• View in gallery

    Representative angiographic images of the anterior segment of the right eye of an 8-year-old female goat with a light-brown iris obtained after IV injection of a 10-mL bolus of ICG (end of injection = time 0). A—Filling of the major arterial circle of the iris and radial iris arteries (arterial phase) at 12 seconds after injection. Notice the major arterial circle of the iris (arrowhead), radial ciliary artery (long arrow), and radial iris artery (short arrow). B—Filling of terminal loops and initial filling of the radial iris veins (capillary phase) at 14 seconds after injection. C—Complete filling of the radial iris veins (venous phase) at 18 seconds after injection. D—Magnified view of the region in panel B containing the capillary terminal loops. Notice the bifurcation of the major arterial circle of the iris (arrowhead) prior to entry into the iris base and the broad terminal capillary loops (dashed arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

  • View in gallery

    A representative standard color image (A) and anterior segment angiographic images with ICG (B through D) and SF (E through G) of the left eye of a 7-year-old female goat with a light-brown iris. Angiographic images were obtained at 11 seconds (B and E [arterial phase]), 13 seconds (C and F [capillary phase]), and 18 seconds (D and G [venous phase]) after injection of dye. The radial iris arteries (arrowhead), broad capillaries (short arrow), and radial iris veins (long arrow) are evident. Notice the improved capacity for visual assessment of the iris vasculature in images obtained by use of ICG, compared with that for images obtained by use of SF. Bar = 5 mm in panel A and 2.5 mm in panels B through G.

  • View in gallery

    Representative images of the anterior segment of the left eye of a 6-year-old female sheep with a light-brown iris (A through C) and the right eye of a 3-year-old female sheep with a light-brown iris (D) obtained after injection of ICG. Notice filling of the major arterial circle of the iris deep within the iris stroma and the relatively straight radial iris arteries (arterial phase) at 16 seconds after injection (A), initial filling of the radial iris veins (capillary phase) at 18 seconds after injection (B), and progressive filling of the radial iris veins (venous phase) at 22 seconds after injection (C). Notice the major arterial circle of the iris (arrowhead), radial iris artery (short arrow), and radial iris veins (long arrow). Panel D represents a magnified view of the anterior segment, with the accordion-like (zig-zag) pattern of the complete major arterial circle of the iris deep within the iris stroma (arrowhead) and readily apparent perfusion of the dorsal corpora nigra (dashed arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

  • View in gallery

    A representative standard color image (A) and anterior segment angiographic images with ICG (B through D) and SF (E through G) of the left eye of a 2-year-old female sheep with a heterochromic iris. Angiographic mages were obtained at 14 seconds (B and E [arterial phase]), 18 seconds (C and F [capillary phase]), and 22 seconds (D and G [venous phase]) after injection of dye. The radial iris arteries (arrowhead) and radial iris veins (arrow) are evident. Notice the improved capacity for visual assessment of the iris vasculature and decreased amount of pigment masking in images obtained by use of ICG, compared with results for images obtained by use of SF. Bar = 5 mm in panel A and 2.5 mm in panels E through G.

  • View in gallery

    Representative angiographic images of the anterior segment of the left eye of a 9-year-old female alpaca with a brown iris (A through C) and the left eye of a 3-year-old female alpaca with a brown iris (D) obtained after IV injection of a 10-mL bolus of ICG. A—Initial filling of the major arterial circle of the iris and radial ciliary arteries (arterial phase) at 29 seconds after injection. Notice the major arterial circle of the iris (arrowhead) and the radial ciliary arteries (arrows). B—Progressive filling of the radial iris arteries and capillary terminal loops (capillary phase) at 31 seconds after injection. C—Filling of the radial iris veins (venous phase) at 45 seconds after injection. D—Magnified view of the anterior segment. Notice the bifurcation of the major arterial circle of the iris prior to entry within the iris base (asterisks) and the unique branching patterns of the radial ciliary arteries (arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

  • View in gallery

    A representative standard color image (A) and anterior segment angiographic images with ICG (B) and SF (C) of the right eye of a 3-year-old female alpaca with a brown iris. Angiographic images were obtained 1 minute after injection of dye. Notice the inability to visually assess the iris vasculature in the angiographic images obtained with SF. There is extravasation of SF into the iris stroma and aqueous humor (asterisk). Bar = 5 mm.

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Comparison of indocyanine green and sodium fluorescein for anterior segment angiography of ophthalmically normal eyes of goats, sheep, and alpacas performed with a digital single-lens reflex camera adaptor

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  • 1 Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA 01536.
  • | 2 Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA 01536.
  • | 3 Biomedical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA 01536.
  • | 4 Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA 01536.

Abstract

OBJECTIVE To compare results of anterior segment angiography of ophthalmically normal eyes of goats, sheep, and alpacas performed by use of indocyanine green (ICG) and sodium fluorescein (SF).

ANIMALS 10 female goats (mean ± SD age, 6.8 ± 1.7 years), 10 female sheep (3.0 ± 2.2 years), and 10 alpacas (7 females and 3 males; 6.8 ± 3.8 years).

PROCEDURES A catheter was aseptically placed into a jugular vein. Each animal was anesthetized and properly positioned, and 0.25% ICG was administered. Images were obtained by use of an adaptor system consisting of a modified digital single-lens reflex camera, camera adaptor, and camera lens. Images were obtained at a rate of 3 images/s for the 60 seconds immediately after ICG administration and then at 2, 3, 4, and 5 minutes after administration. Ten minutes later, 10% SF was administered IV and images were obtained in a similar manner.

RESULTS Angiography with ICG provided visual examination of the arterial, capillary, and venous phases in all species. Visual examination of the iris vasculature by use of SF was performed in goats and sheep but was not possible in the alpacas because of iridal pigmentation. Extravasation of SF was a common finding in sheep and alpacas but not in goats. No adverse events were detected.

CONCLUSIONS AND CLINICAL RELEVANCE Quality angiographic images of the anterior segment were obtainable after IV administration of ICG to goats, sheep, and alpacas. This may provide a useful imaging modality for ocular research in these animal species.

Abstract

OBJECTIVE To compare results of anterior segment angiography of ophthalmically normal eyes of goats, sheep, and alpacas performed by use of indocyanine green (ICG) and sodium fluorescein (SF).

ANIMALS 10 female goats (mean ± SD age, 6.8 ± 1.7 years), 10 female sheep (3.0 ± 2.2 years), and 10 alpacas (7 females and 3 males; 6.8 ± 3.8 years).

PROCEDURES A catheter was aseptically placed into a jugular vein. Each animal was anesthetized and properly positioned, and 0.25% ICG was administered. Images were obtained by use of an adaptor system consisting of a modified digital single-lens reflex camera, camera adaptor, and camera lens. Images were obtained at a rate of 3 images/s for the 60 seconds immediately after ICG administration and then at 2, 3, 4, and 5 minutes after administration. Ten minutes later, 10% SF was administered IV and images were obtained in a similar manner.

RESULTS Angiography with ICG provided visual examination of the arterial, capillary, and venous phases in all species. Visual examination of the iris vasculature by use of SF was performed in goats and sheep but was not possible in the alpacas because of iridal pigmentation. Extravasation of SF was a common finding in sheep and alpacas but not in goats. No adverse events were detected.

CONCLUSIONS AND CLINICAL RELEVANCE Quality angiographic images of the anterior segment were obtainable after IV administration of ICG to goats, sheep, and alpacas. This may provide a useful imaging modality for ocular research in these animal species.

The dyes ICG and SF are commonly used in human ophthalmology for angiographic purposes. Largely because of equipment costs, angiography with ICG and SF is not performed routinely in veterinary medicine.

Sodium fluorescein has been used for angiography in veterinary medicine primarily as a means to examine the vascular supply of the eyes of dogs, specifically the retinal vasculature.1–6 Similarly, its use in large animal species has been limited to the posterior segment.7,8 Although SF may be used for angiography of the anterior segment to assess vasculature of the iris, its diagnostic use may be limited because of the degree of pigmentation and vessel depth within the iris stroma.1,9–12 Peak absorbance for SF is approximately 485 nm, and peak emission is approximately 525 nm. Pigmented structures of the eyes (eg, melanin within the iris stroma) absorb and scatter light at these wavelengths. This causes deeper vessels to be obscured and thus prevents their examination. In addition, only 60% to 80% of SF is protein bound. Unbound SF can leak through gap junctions within normal blood vessels, which results in diffuse fluorescence that can obscure visual examination of the iris vasculature.1,9

Indocyanine green is an alternative angiographic dye that differs from SF in that its peak absorbance is at 805 nm and peak fluorescence (emission) is at 835 nm.1,9,11 These longer wavelengths can readily penetrate pigmented structures of the iris stroma and are less susceptible to scattering. Indocyanine green also is 98% protein bound after administration, which greatly reduces extravasation.1,12,13 Additionally, ICG has a relatively short half-life (3 to 4 minutes in humans,13 7 minutes in horses,14 and 9 minutes in sheep15). Limited extravasation, plus its short half-life, allows angiography with ICG to be repeated in a short time frame.1

Although the use of ICG for angiography in veterinary medicine has been somewhat limited, it has been performed in various species, including dogs,12,16,17 cats,18,19 and rhesus monkeys.20 To our knowledge, there are no studies that have been conducted on anterior segment angiography by use of SF or ICG in any large animal species. As a result, there are no standard protocols or reference parameters for conducting anterior segment angiography in large animal species. The purpose of the study reported here was to establish a standard technique for performing angiography with ICG and SF of the anterior segment in ophthalmically normal eyes of goats, sheep, and alpacas. Furthermore, we sought to compare the potential use of these angiographic dyes in these species.

Materials and Methods

Animals

Healthy adult goats and sheep owned by Tufts University and used for research purposes and healthy client-owned alpacas were evaluated for the study. There were 10 female Alpine-Saanen-cross goats (mean ± SD age, 6.8 ± 1.7 years; mean body weight, 76.6 ± 12.4 kg), 10 female Dorset sheep (mean age, 3.0 ± 2.2 years; mean body weight, 129.8 ± 58.1 kg), and 10 (7 females and 3 males) Huacaya alpacas (mean age, 6.8 ± 3.8 years; mean body weight, 76.3 ± 15.1 kg). Each animal was deemed to be free of ocular disease on the basis of results of a complete ocular examination performed on each animal 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 Consent was obtained from the owners of the alpacas prior to inclusion in the study. The study was approved by the Cummings School of Veterinary Medicine at Tufts University Institutional Animal Care and Use Committee and Clinical Studies Review Committee. All protocols conformed to the Association for Research in Vision and Ophthalmology statement for use of animals in vision research.

Experimental procedures

Each animal was anesthetized once for data collection. Animals were manually restrained, and a jugular vein was cannulated with a 14-gauge catheter by use of a sterile technique. The anesthesia protocol differed among species. Goats received tiletaminezolazepame (3.5 mg/kg, IV), whereas sheep received ketamine hydrochloridef (5.5 m/kg, IV) and midazolamg (0.3 mg/kg, IV). Alpacas received an IM injection of a combination of ketamine (4 mg/kg) and xylazine hydrochlorideh (0.4 mg/kg) as well as butorphanol tartratei (0.04 mg/kg, as needed). Animals were placed to ensure proper positioning of the head and eyes. Heart rate and respiratory rate were measured for each animal before anesthesia, at regular intervals during anesthesia, and after anesthesia.

A Castroviejo lid speculum was used to aid in retraction of the eyelids, and globes were irrigated with approximately 1 mL of eyewashj every 30 to 60 seconds by use of a 20-mL syringe and 25-gauge cannula.

Angiography

Angiography was initiated within 10 minutes after anesthesia was induced. Imaging of the right or left globe of each animal was chosen arbitrarily. Angiographic images were obtained by use of a dSLR camera imaging system. The system consisted of a modified (full spectrum) dSLR camera,k dSLR camera adaptor, and camera lens,l as described elsewhere.21 Exposure was provided by use of 2 solitary light-emitting diodes that were housed within the adaptor, with the nominal wavelength approximating the peak absorption of the angiographic dyes used (780 nm for ICG and 475 nm for SF).

A 10-mL volume 0.25% ICGm was administered rapidly through the catheter as an IV bolus. Immediately after bolus administration was completed (time 0), images were obtained at a rate of 3 images/s for 1 minute; images also were obtained at 2, 3, 4, and 5 minutes after dye injection. Ten minutes later, a 10-mL volume of 10% SFn was administered rapidly as an IV bolus, and images were obtained in a similar manner.

For angiography with ICG, images were obtained before dye injection with the appropriate excitationo and barrierp filters inserted within the illumination and optical pathways of the adaptor, respectively, to determine the degree of background autofluorescence or pseudofluorescence (if any). Camera settings included a shutter speed of 0.01 seconds, effective aperture of f/8, and sensitivity setting (International Standards Organization) of 6,400, as described elsewhere.16

For angiography with SF, images were obtained before dye injection with the appropriate excitationq and barrierr filters inserted within the illumination and optical pathways of the adaptor, respectively, to determine the degree of background autofluorescence or pseudofluorescence (if any). Camera settings included a shutter speed of 0.033 seconds, effective aperture of f/8, and sensitivity setting (International Standards Organization) of 800, as described elsewhere.22

Angiographic measurements obtained included the time to onset of the arterial, capillary, and venous phases as well as arterial and capillary phase intervals, as described elsewhere.23 Briefly, time to onset of the arterial, capillary, and venous phases was identified by the initial filling of dye within the major arterial circle of the iris, pupillary capillaries, and iridal veins, respectively. Phase intervals were defined as the time from the onset of one phase to the onset of the next phase. All time measurements were performed by use of the images after completion of the study; 1 investigator (CGP) determined time measurements (in duplicate), and the mean value was calculated. All images were converted to black and white by use of a black-and-white adjustment tool and graphic image editing software.s Angiographic techniques were subjectively compared to determine their ability to allow visual examination of the iris vasculature and to determine recognizable leakage of dye from the iris vasculature or within the anterior chamber (or both).

Statistical analysis

Time to onset of the arterial, capillary, and venous phases for each angiographic dye were compared via a Wilcoxon signed rank test by use of commercial statistical software.t Data were reported as mean ± SD. Values of P < 0.05 were considered significant.

Results

Animals

All animals in the study recovered uneventfully after anesthesia. There were no adverse reactions (ie, nystagmus) associated with administration of the anesthetic drugs or either angiographic dye.

Goats

The right eye was imaged in 5 goats, and the left eye was imaged in the other 5 goats. Iris pigmentation of the eyes ranged from moderately pigmented (light brown [n = 8]) to heavily pigmented (dark brown [2]).

Anterior segment angiography with ICG allowed clear visual examination of the iris vasculature and its hemodynamics (arterial, capillary, and venous phases) in all 10 goat eyes, regardless of iridal pigmentation (Figure 1). However, angiography with SF allowed clear assessment of only 9 of 10 eyes. The 1 eye that did not allow clear visual assessment was heavily pigmented (dark brown). Significant differences were detected in the time to onset of the arterial, capillary, and venous phases between the 2 angiographic dyes (Table 1). Mean ± SD time to onset of the arterial phase after injection of ICG and SF was 6.1 ± 1.6 seconds and 7.4 ± 1.7 seconds, respectively, with intervals of 3.4 ± 1.3 seconds and 3.2 ± 1.3 seconds, respectively. Fluorescence of the major arterial circle of the iris was rapid and uniform. The major arterial circle of the iris appeared to be located deep within the iris stroma and formed a complete arterial circle. The major arterial circle of the iris branched into superior and inferior components prior to entry within the iris base. Fluorescence of the radial ciliary arteries and radial iris arteries was evident soon thereafter. The radial ciliary arteries were large-caliber vessels that coursed directly toward the iris base as they branched off the outer aspect of the major arterial circle of the iris. The radial iris arteries branched from the inner aspect of the major arterial circle of the iris and had a tortuous course centripetally toward the pupillary edge. Fluorescence of the radial iris arteries was uniform. Prominent arterial networks that supplied the regions of the dorsal and ventral corpora nigra were seen; however, no vasculature was observed specifically within this region.

Figure 1—
Figure 1—

Representative angiographic images of the anterior segment of the right eye of an 8-year-old female goat with a light-brown iris obtained after IV injection of a 10-mL bolus of ICG (end of injection = time 0). A—Filling of the major arterial circle of the iris and radial iris arteries (arterial phase) at 12 seconds after injection. Notice the major arterial circle of the iris (arrowhead), radial ciliary artery (long arrow), and radial iris artery (short arrow). B—Filling of terminal loops and initial filling of the radial iris veins (capillary phase) at 14 seconds after injection. C—Complete filling of the radial iris veins (venous phase) at 18 seconds after injection. D—Magnified view of the region in panel B containing the capillary terminal loops. Notice the bifurcation of the major arterial circle of the iris (arrowhead) prior to entry into the iris base and the broad terminal capillary loops (dashed arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Table 1—

Mean ± SD time (seconds) to onset of the arterial, capillary, and venous phases and arterial and capillary phase intervals after IV injection of ICG and SF for anterior segment angiography of eyes of 10 goats.*

 ICGSF
PhaseOnsetIntervalOnsetInterval
Arterial6.1 ± 1.6a3.4 ± 1.37.4 ± 1.7b3.2 ± 1.3
Capillary9.5 ± 2.5a1.7 ± 0.510.6 ± 2.6b1.5 ± 0.4
Venous11.2 ± 3.0a12.1 ± 2.7b

Represents results for 5 right eyes and 5 left eyes.

Within a row, values with different superscript letters differ significantly (P < 0.05).

— = Not applicable.

The capillary phase began 9.5 ± 2.5 seconds and 10.6 ± 2.6 seconds after injection of ICG and SF, respectively, with a capillary phase interval of 1.7 ± 0.5 seconds and 1.5 ± 0.4 seconds, respectively. Capillaries formed abrupt small to large terminal loops at the pupillary edge (Figure 1).

Time to onset of the venous phase was 11.2 ± 3.0 seconds and 12.1 ± 2.7 seconds after injection of ICG and SF, respectively. Centrifugal movement of dye within tortuous radial veins was evident; many of these veins crossed atop adjacent radial iris arteries as they approached the iris base.

Subjective comparisons of angiographic techniques revealed that use of ICG provided greater vascular detail and image contrast, compared with results after use of SF (Figure 2). Additionally, SF allowed visual examination of the iris vasculature in only 9 of 10 eyes. No evidence of extravasation of ICG or SF was observed within the iris stroma or aqueous humor.

Figure 2—
Figure 2—

A representative standard color image (A) and anterior segment angiographic images with ICG (B through D) and SF (E through G) of the left eye of a 7-year-old female goat with a light-brown iris. Angiographic images were obtained at 11 seconds (B and E [arterial phase]), 13 seconds (C and F [capillary phase]), and 18 seconds (D and G [venous phase]) after injection of dye. The radial iris arteries (arrowhead), broad capillaries (short arrow), and radial iris veins (long arrow) are evident. Notice the improved capacity for visual assessment of the iris vasculature in images obtained by use of ICG, compared with that for images obtained by use of SF. Bar = 5 mm in panel A and 2.5 mm in panels B through G.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Sheep

The right eye was imaged in 5 sheep, and the left eye was imaged in the other 5 sheep. Iris pigmentation varied and included 2 lightly pigmented (yellow), 3 moderately pigmented (light brown), 3 heavily pigmented (dark brown), and 2 variably pigmented (heterochromia) eyes. Anterior segment angiography with ICG allowed clear visual examination of the iris vasculature in all 10 sheep eyes (Figure 3). However, angiography with SF allowed clear assessment of the iris vasculature in only 8 eyes. The 2 eyes that did not allow clear visual assessment were heavily pigmented (dark brown). Significant differences were detected in the time to onset of the arterial, capillary, and venous phases between the 2 angiographic dyes (Table 2).

Table 2—

Mean ± SD time (seconds) to onset of the arterial, capillary, and venous phases and arterial and capillary phase intervals after IV injection of ICG and SF for anterior segment angiography of eyes of 10 sheep.*

 ICGSF
PhaseOnsetIntervalOnsetInterval
Arterial13.1 ± 2.3a2.6 ± 0.814.9 ± 2.2b2.8 ± 0.3
Capillary15.6 ± 2.6a1.2 ± 0.317.7 ± 2.1b1.3 ± 0.3
Venous16.8 ± 2.7a18.9 ± 2.3b

Represents results for 5 right eyes and 5 left eyes.

See Table 1 for remainder of key.

The arterial phase began 13.1 ± 2.3 seconds and 14.9 ± 2.2 seconds after injection of ICG and SF, respectively, and persisted for 2.6 ± 0.8 seconds and 2.8 ± 0.3 seconds, respectively. Fluorescence of the major arterial circle of the iris was rapid, with filling of the radial ciliary arteries and radial iris arteries evident soon thereafter. The major arterial circle of the iris formed a complete arterial circle and had an accordion-like (ie, zig-zag) filling pattern deep within the stroma of the iris base (Figure 3). Radial ciliary arteries emanated off the tips of the major arterial circle of the iris close to the iris base on its outer surface. Radial iris arteries branched off the inner aspect of the major arterial circle of the iris and were relatively straight as they coursed toward the pupillary zone. In 4 eyes, vascularization within the upper and lower corpora nigra was clearly evident.

The capillary phase began 15.6 ± 2.6 seconds and 17.7 ± 2.1 seconds after injection of ICG and SF, respectively, with a capillary phase interval of 1.2 ± 0.3 seconds and 1.3 ± 0.3 seconds, respectively (Figure 3). The venous phase began 16.8 ± 2.7 seconds and 18.9 ± 2.3 seconds after injection of ICG and SF, respectively. Centrifugal movement of dye within radial veins toward the iris base was seen during the venous phase. Radial veins coursed in close proximity to adjacent radial iris arteries and subjectively appeared to be large-caliber vessels.

Figure 3—
Figure 3—

Representative images of the anterior segment of the left eye of a 6-year-old female sheep with a light-brown iris (A through C) and the right eye of a 3-year-old female sheep with a light-brown iris (D) obtained after injection of ICG. Notice filling of the major arterial circle of the iris deep within the iris stroma and the relatively straight radial iris arteries (arterial phase) at 16 seconds after injection (A), initial filling of the radial iris veins (capillary phase) at 18 seconds after injection (B), and progressive filling of the radial iris veins (venous phase) at 22 seconds after injection (C). Notice the major arterial circle of the iris (arrowhead), radial iris artery (short arrow), and radial iris veins (long arrow). Panel D represents a magnified view of the anterior segment, with the accordion-like (zig-zag) pattern of the complete major arterial circle of the iris deep within the iris stroma (arrowhead) and readily apparent perfusion of the dorsal corpora nigra (dashed arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Subjective comparisons indicated that use of ICG provided clear visual assessment of the iris blood vessels in all 10 sheep eyes, whereas use of SF provided clear visual assessment of the iridal vasculature and the angiographic phases in only 8 sheep eyes (Figure 4). No evidence of ICG extravasation was detected. Conversely, 3 eyes had extravasation of SF into the iris stroma, and all 10 eyes had leakage of SF into the aqueous humor.

Figure 4—
Figure 4—

A representative standard color image (A) and anterior segment angiographic images with ICG (B through D) and SF (E through G) of the left eye of a 2-year-old female sheep with a heterochromic iris. Angiographic mages were obtained at 14 seconds (B and E [arterial phase]), 18 seconds (C and F [capillary phase]), and 22 seconds (D and G [venous phase]) after injection of dye. The radial iris arteries (arrowhead) and radial iris veins (arrow) are evident. Notice the improved capacity for visual assessment of the iris vasculature and decreased amount of pigment masking in images obtained by use of ICG, compared with results for images obtained by use of SF. Bar = 5 mm in panel A and 2.5 mm in panels E through G.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Alpacas

The right eye was imaged in 4 alpacas, and the left eye was imaged in the other 6 alpacas. All eyes were heavily pigmented (dark brown). Anterior segment angiography with ICG allowed visual examination of the iris vasculature in all alpaca eyes (Figure 5). However, angiography with SF did not allow clear visual assessment of the iris vasculature in all 10 eyes. Only fluorescence within the pupillary opening, which was used to approximate the onset of the arterial phase, and dye extravasation were observed. A significant difference was detected in time to onset of the arterial phase between the 2 angiographic dyes (Table 3).

Figure 5—
Figure 5—

Representative angiographic images of the anterior segment of the left eye of a 9-year-old female alpaca with a brown iris (A through C) and the left eye of a 3-year-old female alpaca with a brown iris (D) obtained after IV injection of a 10-mL bolus of ICG. A—Initial filling of the major arterial circle of the iris and radial ciliary arteries (arterial phase) at 29 seconds after injection. Notice the major arterial circle of the iris (arrowhead) and the radial ciliary arteries (arrows). B—Progressive filling of the radial iris arteries and capillary terminal loops (capillary phase) at 31 seconds after injection. C—Filling of the radial iris veins (venous phase) at 45 seconds after injection. D—Magnified view of the anterior segment. Notice the bifurcation of the major arterial circle of the iris prior to entry within the iris base (asterisks) and the unique branching patterns of the radial ciliary arteries (arrow). Bar = 5 mm in panels A through C and 2.5 mm in panel D.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Table 3—

Mean ± SD time (seconds) to onset of the arterial, capillary, and venous phases and arterial and capillary phase intervals after IV injection of ICG and SF for anterior segment angiography of eyes of 10 alpacas.*

 ICGSF
PhaseOnsetIntervalOnsetInterval
Arterial25.6 ± 4.6a3.8 ± 2.126.8 ± 4.5bND
Capillary29.4 ± 5.7l.4 ± 0.3NDND
Venous30.7 ± 5.9ND

Represents results for 4 right eyes and 6 left eyes.

ND = Not determined.

See Table 1 for remainder of key.

The arterial phase began 25.6 ± 4.6 seconds after injection of ICG and persisted for 3.8 ± 2.1 seconds. Fluorescence of the major arterial circle of the iris was rapid and revealed the presence of an incomplete arterial circle (Figure 5). Bifurcation into superior and inferior components occurred prior to entry within the iris base, and its location within the iris stroma appeared more superficial, compared with the location for the other ungulates evaluated in this study. Long radial ciliary arteries emanated from the outer aspect of the major arterial circle of the iris, and the radial ciliary arteries bifurcated numerous times as they coursed toward the iris base. Radial iris arteries had a branching pattern that differed among alpacas; they often had a straight course toward the pupillary zone. In many alpacas, complex vascular networks surrounding the corpora nigra were seen both dorsally and ventrally. No blood vessels were detected within the corpora nigra.

The capillary phase began 29.4 ± 5.7 seconds after injection of ICG and persisted for 1.4 ± 0.3 seconds (Figure 5). The venous phase was 30.7 ± 5.9 seconds after ICG injection; it highlighted centrifugal movement of dye within relatively straight iridal veins, many of which paralleled adjacent radial iris arteries. Extravasation of ICG was not detected in any alpaca eyes.

Anterior segment angiography with SF did not allow clear visual assessment of the iris vasculature in all 10 alpaca eyes (Figure 6). Fluorescence of SF was observed within the pupillary opening at a mean ± SD of 26.8 ± 4.5 seconds after SF injection. Leakage of SF within the iris stroma was observed in 3 eyes, whereas all 10 eyes had various degrees of SF leakage within the aqueous humor.

Figure 6—
Figure 6—

A representative standard color image (A) and anterior segment angiographic images with ICG (B) and SF (C) of the right eye of a 3-year-old female alpaca with a brown iris. Angiographic images were obtained 1 minute after injection of dye. Notice the inability to visually assess the iris vasculature in the angiographic images obtained with SF. There is extravasation of SF into the iris stroma and aqueous humor (asterisk). Bar = 5 mm.

Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.311

Discussion

Analysis of results of the study reported here revealed that quality images of the anterior segment of the eyes of goats, sheep, and alpacas were obtainable by use of angiography with ICG and SF and an adaptor imaging system. Use of this adaptor has been validated in previous studies,16,18,21–24 which has permitted investigators to use a standard dSLR camera to acquire high-resolution images of both the anterior and posterior segments of the globe and to conduct angiography with ICG and SF. The adaptor has been designed to address the cost-prohibitive issues of performing angiographic studies in veterinary medicine without sacrificing image quality. Additionally, the portability and light weight of the adaptor allow it to be easily handled and available for convenient use on animals in stalls and or in remote locations.

Compared with results for SF, ICG provided superior image detail and contrast in the large animal species of the present study, regardless of iris pigmentation. There was no difference in the vascular pattern observed between the 2 dyes. The amount of melanin present within the iris stroma likely hindered clear visual assessment of the iris vasculature by use of SF and resulted in poorer image contrast and detail. In heavily pigmented irises, there often was complete masking of SF fluorescence. In addition to the superior image quality, no extravasation of dye within the iris stroma or into the anterior chamber was observed after injection of ICG. In contrast, extravasation of SF within the iris stroma or aqueous humor (or both) was a common finding, particularly in sheep and alpacas. Anterior segment angiography with SF for dogs,16,22 cats,18,23 and humans25 has revealed similar findings, including reduced visibility of the iris vasculature within heavily pigmented eyes and dye leakage within the iris stroma or aqueous humor (or both).16,18,22,23,25 Interestingly, there was no extravasation of SF within the iris stroma or aqueous humor of goats in the present study. This observation presumably reflected differences at the cellular level (ie, intercellular junctions and spaces) of the iris vasculature within this species.

Superior image quality for angiography with ICG, compared with angiography with SF, is believed to reflect the unique metabolic and spectral properties of ICG. Indocyanine green has a relatively high molecular weight (775 Da), with both lipophilic and hydrophilic properties.26 By comparison, SF has a lower molecular weight and is primarily hydrophilic.27 The lipophilic and hydrophilic properties of ICG give the molecule its most important quality: protein-binding affinity.28 After IV injection, ICG is 98% protein bound, whereas SF is only 60% to 80% protein bound. The study reported here, as well as previous studies, revealed extravasation of SF within the iris stroma and aqueous humor to be a common finding, which ultimately hinders its potential diagnostic use. These observations are likely the product of increased proportions of unbound SF molecules in addition to the relatively small molecular size of SF.29

In addition to its molecular properties, ICG has advantageous spectral properties for use in anterior segment angiography, compared with results for use of SF. The excitation and emission spectra of SF are within the visible spectrum, whereas the excitation and emission spectra of ICG are within the near infrared spectrum. The longer wavelengths of the near infrared spectra (790 to 835 nm) allow for greater tissue penetration because only 10% of this light is absorbed by hemoglobin and melanin.30 This trait is especially useful for anterior segment angiography of pigmented irises, which comprised most of the eyes evaluated in the present study. Although excitation and emission within the near infrared spectrum is beneficial for visual assessment of the vasculature of pigmented irises, specific (and often expensive) equipment is required for proper use of ICG. The adaptor used in the study reported here provided the diagnostic utility of a more affordable alternative to expensive conventional hardware.

To our knowledge, the study reported here was the first in which the iris vasculature of these large animal species has been assessed in vivo. Studies involving ex vivo vascular corrosion casts have been performed for goats31 and sheep31,32 and have revealed vascular patterns similar to those described in the present study. Some notable characteristics of the iris vasculature include the relatively deep major arterial circle of the iris of sheep and goats, bifurcation of the major arterial circle of the iris into its superior and inferior components prior to entering the iris base of goats and alpacas, and large radial ciliary arteries observed in alpacas. A characteristic that was evident throughout was the prominent, often complex network of vessels associated with the corpora nigra in these ungulate species. Furthermore, the study reported here was the first in which vascularization within the corpora nigra of sheep species has been reported.

Comparing filling times among species revealed that goats had the earliest mean onset for visual assessment with both ICG and SF, whereas alpacas had the latest onset. These variations in time could represent anatomic and physiologic differences among the 3 species; however, they could also relate to differences in the anesthetics used. Heart rates of the animals differed considerably among and within species before, during, and after anesthesia, which likely was a result of variation in temperament. The wide reference range for heart rates of the species evaluated could lead to variations in filling times, and future studies may detect differences if other anesthetic regimens are used. Although differences in filling times may prove interesting, actual time values are of little use in human medicine. Instead, filling patterns, dye leakage, and the presence of filling defects are more clinically important in humans.25

The amount of dye used for each injection was standardized across species to allow development of a standard protocol independent of weight as well as to limit costs. Investigators of previous studies that performed anterior segment angiography on dogs16,22 and cats23 using the equipment described in the present study determined the dye dose on the basis of body weight (eg, 1 mg/kg for ICG and 20 mg/kg for SF). If similar doses had been used for the large animal species of the present study, it would have prohibitively increased the cost (from approx $150/animal to $600/animal). To increase the feasibility of these studies in large animal species, a standard protocol of 25 mg of ICG and 1,000 mg of SF was administered IV to each animal, regardless of body weight. On the basis of mean body weights, the mean doses of ICG and SF were 0.33 and 13 mg/kg for goats, respectively; 0.19 and 7 mg/kg for sheep, respectively; and 0.34 and 13 mg/kg for alpacas, respectively. The reduced doses, particularly for SF, could have potentially decreased the ability for investigators to perform visual assessment of the iris vasculature in more heavily pigmented eyes.

Limitations of the present study included the small sample size for each species as well as the fact that only female goats and sheep were evaluated. In addition, another potential limitation was that ICG was administered first, followed by SF, for each eye evaluated. This order was chosen because of the anticipated leakage of SF within the iris stroma and aqueous humor, which would have prevented visual assessment after injection of ICG if SF had been administered first. However, it is possible that administration of ICG before administration of SF might have altered the SF fluorescence and extravasation of SF that was detected. Another limitation of the study was the wide range of heart rates both within and among species. This variation was likely related to differences in temperament, responses to anesthetics, and differences in anesthetic regimens. These variables likely impacted cardiac output and heart rate, which thereby affected timing of the angiographic phases.

Although anterior segment angiography is widely used for the diagnosis and treatment of a variety of diseases in human medicine, its use in veterinary medicine has been limited because of the relatively high cost of equipment required to obtain quality images. The camera adaptor used in the present study, in addition to a modified dSLR camera, may provide a more affordable alternative. The lightweight nature of the adaptor would allow for portability and increased availability in clinical settings such as barns and stables. Availability of a sensitive and affordable diagnostic tool for the evaluation of the iris vasculature will allow earlier detection and treatment of various ophthalmic conditions. Furthermore, the data obtained by use of these angiographic techniques in the present study can be used to design future studies for the evaluation of vascular changes that occur in numerous vision-threatening diseases. Overall, anterior segment angiography with ICG appeared to be a useful imaging technique for visual assessment of the eyes of goats, sheep, and alpacas.

Acknowledgments

Supported by the Cummings School of Veterinary Medicine at Tufts University Companion Animal Fund.

Presented in abstract form at the 46th Annual Conference of the American College of Veterinary Ophthalmologists, Coeur d'Alene, Idaho, October 2015.

Dr. Pirie is lead inventor of the camera adaptor described in this report, for which Tufts University holds the patent.

The authors thank Dr. Bruce Barton for assistance with the statistical analysis, Kimberly Flink for technical assistance, and Scott Brundage for assistance with handling of the animals.

ABBREVIATIONS

dSLR

Digital single-lens reflex

ICG

Indocyanine green

SF

Sodium fluorescein

Footnotes

a.

Ful-Glo, Akorn Inc, Lake Forest, Ill.

b.

Kowa SL-15 portable slit-lamp biomicroscope, Kowa Co Ltd, Tokyo, Japan.

c.

TonoVet, iCare, Vantaa, Finland.

d.

Welch Allyn binocular indirect ophthalmoscope, Welch Allyn Distributors, Skaneateles Falls, NY.

e.

Telazol, Zoetis, Florham Park, NJ.

f.

Putney Inc, Portland, Me.

g.

West-Ward Pharmaceutical, Eatontown, NJ.

h.

AnaSed LA, MWI Animal Health, Boise, Idaho.

i.

Torbugesic, Pfizer, New York, NY.

j.

Major Pharmaceuticals, Livonia, Mich.

k.

Canon 7D, Canon, Tokyo, Japan.

l.

Canon EF-S 60 mm f/2.8 macro lens, Canon, Tokyo, Japan.

m.

IC-Green, Akorn Inc, Lake Forest, Ill.

n.

Ak-fluor, Akorn Inc, Lake Forest, Ill.

o.

769/41 nm Bright Line, Semrock Inc, Rochester, NY.

p.

832/37 nm BrightLine, Semrock Inc, Rochester, NY.

q.

MF479/40 nm, Thorlabs Inc, Newton, NJ.

r.

MF525/39 nm, Thorlabs Inc, Newton, NJ.

s.

Adobe CS6, Adobe Systems Inc, San Jose, Calif.

t.

SAS, version 9.2, SAS Institute Inc, Cary, NC.

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Contributor Notes

Address correspondence to Dr. Pirie (chris.pirie@tufts.edu).