• View in gallery

    Representative anterior segment angiographic images of the left eye of an ophthalmologically normal 4-year-old Beagle with a brown iris following IV administration (cephalic vein) of SF (20 mg/kg; A, C, E, and G) and MB-102 (20 mg/kg; B, D, F, and H). Images were obtained by use of narrowband filter combinations tailored to each angiographic dye. Images depict the arterial phase at 11 seconds (A and B), capillary phase at 12 seconds (C and D), venous phase at 13 seconds (E and F), and 5 minutes (G and H) after dye administration. Notice the extravasation of dye and presence of SF (G) and MB-102 (H), which appears as a vertical fluorescent column within the anterior chamber (arrow).

  • View in gallery

    Representative posterior segment angiographic images of the right eye of an ophthalmologically normal 4-year-old Beagle with a brown iris following IV administration of SF (A, C, E, G and I)and MB-102 (B, D, F, H and J). Images depict the choroidal and arterial phase at 7 seconds (A and B), arteriovenous phase at 9 seconds (C and D), early venous phase at 11 seconds (E and F), late venous phase at 16 seconds (G and H), and 10 minutes (I and J) after dye administration. Notice the superior contrast and visualization of the retinal vasculature with MB-102, particularly in the late venous phase (H). Extravasation at 10 minutes after SF (I) and MB-102 (J) administration hinders clear visualization of the retinal vasculature. See Figure 1 for remainder of key.

  • View in gallery

    Representative posterior segment angiographic images of an ophthalmoscopically normal 4-year-old Beagle (A) and an ophthalmoscopically normal 5-year-old Beagle (B) following IV administration of MB-102 (20 mg/kg). Images depict the arteriovenous phase at 7 and 8 seconds after dye administration as obtained with a broadband (A) and narrowband (B; tailored to MB-102) filter combination. Notice the superior image quality and image contrast with use of the narrowband filter combination.

  • View in gallery

    Representative anterior segment angiographic images of the right eye of a 4-year-old Beagle with primary open angle glaucoma and a dark brown iris obtained at 15 seconds following IV administration of SF (20 mg/kg; A) and MB-102 (40 mg/kg; B). Images, obtained with narrowband filter combination tailored to the administered dye, depict disruption of the terminal capillaries to a similar degree, as shown by dye leakage at the pupillary border of the iris (arrow).

  • View in gallery

    Representative posterior angiographic images of the right eye of a 1-year-old mixed-breed dog with retinal disease following IV administration of SF (20 mg/kg; A and C) and MB-102 (40 mg/kg; B and D). Images obtained during the late venous phase (A and B) depict the beginning of 2 window defects (arrows), which become more obvious at 1 minute after dye administration (C and D). Notice that the window defects are more apparent with the use of MB-102 owing to less background fluorescence and superior image contrast.

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Effectiveness of MB-102, a novel fluorescent tracer agent, for conducting ocular angiography in dogs

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  • 1 1Department of Clinical Sciences, Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA 01536.
  • | 2 2Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.
  • | 3 3MediBeacon Inc, St Louis, MO 63132.

Abstract

OBJECTIVE

To evaluate the effectiveness of a novel fluorescence tracer agent, MB-102, for conducting ocular angiography in dogs.

ANIMALS

10 ophthalmologically normal dogs (2 to 4 years old) and 10 dogs with retinal degeneration or primary open-angle glaucoma (< 6 years old).

PROCEDURES

While anesthetized, all dogs received sodium fluorescein (20 mg/kg, IV) or MB-102 (20 or 40 mg/kg, IV) first and then the other dye in a second treatment session 2 days later in a randomized crossover design. Anterior fluorescence angiography was performed on one eye and posterior fluorescence angiography on the other. Imaging was performed with a full-spectrum camera and camera adaptor system. Filter sets that were tailored to match the excitation and emission characteristics of each angiographic fluorescent agent were used.

RESULTS

All phases and phase intervals during anterior and posterior segment angiography were identified, regardless of the dye used. However, agent fluorescence and visualization of the iridal blood vessels were hindered in some dogs, irrespective of agent, owing to the degree of iridal pigmentation present. No significant difference was noted between the 2 dyes in any phase or phase interval, and slight improvement in image contrast was observed with MB-102 during the venous phases owing to a reduction of vessel wall staining in both normal and diseased eyes.

CONCLUSIONS AND CLINICAL RELEVANCE

Results suggested that MB-102 would be useful for conducting ocular angiography in dogs.

Fluorescence angiography, a procedure that involves IV administration of a fluorescent dye and imaging of the ocular vasculature, has been used to assess the integrity of this vasculature in dogs with various disease states.1–5 Sodium fluorescein is the most commonly used dye for such purposes. It has a low molecular weight (376 Da), is hydrophilic, and achieves peak absorption (485 to 500 nm) and emission (520 to 530 nm) within the visible spectrum.6,7

Despite the popularity of SF for conducting fluorescence angiography, the dye has several drawbacks. First, it has a short half-life, is metabolized to nonfluorescent products, and is susceptible to photobleaching, so the window for observing dye fluorescence within the ocular vasculature is only 10 to 30 minutes following injection.8,9 Second, adverse effects are common, particularly in humans. Sodium fluorescein is soluble only at alkaline pH, and IV administration causes pain at the injection site, vein inflammation, nausea, and vomiting in approximately 10% of humans.10–14 More serious adverse reactions, including sickle cell crises,15 seizures,16 and life-threatening anaphylaxis,17–19 are frequent enough (approx 1% of human subjects20,21) that a crash cart must be present during administration, and an estimated 1 in 220,000 humans die as a result of SF injection.22 Complicating the situation, adverse reactions are more likely to occur in diabetic and hypertensive patients, who are most likely to require flourescence angiography.11 For these individuals, use of a lower dose of SF mitigates but does not eliminate the risk of adverse effects and results in reduced image quality. Although oral delivery eliminates the problem of pain at the injection site, it does not reduce the risk of severe adverse effects such as anaphylaxis.23,24 Similar adverse effects have been reported within the veterinary literature, although not to the same frequency. Emesis can occur in approximately 15% of dogs following IV administration, and anaphylaxis has been reported in cats.25,26 Given these drawbacks, a longer-lasting, safer alternative to SF is needed.

MB-102 is a biocompatible fluorescent dye that was initially developed for the study of glomerular filtration rate in humans.27–29 Unlike SF, MB-102 is soluble at a physiologic pH, does not bind proteins in the blood, and is not metabolized, so it has a considerably longer half-life than does SF. Although MB-102 has a molecular weight (372 Da) similar to that of SF, its peak absorption and emission characteristics differ, occurring at 445 and 560 nm, respectively. With its emission spectrum occurring at slightly longer wavelengths than that of SF, MB-102 may be advantageous for highlighting specific pathological changes not readily identified with SF. However, the main advantage may be that MB-102 is not metabolized in vivo and remains intact, and preclinical toxicity testing in rats, rabbits, and dogs has failed to demonstrate any adverse effects30,31 (highest dose evaluated in dogs, 220 mg/kg32). In addition, to date no serious adverse effects of MB-102 administration have been recorded in human clinical studies28,29 of glomerular filtration rate. Therefore, we expected that MB-102 would have an enhanced safety profile relative to the known concerns with SF. The purpose of the study reported here was to assess the use of MB-102 for visualizing ocular vasculature in ophthalmologically healthy and diseased eyes of dogs and compare results with those of standard SF angiography.

Materials and Methods

Animals

Two groups of dogs were used in the study and were evaluated at different institutions. Procedures involving ophthalmologically normal dogs were conducted at Tufts University Cummings School of Veterinary Medicine; this group (referred to as the normal group) included 10 Beagles owned by Tufts University for teaching and research purposes. Procedures involving dogs with retinal disease, retinal degeneration, or both were conducted at Michigan State University; this group (referred to as the diseased group) included 10 purpose-bred dogs (5 with hereditary retinal degeneration [PDE6A mutation and mild to advanced stage of disease] and 5 with primary open angle glaucoma [ADAMTS10 mutation and mild to moderate-severe stage of disease]).

All dogs received complete ophthalmic and physical examinations prior to ocular angiography. Ophthalmic examination included evaluation of menace responses and dazzle and pupillary light reflexes as well as fluorescein staininga of the ocular surface, slit-lamp biomicroscopy,b applanation tonometry,c and indirect ophthalmoscopy.d Fluorescein staining of the ocular surface was performed approximately 2 hours prior to angiographic imaging.

The study protocols were approved by the Institutional Animal Care and Use Committee at Tufts University and Michigan State University and conformed to the statement of the Association for Research in Vision and Ophthalmology regarding use of animals in vision research.

In vitro and in vivo MB-102 dose determination

To determine a suitable dose of MB-102 for use in dogs, in vitro and in vivo tests were performed. For the in vitro test, serial dilutions of MB-102e mixed in canine blood samples (equivalent dose range, 0.5 to 30 mg/kg) were performed and SFf was mixed in canine blood samples to yield the equivalent conventional dose (20 mg/kg). The photographic setup and broadband filter combination as described subsequently for angiographic imaging was then used to obtain images of these samples. Subjective analysis of the amount of fluorescence within the acquired images was performed, and the mean intensity and percentile of the intensity histogram were determined by use of commercially available software.g On the basis of the findings, a 20-mg/kg dose of MB-102 was considered appropriate (data not shown).

A dose escalation trial of MB-102 (5, 10, and 20 mg/kg, IV) was subsequently performed by use of 3 separate ophthalmologically normal dogs owned by Tufts University for teaching and research purposes, followed by angiographic imaging as described subsequently. Initial results indicated that a 20-mg/kg dose of MB-102 yielded results comparable to those achieved with a 20-mg/kg dose of SF (data not shown).

Study design

In a randomized crossover design, dogs in the normal group were assigned to receive SF (20 mg/kg, IV) or MB-102 (20 mg/kg, IV) first and then the opposite treatment 2 days later. This washout period was considered appropriate because the reported half-life following IV administration of MB-102 in dogs with healthy renal function is approximately 1 hour,30,31 and this half-life could be expected to be longer in dogs with reduced renal function. The order of dye administration was assigned by means of a coin toss. In a similar design, dogs in the diseased group were assigned to receive SF (20 mg/kg, IV) or MB-102 at a higher dose (40 mg/kg, IV).

Anterior and posterior segment angiography

In preparation for angiography, all dogs received butorphanol tartrateh (0.2 mg/kg, IM), maropitant citratei (1 mg/kg, SC), and diphenhydramine hydrochloridej (2 mg/kg, SC). One eye of each dog was randomly chosen to have the posterior segment imaged, and the other eye underwent anterior segment imaging. For eyes undergoing posterior segment imaging, a single drop of 1% tropicamide solutionk was applied to induce pupillary dilation. A cephalic catheter (left or right) was placed in all dogs, and anesthesia was induced with propofoll (4 mg/kg, IV). The same cephalic vein was catheterized regardless of the dye used. Dogs were subsequently endotracheally intubated, and anesthesia was maintained with isoflurane in oxygen.

To aid in globe positioning, 2 stay sutures were placed within the bulbar conjunctiva by use of 5-0 nylon suture. During imaging, all globes were continuously lubricated with sterile ophthalmic saline (0.64% NaCl) solution.m

The same angiographic imaging protocol was used, regardless of the angiographic dye or filter set used. The assigned dye was administered IV as a bolus into the cephalic catheter, and imaging was performed at a rate of 3 images/s for a total of 30 seconds. Thereafter, imaging was performed at 1, 2, 3, 4, 5, 10, 15, 20, 25, and 30 minutes. Once imaging was complete, the stay sutures were removed and dogs were allowed to recover. All dogs were monitored during their recovery period and reexamined (complete physical and ophthalmic examination) after recovery to determine whether any sustained adverse effects had occurred.

Fluorescence detection and photography

A liquid chromatography instrumentn with UV and fluorescence detectors was used to obtain high-quality normalized fluorescence spectra of each angiographic dye. This information was used to select appropriate filter combinations (broadband or narrowband) required for conducting ocular angiography with SF and MB-102. Angiographic imaging was performed with a digital single-lens reflex camera adaptor system consisting of a full-spectrum digital camera,o camera adaptor, 60-mm lens,p and indirect ophthalmic lens (for posterior segment imaging).q Illumination for visualization and focusing was provided by a solitary white light–emitting diode. Dye excitation and exposure were provided by a high-brightness, light-emitting diode with a dominant wavelength of 465 nm and FWHM of 24 nm. Camera settings were maintained between angiographic dyes, with a shutter speed of 1/30 second and aperture of f/8. For anterior segment imaging, an International Organization of Standardization (ISO) setting of 400 (broadband filter) or 640 (narrowband filter) was used, whereas for posterior imaging, a setting of 200 (broadband filter) or 400 (narrowband filter) was used (to maintain a comparable level of exposure).

One imaging filter combination (broadband or narrowband) was used for each dog. The filter combination used for each dog was randomized by means of a coin toss. Filter combinations included both a broadband filter set to allow for reduced illumination requirements and improved capturing of dye fluorescence, and a narrowband filter set tailored to match the emission characteristics of each angiographic dye (maximizing image contrast; Supplementary Appendix S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.81.5.428). The broadband filter set used for both dyes consisted of an excitation filterr and barrier filter.s The narrowband filter set used for SF also consisted of an excitation filterr and barriert filter combination, and the set used for MB-102 consisted of a different excitation filterr and barrier filteru combination.

Angiographic evaluations

For anterior segment imaging, times to onset of the arterial, capillary, and venous phases were identified by the initial filling of the dye within the major arterial circle, pupillary capillaries, and iridal veins, as described previously.32 Phase intervals were defined as the time elapsed from the beginning of one phase to the beginning of the next. For posterior segment imaging, all measurements were obtained in accordance with previously reported methods.2 Transit times between dye injection and onset of choroidal fluorescence (choroidal phase) and the retinal arterial, retinal arteriovenous, retinal early venous, and retinal late venous phases were recorded. All time measurements were performed in duplicate by the same observer (CGP), and the mean of the 2 measurements was recorded.

The ability of the angiographic dyes to provide clear visualization of the vasculature and the degree of dye extravasation were subjectively assessed and compared between dyes. For comparison purposes, all images were converted to black and white by use of a black-and-white adjustment tool (entire image) via image editing software.g

Statistical analysis

Statistical softwarev was used to compare times to onset of each phase and phase intervals between angiographic dyes with the Wilcoxon rank sum test. Values of P < 0.05 were considered significant.

Results

Animals

The 10 dogs in the normal group included 6 castrated males and 4 spayed females with a median age of 4.25 years (range, 3.25 to 4.75 years) and median body weight of 11.25 kg (range, 10.2 to 12.1 kg). Iris pigmentation was moderate (brown) in all eyes imaged.

The 10 dogs in the diseased group included 5 sexually intact males and 5 sexually intact females with a median age of 3.1 years (range, 0.6 to 5.5 years) and median body weight of 10.9 kg (range, 3.7 to 14.1 kg). Iris pigmentation was heavy (dark brown) in all eyes imaged.

No adverse effects were identified following IV administration of SF or MB-102 at any administered dose. Staining of the skin was commonly noted shortly after administration of SF, and discoloration of the urine was noted up to 24 hours after administration. No skin or urine discoloration was observed after administration of MB-102.

Normal group

Anterior segment angiography—Representative images depicting anterior segment angiographic phases and late-stage dye extravasation within the aqueous humor were obtained for ophthalmologically normal dogs (Figure 1) No significant differences between SF (20 mg/kg, IV) and MB-102 (20 mg/kg, IV) were identified in times to onset of the arterial, capillary, and venous phases or phase intervals (Table 1). Dye fluorescence and clear visualization of the iridal blood vessels were hindered, regardless of the dye used, owing to the degree of iridal pigmentation present (ie, brown irises) in 3 of the evaluated eyes.

Figure 1—
Figure 1—

Representative anterior segment angiographic images of the left eye of an ophthalmologically normal 4-year-old Beagle with a brown iris following IV administration (cephalic vein) of SF (20 mg/kg; A, C, E, and G) and MB-102 (20 mg/kg; B, D, F, and H). Images were obtained by use of narrowband filter combinations tailored to each angiographic dye. Images depict the arterial phase at 11 seconds (A and B), capillary phase at 12 seconds (C and D), venous phase at 13 seconds (E and F), and 5 minutes (G and H) after dye administration. Notice the extravasation of dye and presence of SF (G) and MB-102 (H), which appears as a vertical fluorescent column within the anterior chamber (arrow).

Citation: American Journal of Veterinary Research 81, 5; 10.2460/ajvr.81.5.428

Table 1—

Summary statistics for time to onset of the arterial, capillary, and venous phases and arterial and capillary phase intervals following IV administration of SF (20 mg/kg) and MB-102 (20 mg/kg) for anterior segment and posterior segment angiography of the eyes of 10 ophthalmologically normal dogs.

 SFMB-102
Variable, by ocular segmentMean ± SDMedian (range)Mean ± SDMedian (range)
Anterior segment
  Onset of fluorescence within the pupil (s)9.0 ± 1.29.3 (6.3–10.3)7.9 ± 1.38.0 (6.0–10.0)
  Arterial phase (s)12.2 ± 1.312.0 (10.6–15.0)11.9 ± 1.612.2 (8.0–13.6)
  Arterial phase interval (s)1.3 ± 0.31.2 (1.0–1.7)1.4 ± 0.61.2 (1.0–2.7)
  Capillary phase (s)13.5 ± 1.513.3 (11.6–16.6)13.3 ± 1.713.3 (9.6–16.0)
  Capillary phase interval (s)1.0 ± 0.01.0*1.0 ± 0.31.0 (1.0–1.3)
  Venous phase (s)14.5 ± 1.514.3 (12.6–17.6)14.3 ± 1.714.3 (10.6–17.0)
  Extravasation within the iris stroma (min)1.0 ± 0.01.0*NA NA 
  Extravasation within the aqueous humor (min)2.3 ± 0.82.5 (1.0–3.0)2.2 ± 1.02.0 (1.0–4.0)
Posterior segment
  Arterial phase (s)7.0 ± 1.07.3 (5.3–8.6)6.7 ± 1.06.2 (5.6–8.6)
  Arterial phase interval (s)2.7 ± 0.82.3 (2.0–4.3)2.8 ± 1.02.7 (1.3–4.6)
  Arteriovenous phase (s)9.7 ± 1.19.5 (8.6–12.3)9.5 ± 1.89.5 (7.3–12.6)
  Arteriovenous phase interval (s)2.4 ± 1.12.0 (1.7–5.3)2.6 ± 1.02.4 (1.3–4.4)
  Early venous phase (s)12.1 ± 2.011.3 (11.0–17.6)12.1 ± 2.511.5 (9.6–16.6)
  Early venous phase interval (s)5.0 ± 0.75.2 (3.7–6.0)5.6 ± 1.15.6 (4.3–7.3)
  Late venous phase (s)17.1 ± 2.216.7 (15.3–23.0)17.7 ± 3.416.2 (14.3–23.6)
  Extravasation obscuring the vasculature (min)10.0 ± 0.010.0*10.0 ± 2.410.0 (5.0–15.0)

All values were the same, so there was no range.

NA = Not applicable because no extravasation occurred.

No significant (P ≥ 0.05) difference was detected between dyes for any variable.

Visualization of the iridal vasculature by use of both filter combinations was comparable between angiographic dyes. Image contrast was superior for both dyes when the narrowband filter sets were used. Dye extravasation was noted as early as 1 minute following IV administration for both SF and MB-102 and continued thereafter. The degree of dye extravasation within the aqueous humor was also comparable between dyes.

Posterior segment angiography—Representative images depicting posterior segment angiographic phases and late-stage dye extravasation within the posterior segment were obtained (Figure 2) No significant differences were identified between SF (20 mg/kg, IV) and MB-102 (20 mg/kg, IV) in times to onset of the choroidal, retinal arterial, retinal arteriovenous, retinal early venous, and retinal late venous phases or in phase intervals (Table 1). Clear visualization of all retinal phases and their respective phase intervals was noted with both SF and MB-102. Use of the narrowband filter set (for both dyes) provided superior image quality and contrast, compared with that achieved with the broadband filter set (Figure 3) The ability to observe each phase was comparable between dyes; however, superior image contrast was observed with MB-102, particularly during the early to late venous phases and as dye left the choriocapillaris. On the other hand, when the narrowband filter combination was used, exposure was subjectively considered to be slightly less with MB-102 than with SF.

Figure 2—
Figure 2—

Representative posterior segment angiographic images of the right eye of an ophthalmologically normal 4-year-old Beagle with a brown iris following IV administration of SF (A, C, E, G and I)and MB-102 (B, D, F, H and J). Images depict the choroidal and arterial phase at 7 seconds (A and B), arteriovenous phase at 9 seconds (C and D), early venous phase at 11 seconds (E and F), late venous phase at 16 seconds (G and H), and 10 minutes (I and J) after dye administration. Notice the superior contrast and visualization of the retinal vasculature with MB-102, particularly in the late venous phase (H). Extravasation at 10 minutes after SF (I) and MB-102 (J) administration hinders clear visualization of the retinal vasculature. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 5; 10.2460/ajvr.81.5.428

Figure 3—
Figure 3—

Representative posterior segment angiographic images of an ophthalmoscopically normal 4-year-old Beagle (A) and an ophthalmoscopically normal 5-year-old Beagle (B) following IV administration of MB-102 (20 mg/kg). Images depict the arteriovenous phase at 7 and 8 seconds after dye administration as obtained with a broadband (A) and narrowband (B; tailored to MB-102) filter combination. Notice the superior image quality and image contrast with use of the narrowband filter combination.

Citation: American Journal of Veterinary Research 81, 5; 10.2460/ajvr.81.5.428

To produce more comparable exposure results between the 2 angiographic dyes, 5 and 3 dogs were subsequently imaged following IV administration of MB-102 at a dose of 40 and 60 mg/kg, respectively. Results for timing of the various angiographic phases and for phase intervals were again similar to those noted for the lower dose. However, exposure when MB-102 was administered at a dose of 40 mg/kg was more comparable to that of SF, particularly when the narrowband filter combinations were used, and so a 40-mg/kg dose of MB-102 was used subsequently for dogs in the diseased group. No detectable difference in exposure or fluorescence was observed when the dose of MB-102 was increased to 60 mg/kg (data not shown).

Diseased group

Anterior segment angiography—Anterior segment angiography in dogs with retinal disease, retinal degeneration, or both failed to provide clear visualization of the iris vasculature and its phases, regardless of the dye used, owing to the substantial amount of iridal pigmentation present. Data regarding time to onset of fluorescence following IV administration of each dye were summarized (Table 2). As noted for the normal group, dye extravasation within the anterior chamber was observed in the later monitoring period after imaging was complete for both SF and MB-102. The degree of extravasation within the aqueous humor was comparable between dyes. In 3 dogs with primary open angle glaucoma (ADAMTS10 mutation; mild to moderate stage of disease), progressive stromal leakage within the pupillary border was observed and believed to represent disruption of the terminal capillaries or early formation of a preiridal fibrovascular membrane (Figure 4). Leakage was noted at a mean of 15.7 and 16.0 seconds after administration of SF and MB-102, respectively. The degree of leakage was comparable between dyes.

Figure 4—
Figure 4—

Representative anterior segment angiographic images of the right eye of a 4-year-old Beagle with primary open angle glaucoma and a dark brown iris obtained at 15 seconds following IV administration of SF (20 mg/kg; A) and MB-102 (40 mg/kg; B). Images, obtained with narrowband filter combination tailored to the administered dye, depict disruption of the terminal capillaries to a similar degree, as shown by dye leakage at the pupillary border of the iris (arrow).

Citation: American Journal of Veterinary Research 81, 5; 10.2460/ajvr.81.5.428

Table 2—

Summary statistics for time to onset of the arterial, capillary, and venous phases and arterial and capillary phase intervals following IV administration of SF (20 mg/kg) and MB-102 (40 mg/kg) for anterior segment and posterior segment angiography of the eyes of 10 dogs with ophthalmic disease (hereditary retinal degeneration [n = 5] or primary open angle glaucoma [5]).

 SFMB-102
Variable, by ocular segmentMean ± SDMedian (range)Mean ± SDMedian (range)
Anterior segment
Onset of fluorescence within the pupil (s)9.7 ± 2.49.7(5.6–14.0)9.6 ± 2.99.6(5.0–14.3)
Extravasation within the iris stroma* (min)15.7 ± 2.414.0 (14.0–19.0)16.0 ± 0.816.0 (15.0–17.0)
Extravasation within the aqueous humor (min)2.8 ± 2.62.0(1.0–10.0)2.9 ± 2.62.0 (1.0–10.0)
Posterior segment
Arterial phase (s)6.3 ± 1.36.5 (2.6–9.6)6.8 ± 2.66.5 (3.3–12.0)
Arterial phase interval (s)2.3 ± 0.52.4(1.4–2.7)2.0 ± 0.42.0 (1.3–3.0)
Arteriovenous phase (s)8.5 ± 2.18.5 (5.0–12.3)8.7 ± 3.98.3 (4.6–15.0)
Arteriovenous phase interval (s)2.3 ± 1.81.7(1.3–7.7)2.7 ± 1.22.2 (1.7–6.0)
Early venous phase (s)10.8 ± 3.510.5 (7.0–20.0)1 1.4 ±3.910.3 (6.6–21.0)
Early venous phase interval (s)4.9 ± 1.64.6 (3.0–9.0)5.2 ± 1.64.7 (3.0–9.0)
Late venous phase (s)15.7 ±4.813.8 (12.3–29.0)16.6 ±5.315.2 (1.3–30.0)
Extravasation obscuring the vasculature (min)7.7 ± 4.210.0 (1.0–15.0)7.8 ±4.110.0(1.0–15.0)

Only 3 dogs had extravasation within the iris stroma with both dyes.

No significant (P ≥ 0.05) difference was detected between dyes for any variable.

Posterior segment angiography—Clear visualization of the retinal vasculature and its phases was achieved with both angiographic dyes. As noted for dogs in the normal group, slight improvement in image contrast was observed for MB-102 during the early to late venous phases, owing to a reduction in the amount of vessel wall staining. Dye extravasation was observed, beginning as early as 5 minutes following IV administration, and was comparable between dyes. Reasonable visualization of retinal vessels was achieved at a mean of 7.7 and 7.8 minutes after administration of SF and MB-102, respectively.

Noteworthy findings in dogs with hereditary retinal degeneration (PDE6A mutation) included slight retinal vessel attenuation (2/5 dogs), tapetal hyperreflectivity contributing to an overall increase in background auto- or pseudofluorescence within the superior fundus (1/5 dogs), and 2 focal chorioretinal scars appearing as window defects (1/5 dogs). Findings and the ability to observe the aforementioned changes were comparable between dyes. Because of less vessel wall staining, the noted window defects appeared slightly more obvious when MB-102 was used instead of SF (absence of background fluorescence from dye present within the choriocapillaris; Figure 5). Use of the narrowband filter set, specifically with SF, allowed some auto- or pseudofluorescence to occur. However, this was not observed with use of the narrowband filter set for MB-102, which provided superior image contrast.

Figure 5—
Figure 5—

Representative posterior angiographic images of the right eye of a 1-year-old mixed-breed dog with retinal disease following IV administration of SF (20 mg/kg; A and C) and MB-102 (40 mg/kg; B and D). Images obtained during the late venous phase (A and B) depict the beginning of 2 window defects (arrows), which become more obvious at 1 minute after dye administration (C and D). Notice that the window defects are more apparent with the use of MB-102 owing to less background fluorescence and superior image contrast.

Citation: American Journal of Veterinary Research 81, 5; 10.2460/ajvr.81.5.428

Noteworthy findings for dogs with primary open angle glaucoma included modeled and delayed filling of the choriocapillaris (3/5 dogs). Observations indicated this modeling was comparable between MB-102 and SF.

Discussion

In the present study, the effectiveness of MB-102 for conducting ocular angiography in dogs was investigated. Overall, use of MB-102 resulted in visualization and vascular details in both ophthalmologically normal and diseased eyes comparable to results achieved with the use of SF, making MB-102 a viable alternative. During the venous phases, particularly during posterior segment imaging, slight improvement in image contrast was noted with the use of MB-102. Fluorescein, which is partially lipophilic in vivo,33,34 has a tendency to attach to cells during systemic distribution. On the other hand, MB-102, which is essentially inert and noninteractive in vivo,30,31 would be expected to have less vessel wall staining and less background than SF, and these properties may aid in detection of new or abnormal vessel formation associated with active leakage.

No significant differences in dye transient times were identified between MB-102 and SF for both dog groups in the present study. Although differences in transit times have been reported for various angiographic dyes and are considered relevant, detection of filling patterns, leakage, or the presence of filling defects is considered of greater clinical importance.35 Use of MB-102 provided superior visualization of the pathological changes (eg, chorioretinal scarring producing a window defect) present in the evaluated diseased eyes and may expand upon the diagnostic capabilities of fluorescence angiography in various ophthalmic conditions. Furthermore, the longer half-life of MB-102 versus SF might allow better assessment of the impact of a disease process or therapeutic intervention on the ocular vasculature in real time.

Dye extravasation was common in the study reported here, and the degree of extravasation was comparable between SF and MB-102. Previous research32 into SF for conducting ocular angiography in dogs yielded similar findings. The magnitude of dye extravasation was likely related to the combined effect of the small molecular weight and poor to absent protein binding of both SF and MB-102 as well as anatomic differences within the ocular vasculature of dogs. Although extravasation of SF in ophthalmologically normal humans has been reported as a non-specific finding, particularly in people more than 50 years of age, the degree of leakage is often minimal. However, in younger people, leakage is considered abnormal and indicative of pathological change.36 Unlike SF, MB-102 has negligible protein binding in blood. Because it is the unbound portion of the dye that is known to extravasate, MB-102 may offer superior diagnostic capabilities for detection of subtle disruptions within the ocular vasculature, particularly early in the disease process. Earlier detection could allow earlier therapeutic interventions and improved patient outcomes, and additional research is needed to explore this diagnostic potential.

In the present study, 2 filter combinations (broadband vs narrowband) were evaluated when comparing MB-102 with SF. The broadband filter combination permitted greater transmission of light, lower illumination and retinal exposure to light, and a direct side-by-side comparison of SF to MB-102. However, the narrowband filter combinations, which were tailored to the spectral characteristics of each dye used, resulted in greater image quality and superior image contrast, characteristics that could aid in detection of pathological changes. With use of the broadband filter combination, MB-102 at a dose of 20 mg/kg yielded exposure results comparable to those achieved with the conventional dose of SF (20 mg/kg) in dogs. However, with use of the narrowband filter combinations, MB-102 at a dose of 40 mg/kg yielded exposure results more comparable to those of SF at a dose of 20 mg/kg, despite observations from preliminary testing. The varying exposure results for MB-102 and the need to increase the dose (from 20 to 40 mg/kg) for the narrowband filter combinations likely reflected differences in the FWHM and the transmittance of light by the commercially available emission filters that were used in the study (SF, FWHM = 39 nm; MB-102, FWHM = 24 nm). As such, the dose of MB-102 used in future works may need to be altered depending on the transmission characteristics of the filter sets used.

No clinically important adverse effects were noted following IV administration of either dye at any dose used in the present study. However, as commonly observed, discoloration of the skin and urine was noted following use of SF. No discoloration of the skin or urine was observed following use of MB-102, and this may represent an advantage of MB-102 versus SF, particularly for use in humans.

Limitations of the present study included the small numbers of ophthalmologically normal dogs and dogs with ophthalmic disease. Additionally, no blood pressure or ECG monitoring was performed during dye administration. Although no adverse effects were observed, this possibility could not be ruled out. Nevertheless, our findings suggested that MB-102 would be a viable fluorescent tracer agent for conducting ocular angiography in dogs. Given its unique spectral and metabolic characteristics (longer half-life than SF) and the observed safety profile, MB-102 should allow an expansion in how fluorescence angiography may be used to diagnose and monitor various ophthalmic diseases in dogs.

Acknowledgments

Funded by NIH grant No. 1R43EY027207-01.

Drs. Rogers and Dorshow are employees of MediBeacon Incorporated, the manufacturers of MB-102. The authors declare that there were no other conflicts of interest.

The authors thank Dr. Lisa Balbes for editorial assistance.

ABBREVIATIONS

FWHM

Full width at half maximum

SF

Sodium fluorescein

Footnotes

a.

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

b.

SL-17 portable slit-lamp biomicroscope, Kowa Co Ltd, Tokyo, Japan.

c.

TonoVet, iCare, Vantaa, Finland.

d.

Direct ophthalmoscope, Welch Allyn Distributors, Skaneateles Falls, NY.

e.

MediBeacon Inc, St Louis, Mo.

f.

AK-Fluor, Akorn Inc, Lake Forest, Ill.

g.

Creative Cloud, Adobe Systems Inc, San Jose, Calif.

h.

Torbugesic-SA, Zoetis, Florham Park, NJ.

i.

Cerenia, Pfizer, New York, NY.

j.

Baxter Healthcare Corp, Deerfield, Ill.

k.

Tropicacyl, Akorn Inc, Lake Forest, Ill.

l.

PropoFlow, Baxter Healthcare Corp, Deerfield, Ill.

m.

BSS, Alcon Laboratories Inc, Fort Worth, Tex.

m.

Acquity H-class analytical UPLC/HPLC instrument, Waters Corp, Milford, Mass.

o.

7D, Canon Inc, Tokyo, Japan.

p.

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

q.

60D, Volk Optical Inc, Mentor, Ohio.

r.

447/60-nm Bright Line, Semrock, Rochester, NY.

s.

565/133-nm Bright Line, Semrock, Rochester, NY.

t.

525/39-nm Bright Line, Semrock, Rochester, NY.

u.

565/24-nm Bright Line, Semrock, Rochester, NY.

v.

SAS, version 9.4, SAS Institute Inc, Cary, NC.

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Supplementary Materials

Contributor Notes

Dr. Pirie's present address is the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

Address correspondence to Dr. Dorshow (rbdorshow@medibeacon.com).