2.1.

Animals and Tumor Cells

Five male New Zealand white rabbits weighing 3.0 to 4.0 kg were used in the present study, which was approved by the Animal Care and Use Committee of Kosin University. Animals were housed in separate cages at a controlled temperature of $22\pm 2°\mathrm{C}$ with $55\pm 5\%$ humidity. VX2 cells derived from rabbit skin SCC were used for tumor inoculation. All cells were obtained as a subculture from the femoral muscle of a study rabbit. Briefly, a thin (30 g) section of a tumor was cut, washed, suspended in Eagle’s Minimum Essential Medium, and centrifuged at $220\times \mathrm{g}$ for 5 min.

2.2.

Operative Procedures

After the injection of general anesthesia (ketamine $10\mathrm{mg}/\mathrm{kg}$, xylazine $3\mathrm{mg}/\mathrm{kg}$, subcutaneously), rabbits were secured in a supine position, and their conjunctival areas were exposed under visual guidance [Fig. 1(a)]. Using a 27-gauge needle, 0.4 ml of a suspension of VX2 cells ($1\times {10}^7$) was injected into the subconjunctival space directly over the pars plana in the five rabbits [Fig. 1(c)] located 2.75 mm from the 9 o’clock limbus of the right eye. OCT evaluation was performed before tumor injection [Fig. 1(b)] and after tumor injection [Fig. 1(d)], respectively. Antibiotics were administered during all procedures.

Fig. 1

Photographic and optical coherence tomographic (OCT) confirmation for VX2 tumor inoculation. (a) VX2 tumor inoculation, (b) OCT image of a normal conjunctiva before inoculation, (c) photo right after inoculation, (d) OCT image right after inoculation, (e) photo after three days, (f) OCT image after five days, (g) photo after seven days, and (h) OCT image after seven days. OCT images were taken by a system built in-house.

After the implantation, a slit lamp examination (SLE) [Fig. 1(e)] coupled with OCT were used to confirm initial findings and evaluate tumor nest formation [Fig. 1(f)]. On the seventh day after implantation, a second SLE [Fig. 1(g)] and OCT were performed [Fig. 1(h)]. At this point, if a tumor islet was found, the tumor was evaluated every three days thereafter. When tumors grew beyond a size of 5 mm, and if we were unable to evaluate OCT image of tumor due to the limited imaging depth, ultrasonographic examination was performed to measure tumor size and depth of tumor invasion. Animals were euthanized for histologic characterization of the tumor and implantation site. If no tumor cells were detected, animals were allowed to recover from anesthesia. SLE and OCT were repeated after 2 weeks, and animals were euthanized at this point, regardless of whether or not a tumor was detected. Two ophthalmologists, who were not aware of the previous tumor inoculation, conducted a blind examination of rabbit eyes using OCT and slit lamp independently.

2.3.

OCT System

We built an 850-nm spectrometer-based OCT system. A broadband light source (Broadlighter D855, Superlum, Cork, Ireland) with a center wavelength of 850 nm and a full-width-at-half-maximum of 100 nm was used. Interference fringe pattern was collected by a line scan camera (Sprint spL4096-140 km, Basler, Exton, Pennsylvania) with a line rate of 140 kHz and 4096 pixels. A two-axis scanner was customized using two galvanometers (6220H, Cambridge Technology, Cambridge, Massachusetts). B-mode images were acquired at 10 fps for 1024-lateral pixels. Further, a point spread function was measured and demonstrated a depth resolution of 4 μm in air, a roll-off of $12\mathrm{dB}/\mathrm{mm}$, and a signal-to-noise ratio of 103 dB. We also used a commercial OCT system (Spectralis, Heidelberg Engineering, Heidelberg, Germany).

2.4.

US System

We used a US system with a high-frequency linear transducer (40 to 8 MHz) (Sonix Touch, Ultrasonix, British Columbia, Canada). The depth range was 0.2 to 3 cm, and geometric focus was 6 mm.

2.5.

Pathological Evaluation

After the death of animals, both eyes were harvested together. We performed exenteration to harvest the eye ball. All tumors were not extended to the fornix and the palpebral subconjunctiva at the day of harvest. The resulting tissues were then fixed with formalin, embedded in paraffin, sliced at 5-μm intervals, stained with hematoxylin-eosin, and examined microscopically. Bulbar conjunctivas were evaluated for abnormalities by gross inspection, and pathological examination was performed as needed. Formation of a viable VX2 tumor was defined pathologically as follows: infiltration of lymphocytes beneath the conjunctival epithelium at the site of inoculation and surrounding mass of tumor cells visualized as clear images. This mass was considered to be a viable tumor in the subconjunctival space.

3.1.

Subconjunctival Tumor Formation Under Slit Lamp Examination

Tumor cells were injected into the subconjunctival space of five rabbits. Of the treated rabbits, all five successfully formed tumors over an average period of 14 days prior to euthanization [Fig. 2(c) and 2(d)]. Likewise, of the five rabbits, small nodular lesions were first found in three rabbits (60%) at day 7 and in two rabbits by 14 days, respectively.

Fig. 2

(a) Photo of left conjunctiva after seven days, (b) photo of right conjunctiva after seven days, (c) photo of left conjunctiva after 14 days, (d) photo of right conjunctiva after 14 days, (e) OCT image of left conjunctiva after 14 days, and (f) OCT image of right conjunctiva after 14 days. OCT images were taken by Spectralis, Heidelberg Engineering.

3.2.

OCT Examination

Of the five rabbits used in this study, five (100%) revealed a small low scattering area of the subconjunctiva at day 7, resulting in a mass in the next 14 days [Fig. 2(e)]. The average size of tumor at the seventh day was $100\pm 50\mu \text{m}$. After that the measurement of tumor size was impossible because OCT had limited imaging depth [Fig. 2(e) and 2(f)].

3.3.

Ultrasonographic Examination

All rabbits showed well-marginated hyperechoic nodules of the subconjunctiva by US, which were confirmed by pathology. The average size of tumors at day 14 was $5\pm 3\mathrm{mm}$ [Fig. 3(a)].

Fig. 3

(a) High frequency ultrasound (US) image (20 MHz, 1 cm of image depth), (b) a pathology of conjunctival tumor, and (c) enlarged pathologic image.

3.4.

Pathologic Finding

Pathologic examination of VX2 tumors in subconjunctiva revealed characteristics typical of carcinomas. The tumor characteristics ranged from small clusters of malignant cells infiltrating the conjunctival epithelium and underlying sclera to macroscopically visible nodules as large as 5 mm. Histologically, tumors consisted of epithelioid cells arranged in sheets, solid nests, and glandular patterns. All cells had a high nuclear:cytoplasmic ratio with an eosinophilic cytoplasm. The nuclei had small to inconspicuous nucleoli, and were very mitotically active with mitotic counts as high as 5 to 6 per high-power field [Fig. 3(b) and 3(c)].