Evaluating corneal thickness is important in both clinical and research settings. There are a variety of methods to measure corneal thickness in vivo, such as high-resolution ultrasound biomicroscopy and confocal microscopy.1,2 Inadvertent damage to the cornea and imprecise measurements can occur with these modalities because they both require corneal contact.3 In contrast to these methods, optical coherence tomography can be used. This noncontact imaging modality collects high-resolution, cross-sectional images of the cornea by measuring optical reflections.4 The basis of this modality is interferometry, which generates an interference pattern from 2 beams of light, which in turn creates an axial scan. Thousands of these axial scans are collected transversely, which generate a cross-sectional image of the tissue being measured.5 To obtain images of the tissues, SD-OCT devices use a superluminescent diode, a spectrometer, and the Fourier transform algorithm to achieve rapid, high-resolution scans.6,7
The use of SD-OCT devices has been validated as reliable and repeatable for measuring corneal thickness in humans,8–11 dogs,12,13 cats,14 and horses.15,16 Although such devices are becoming more prevalent in veterinary medicine, normative data for corneal thickness measurements by use of this modality are lacking, particularly for farm animal species. The purpose of the study reported here was to measure corneal thickness (specific measurements of the CET, CST, DMT, and TCT) of healthy goats, sheep, and alpacas by use of a portable SD-OCT device. Intraobserver reliability of this device and associated software was evaluated for each corneal layer and for the TCT.
Materials and Methods
Animals
Healthy adult goats and sheep owned by Tufts University and used for research purposes as well as healthy client-owned alpacas were included in the study. There were 11 female Alpine-Saanen crossbred goats (mean ± SD age, 6.4 ± 2.1 years), 10 female Dorset sheep (mean age, 3.0 ± 2.1 years), and 11 Huacaya alpacas (4 males and 7 females; mean age, 6.5 ± 3.6 years). A complete ocular examination, including detailed examination of the cornea, was performed on all animals by a resident in a veterinary ophthalmology training program (AJL). Ocular examination included fluorescein staining,a slit-lamp biomicroscopy,b rebound tonometry,c and indirect ophthalmoscopy.d All animals were deemed free of ocular disease.
Consent was obtained from alpaca owners prior to inclusion of their animals in the study. The study was approved by the Cummings School of Veterinary Medicine at Tufts University Institutional Animal Care and Use Committee as well as the Clinical Studies Review Committee. All protocols conformed to the Association for Research in Vision and Ophthalmology's statement for use of animals in vision research.
Experimental procedures
Each animal was sedated, and gentle manual restraint was used to ensure proper positioning of the head and globe. The sedation protocol differed among species. Goats received tiletaminezolazepame (3.5 mg/kg, IV), whereas sheep received ketamine hydrochloridef (5.5 mg/kg, IV) and midazolamg (0.3 mg/kg, IV) and alpacas received a combination of ketamine (4 mg/kg), xylazine hydrochlorideh (0.4 mg/kg), and butorphanol tartratei (0.04 mg/kg, as needed) IM. Globes were regularly irrigated with approximately 5 mL of eyewashj applied by use of a 5-mL syringe and 25-gauge cannula every 30 to 60 seconds during data collection.
One investigator (CGP) performed corneal pachymetry on both eyes of each animal in triplicate to obtain corneal thickness measurements. A 6-mm corneal pachymetry protocol was used, which involved a portable SD-OCT devicek and supplemental cornea-anterior module attachment. The aiming circle was aligned at the center of the pupil for each scan. The TCT was automatically measured from eight 6-mm-long radial scans (1,024 axial scans/radial scan). The value was recorded from the center circle (1-mm radius) of the pachymetry display. Built-in software of the SD-OCT machine had an internal quality control score, and only scans with a score ≥ 27 and free of motion artifact were used for data analysis.
Corneal measurements were obtained manually by 1 investigator (AJL) using the integrated caliper function of the SD-OCT software. The CET, CST, DMT, and TCT were measured twice on each image (Figure 1); measurements were obtained > 24 hours apart, and a mean value was calculated. In addition, TCT values were obtained via built-in OCT software, which automatically measured TCT on the images acquired.
Representative SD-OCT image of the cornea of a healthy alpaca. Notice the demarcation between the epithelium, stroma, and Descemet membrane-endothelium. Caliper measurements were obtained for the CET (A), CST (B), DMT (C), and TCT (D).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Representative SD-OCT image of the cornea of a healthy alpaca. Notice the demarcation between the epithelium, stroma, and Descemet membrane-endothelium. Caliper measurements were obtained for the CET (A), CST (B), DMT (C), and TCT (D).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Representative SD-OCT image of the cornea of a healthy alpaca. Notice the demarcation between the epithelium, stroma, and Descemet membrane-endothelium. Caliper measurements were obtained for the CET (A), CST (B), DMT (C), and TCT (D).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Manual measurements of CET, CST, DMT, and TCT; automatic measurement of TCT; patient signalment; and the eye examined were entered into a spreadsheet,l and statistical analysis was performed by use of commercial software.m To test repeatability of manual measurements of corneal layer thickness between 2 time points > 24 hours apart, the ICC and CV were calculated. As ICC approaches 1.00, repeatability approaches exactness, whereas a lower CV is interpreted as better repeatability.17
Results
Goats
Images of the eyes of the goats were obtained (Figure 2). Mean ± SD value for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 96.1 ± 5.0 μ, 486.0 ± 10.3 μm, 36.8 ± 4.8 μm, 616.9 ± 7.1 μm, and 611.5 ± 6.5 μm, respectively. To compare intraoperator reliability of the portable SD-OCT device and the integrated caliper function between 2 time points, the ICC was determined for each layer of the cornea. The ICC for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 0.49, 0.65, 0.26, 0.68, and 0.75, respectively (Table 1). The CV for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 12.42, 7.63, 20.94, 5.10, and 5.28, respectively.
Representative SD-OCT images of the cornea of a healthy goat (A), sheep (B), and alpaca (C).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Representative SD-OCT images of the cornea of a healthy goat (A), sheep (B), and alpaca (C).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Representative SD-OCT images of the cornea of a healthy goat (A), sheep (B), and alpaca (C).
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.80
Mean ± SD thickness,* ICC, and CV between repeated measurements of each layer of the cornea of eyes of goats, sheep, and alpacas determined with a portable SD-OCT device.
| Species | Variable | Thickness (μm) | ICC | CV (%) |
|---|---|---|---|---|
| Goat (n = 11 [22 eyes]) | CET | 96.1 ± 5.0 | 0.49 | 12.42 |
| CST | 486.0 ± 10.3 | 0.65 | 7.63 | |
| DMT | 36.8 ± 4.8 | 0.26 | 20.94 | |
| TCT | ||||
| Manual | 616.9 ± 7.1 | 0.68 | 5.10 | |
| Automatic | 611.5 ± 6.5 | 0.75 | 5.28 | |
| Sheep (n = 10 [20 eyes]) | CET | 111.6 ± 5.7 | 0.57 | 9.09 |
| CST | 599.8 ± 10.0 | 0.73 | 5.52 | |
| DMT | 31.0 ± 4.5 | 0.13 | 20.98 | |
| TCT | ||||
| Manual | 741.1 ± 9.9 | 0.83 | 5.43 | |
| Automatic | 727.8 ± 6.5 | 0.86 | 4.46 | |
| Alpaca (n = 11 [22 eyes]) | CET | 147.4 ± 5.7 | 0.51 | 7.73 |
| CST | 446.1 ± 7.4 | 0.62 | 5.79 | |
| DMT | 44.5 ± 5.0 | 0.36 | 23.37 | |
| TCT | ||||
| Manual | 634.8 ± 6.2 | 0.54 | 4.72 | |
| Automatic | 631.3 ± 3.8 | 0.58 | 4.67 |
Thickness of each layer and the TCT was determined manually by use of the integrated caliper function; in addition, the TCT was calculated automatically by the use of built-in software.
Sheep
Images of the eyes of the sheep were obtained (Figure 2). Mean ± SD value for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 111.6 ± 5.7 μm, 599.8 ± 10.0 μm, 31.0 ± 4.5 μm, 741.1 ± 9.9 μm, and 727.8 ± 6.5 μm, respectively. The ICC for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 0.57, 0.73, 0.13, 0.83, and 0.86, respectively (Table 1). The CV for manually measured CET, CST, DMT, and TCT and automatically measured TCT was 9.09, 5.52, 20.98, 5.43, and 4.46, respectively.
Alpacas
Images of the eyes of the alpacas were obtained (Figure 2). Mean ± SD value for the manually measured CET, CST, DMT, and TCT and automatically measured TCT was 147.4 ± 5.7 μm, 446.1 ± 7.4 μm, 44.5 ± 5.0 μm, 634.8 ± 6.2 μm, and 631.3 ± 3.8 μm, respectively. The ICC for the manually measured CET, CST, DMT, and TCT and automatically measured TCT was 0.51, 0.62, 0.36, 0.54, and 0.58, respectively (Table 1). The CV for the manually measured CET, CST, DMT, and TCT and automatically measured TCT was 7.73, 5.79, 23.37, 4.72, and 4.67, respectively.
Discussion
The present study provided corneal thickness values of the CET, CST, DMT, and TCT for eyes of healthy sedated goats, sheep, and alpacas measured by use of a portable SD-OCT device. On the basis of the ICC calculated for each layer of the cornea, the portable SD-OCT device (and the integrated caliper function in the software of that device) provided manual measurements of corneal thickness for the CET, CST, and TCT with clinically acceptable intraoperator reliability for eyes of healthy goats, sheep, and alpacas (Table 1). However, the ICC for DMT (goat, 0.26; sheep, 0.13; and alpaca, 0.36) was below the accepted threshold for good reliability (ICC range, 0.40 to 0.75). This discrepancy was believed to be the result of 3 factors: minute variations between measurements, the relatively large fixed incremental change created by use of the integrated caliper function, and the comparatively small thickness of the Descemet membrane. For the caliper function, variation of a single unit between measurements equated to a 4-μm difference, which represented variation of 9% to 12% for DMT. A more refined caliper function with smaller incremental changes would likely improve reliability between measurements of the DMT.
Mean ± SD TCT of the eyes of healthy sheep in the present study was 741.1 ± 9.9 μm. Mean age of the sheep was 3 years. This result differs slightly from results in previous reports on corneal thickness in sheep. Investigators of 1 study18 found that the TCT (as measured with confocal microscopy) of a female 2-year-old Pomeranian Coarsewool sheep was 850 μm. In another study,19 investigators analyzed eyes of 48 healthy lambs and adult sheep of 3 breeds (Ripollesa, Manchega, and Rasa Aragonesa); the eyes were harvested at a slaughterhouse, and TCT was measured by use of a digital pachymeter of a noncontact specular microscope in automatic mode. Overall, mean TCT obtained in that study19 was 768.8 ± 72 μm; however, when stratified into 2 age groups, the lambs (3 to 6 months old) had a mean TCT of 699 ± 56 μm, whereas the adult sheep (2 to 5 years old) had a larger mean TCT of 804 ± 87 μm, which indicated a significant effect of age on the TCT in sheep. These aforementioned studies yielded results below the range (TCT, 800 to 2,000 μm) previously reported in another study.20 Unfortunately, the details for that study20 are lacking, with no mention of sample size, age of the animals, or breed; thus, it is difficult to make comparisons. Differences in corneal thickness among these reports likely reflect differences in imaging modalities, the study population of sheep (age and breed), and corneal thickness between live versus dead tissue.
Mean ± SD TCT of eyes of healthy alpacas in the present study was 634.8 ± 6.2 μm. Mean age of the alpacas was 6.5 years. This value is slightly higher than the TCT of 595 μm determined for alpacas by use of ultrasonographic pachymetry.21 In that study,21 increases in age were correlated with increases in corneal thickness. The median age of alpacas in that study was 4 years old, whereas the median age of alpacas in the study reported here was 5 years old. The older alpacas in our study population could have been the reason for the discrepancy. Differences in modality could also have caused differences from values reported in the veterinary literature. In the aforementioned study,21 ultrasonographic pachymetry was used to measure corneal thickness. The speed of sound through the alpaca cornea was estimated; because corneal thickness is determined by velocity and time, estimating the speed of sound may affect the TCT. Although SD-OCT-based systems share the same principles as ultrasonographic pachymetry, differences in the manner in which sound waves move through tissues, compared with the movement for light waves, would likely contribute to differences in TCT measured in the present study and those measured by use of ultrasonographic pachymetry. Direct comparisons between results for SD-OCT and ultrasonographic pachymetry systems are needed to evaluate these differences.
A limitation of the present study was the small number of animals for each species. It would be expected that the ICC will increase if the population of animals examined increases. Another limitation of the study was the use of only female goats and sheep, which was the population available and could have resulted in skewed results. Despite the small numbers of animals and uneven sex representation, there was evidence that manual measurements of corneal thickness by use of SD-OCT were consistent within a single evaluator for most layers of the cornea and the TCT.
In the study reported here, data for the CET, CST, DMT, and TCT of the eyes of healthy goats, sheep, and alpacas were obtained by use of a portable SD-OCT device. Optical coherence tomography provides cross-sectional detail and analysis of the cornea and its specific layers in vivo. This modality can be used in the future as a tool for corneal surgical planning and evaluating diurnal and individual variations in corneal thickness. It can also be used to increase information about inherited and acquired disease processes. Although this device failed to provide good intraoperator reliability for DMT, it did provide clinically acceptable intraoperator reliability for CET, CST, and TCT in these species.
Acknowledgments
Supported in part by the Cummings School of Veterinary Medicine at Tufts University Companion Animal Fund and by Optovue.
Presented in abstract form at the 46th Annual Conference of the American College of Veterinary Ophthalmologists, Coeur d'Alene, Idaho, October 2015.
The authors thank Dr. Bruce Barton for assistance with the statistical analysis, Kimberly Flink for technical assistance, and Scott Brundage for assistance with the animals.
ABBREVIATIONS
| CET | Corneal epithelial thickness |
| CST | Corneal stromal thickness |
| CV | Coefficient of variation |
| DMT | Descemet membrane thickness |
| ICC | Intraclass correlation coefficient |
| SD-OCT | Spectral-domain optical coherence tomography |
| TCT | Total corneal thickness |
Footnotes
Akorn Inc, Lake Forest, Ill.
Kowa SL-15 portable slit-lamp biomicroscope, Kowa Co Ltd, Tokyo, Japan.
TonoVet, Icare, Vantaa, Finland.
Welch Allyn binocular indirect ophthalmoscope, Welch Allyn Inc, Skaneateles Falls, NY.
Telazol, Zoetis Inc, Florham Park, NJ.
Putney Inc, Portland, Me.
West-Ward Pharmaceuticals Corp, Eatontown, NJ.
AnaSed LA, MWI Animal Health, Boise, Idaho.
Torbugesic, Pfizer Inc, New York, NY.
Major Pharmaceuticals, Livonia, Mich.
Optovue iVue SD-OCT, provided by Optovue Inc, Freemont, Calif.
Microsoft Excel 2010, Microsoft Corp, Redmond, Wash.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
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