveitis.³⁶

Fig. 3

In vivo OCT imaging before and after steroid treatment in the acute anterior uveitis rat model. (a) and (b) Representative cross-sectional OCT structural images obtained at AC and at the limbal region at day 0 (baseline), respectively. (c) and (d) Representative cross-sectional OCT structural images obtained at the location similar to (a) and (b) at day 2 (steroid treatment), respectively, showing no significant change in morphology compared to the baseline [(a) and (b)]. The anti-inflammation features in the OCT images [(c) and (d)] are confirmed with a histologic section through the anterior segment of the same rat eye (e). Scale bars: $500\mu \mathrm{m}$.

3.3.

Quantification of Central Corneal Thickness Changes

To quantify the central corneal thickness (CCT) changes in rats with and without steroid treatment, the central cornea was defined to the area [$2.6\mathrm{mm}(\mathrm{X})\times 2.6\mathrm{mm}(\mathrm{Y})$] centered on the pupil. The thickness across this area was determined using a semiautomatic segmentation algorithm.³⁷ A resulting 2-D CCT map was obtained from the segmented 3-D corneal information. Figure 4 shows the average corneal thickness obtained at day 0 and day 2 for the inflamed and steroid-treated animals, respectively. For the rat without steroid treatment [Fig. 4(a)], the CCT at day 2 ($166.79\pm 12.45\mu \mathrm{m}$) is more than 1.3 times thicker than the CCT at day 0 ($121.29\pm 8.16\mu \mathrm{m}$). This is consistent with clinical findings of corneal edema in human patients with uveitis.³⁴ For the rat treated with the steroid, the CCT at day 2 ($74.81\pm 8.23\mu \mathrm{m}$) was not increased when compared with day 0 ($76.32\pm 5.06\mu \mathrm{m}$). This is consistent with the absence of inflammation in the presence of steroids as assessed by histology.

Fig. 4

Changes in central corneal thickness of the peak inflamed rat (a) without and (b) with the steroid treatment.

3.4.

Quantification of Iris Vascular Changes Using OCT Angiograms

We also investigated blood perfusion changes in the rat iris during acute inflammation using OMAG. This angiographic modality was able to delineate functional microvasculature in the anterior segment tissue beds of the rat eyes in vivo.³¹ Figure 5 shows microvascular imaging results for the rat without steroid treatment. An en face (XY) OMAG ($3.57\mathrm{mm}\times 3.57\mathrm{mm}$) of the anterior segment in the rat eye at day 0 (baseline) [Fig. 5(a)] illustrates the complex microvascular anatomy of the anterior segment, consisting of radial iris vessels (solid arrows), long posterior ciliary arteries (LPCA), circumferential ciliary plexus (dotted arrows) in the ciliary process located behind the iris, and limbal plexus (asterisks) supplied by aqueous-containing veins (arrow heads). The OMAG angiogram at day 2 [Fig. 5(c)] shows that the iris vessels become dilated, and the ciliary plexus is obscured when compared to the day 0 images. However, the limbal circulation is almost not affected. Higher-magnification OMAG angiograms of the region from smaller scanned areas ($2.1\mathrm{mm}\times 2.1\mathrm{mm}$) highlight these differences [Figs. 5(b) and 5(d)].

Fig. 5

In vivo OCT-based microangiography (OMAG) imaging of anterior segment vasculature in the anterior uveitis rat model. (a) and (c) Two-dimensional (2-D) en face (XY) OMAG angiograms [$3.57\mathrm{mm}(\mathrm{X})\times 3.57\mathrm{mm}(\mathrm{Y})$] of the anterior segment of the rat eye at day 0 (baseline) and day 2 (peak inflammation), respectively. Compared to (a), in (c), the dilation of the iris vessels, including a long posterior ciliary artery (LPCA) and radial iris vessels [solid arrows in (a)], is evident and the presence of the ciliary plexus [dotted arrows in (a)] is obscured. These results are better appreciated with higher magnification angiographic views (b) and (d) from the smaller scanned area [$2.1\mathrm{mm}(\mathrm{X})\times 2.1\mathrm{mm}(\mathrm{Y})$], indicated as white boxes in (a) and (c), respectively. (e) A normalized intensity line profile across the location 1 on the LPCA [dotted line in (d)]. Its FWHM represents the vessel diameter. In (f), the iris vascular dynamics is quantified with the vessel diameters measured at three locations (1, 2, and 3) on the iris vessels in (b) and (d). Scale bars: $500\mu m$.

To quantify the amount of vascular dilation that occurs with inflammation, we compared iris vessel diameters at the same location for the pre and postinjection OMAG angiograms. Three iris vessels were chosen and marked with numbers in Figs. 5(b) and 5(d), and the vessel diameters at the numbered locations were calculated as a full width at half maximum (FWHM) of the normalized intensity line profile across each vessel.³⁸ Figure 5(e) shows a typical normalized intensity line profile across location 1 (dotted line) on the LPCA in Fig. 5(d). At this point, the FWHM of the LPCA was measured as $119.8\mu \mathrm{m}$, corresponding to the vessel diameter at day 2. The iris vessel diameters of three vessels were compared at day 0 and day 2 [Fig. 5(f)]. On average, the vessel diameters at day 2 are 30 to 40% greater than those at day 0. This is consistent with clinical findings of the iris vessel dilation in human patients with AAU.³⁹

To determine if the iris vascular changes could be detected in the presence of steroid treatment, the anterior microcirculation of the treated rat was visualized with OMAG angiograms ($3.57\mathrm{mm}\times 3.57\mathrm{mm}$) as shown in Fig. 6. In the steroid-treated eye, the vasculature [Fig. 6(b)] exhibits no appreciable difference when compared to the baseline images [Fig. 6(a)], including the qualitative appearance of the iris and limbal vascular caliber and the visibility of the CB. Iris vessel diameters were then quantified using the three numbered iris vessels in Figs. 6(a) and 6(b), and plotted in Fig. 6(c). There was no obvious increase in vessel diameter. Interestingly, we did identify a small but not significant decrease in the vessel diameter of $\sim 10\%$ in the steroid-treated eye. It is well known that the steroid we used (triamcinolone) has specifically profound antiangiogenic, antipermeability, and vasoconstrictive effects in addition to relieving inflammatory responses.⁴⁰ Therefore, we expect that the overall decrease in iris vessel diameters may be primarily due to such vascular effects due to the steroid applied to the inflamed rat eye.

Fig. 6

In vivo OMAG imaging of the anterior segment with the addition of steroid treatment to the acute anterior uveitis rat model. (a) and (b) 2-D en face (XY) OMAG angiograms [$3.57\mathrm{mm}(\mathrm{X})\times 3.57\mathrm{mm}(\mathrm{Y})$] of the anterior segment in the rat eye at day 0 (baseline) and at day 2 (steroid treatment), respectively. Compared to the baseline (a), no appreciable changes in vasculature are observed for the steroid treated eye (b). (c) Comparison of iris vessel diameters at three locations (1, 2, and 3) on the iris vessels in (a) and (b). Scale bars: $500\mu \mathrm{m}$.