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Deep subconjunctival injection of gentamicin for the treatment of bacterial conjunctivitis in macaques (Macaca mulatta and Macaca fascicularis)
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Sylvia I. Gografe, DVM, PhD1, Barbara C. Hansen, PhD2 & Kenneth D. Hansen, MD, JD3
Infectious conjunctivitis occurs in a number of domestic and laboratory animal species and is usually treated topically with eye drops or eye ointments, which must be administered several times a day and sometimes for a prolonged period of time. In aggressive nonhuman primates or other large laboratory animal species, this may require the use of anesthesia or physical restraint before each treatment, which can be stressful to the animals and demanding for personnel. The authors describe a technique for administering deep subconjunctival injections of an antibiotic to laboratory macaques for the treatment of conjunctivitis. Three cases of recurrent conjunctivitis in macaques that responded poorly to other treatment approaches were effectively treated using this technique. This approach is recommended for the treatment of conjunctivitis in macaques and other large animal species. Conjunctivitis and keratoconjunctivitis are relatively common in domestic and some laboratory animals (ref. 1; Orchard, E., Mannheimer Foundation, Homestead, FL, personal communication). In most cases, the pathogenesis of bacterial conjunctivitis is the disruption of host defense mechanisms. Ocular anomalies such as ectropion, misdirected cilia or keratoconjunctivitis sicca, as well as conditions like diabetes mellitus, can predispose an animal to recurring conjunctivitis2,3. In our colony of middle-aged to geriatric macaques, conjunctivitis, often relapsing, is an occasional veterinary medical problem. In a period of 18 months, a cynomolgus macaque (Macaca fascicularis) and two rhesus macaques (Macaca mulatta) in our colony were diagnosed with recurring conjunctivitis. The cynomolgus macaque had type 2 diabetes mellitus but had no prior history of conjunctivitis. In all four of its relapsing episodes, symptoms were restricted to the right eye. One of the rhesus macaques was prediabetic, with normal fasting glucose levels but impaired glucose tolerance, and had metabolic syndrome4, including obesity, elevated insulin levels and elevated
triglyceride levels. It too had no prior history of eye disease. Its first occurrence of conjunctivitis was unilateral, whereas the following two episodes involved both eyes. The second rhesus macaque was healthy upon arrival at our institution with a history of intermittent bilateral conjunctivitis. This monkey had four episodes of conjunctivitis affecting both eyes during the 18-month period. Conjunctivitis is typically treated with topical administration of antibiotic eye drops (four times per d) or ophthalmic ointment (two times per d) into the inferior conjunctival sac. This treatment must be administered multiple times daily and sometimes for a prolonged period of time in order to be effective1,5, requiring substantial technician time and facility resources6. Administering such treatment to nonhuman primate species requires anesthesia or physical restraint, because the monkeys can often be aggressive, posing a risk of injury to technicians and to themselves. Although conscious monkeys can be immobilized for the procedure using various restraint devices, these devices do not fully restrict the monkey’s movement,
1Florida Atlantic University, Boca Raton, FL. 2Departments of Internal Medicine and Pediatrics, College of Medicine, University of South Florida, Tampa, FL. 3International Health Foundation, McLean, VA. Correspondence should be addressed to S.I.G. ([email protected]).
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especially of the head. Furthermore, conscious monkeys tend to squeeze their eyes closed, making consistent administration of eye drops or ointment into the inferior conjunctival sac a challenge. Some individuals cannot tolerate intensive topical treatment3. Eye drops or ointment can elicit an uncomfortable foreign-body sensation in the eyes of the monkeys or can induce blurriness of vision, both of which can cause increased lacrimation that substantially dilutes the antibiotic solution. Thus, even if the animal is trained to facilitate therapeutic interventions under restraint, compliance with treatment may be compromised. We initially treated conjunctivitis in these macaques using the topical antibiotic or systemic antibiotic treatment approaches. While the monkeys were awake and restrained, we irrigated the inferior conjunctival sac with sterile saline (Baxter International, Deerfield, IL) or boric acid eye solution (Bausch and Lomb, Rochester, NY) 1–2 times per day to remove obvious exudates and applied antibiotic ophthalmic ointment (Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment; Akorn Animal Health, Lake Forest, IL or Vetropolycin; Dechra Veterinary Products, Overland Park, KS) to the eye 2–3 times per day. Two of the monkeys were treated with systemic antibiotics (ampicillin (Fort Dodge, Overland Park, KS) or enrofloxacin (Baytril; Bayer HealthCare, Shawnee Mission, KS)) for 6–8 days. These treatment approaches were unsuccessful, and all three monkeys had three or four relapses of conjunctivitis occurring from 8 days to 5 months apart. We sought new treatment options to improve therapeutic outcomes as well as to minimize the possible negative physical or psychological effects on the monkeys. We adapted a treatment approach frequently used in humans with severe or recalcitrant conjunctivitis that involves deep subconjunctival injection of an antibiotic solution for use in macaques. Subconjunctival injection of antibiotics has long been practiced in human medicine and is used by 16% of American and 77% of British practitioners7. It was also used in a previous study to effectively resolve a bacterial corneal ulcer in a rabbit8. Multiple terms are used for such injections, sometimes interchangeably but often without clear anatomical distinctions, including subconjunctival, periorbital or sub-Tenon’s. According to Baum8, whether the injection is subconjunctival or anterior sub-Tenon’s does not make a difference to therapeutic outcome. The treatment approach we used would most accurately be described as a deep subconjunctival injection. This approach was used to treat recurrent conjunctivitis in the same three macaques, and the technique that we used is described here. We found several advantages of this approach over oral or topical routes of antibiotic administration; the injections greatly increase drug concentrations at the treatment site, minimize the risk LAB ANIMAL
of systemic drug-induced side effects and reduce the frequency of dosing1,9,10. Furthermore, the procedure requires the same amount of anesthetic that would be used for instillation of topical ophthalmic medications. To our knowledge, this is the first reported use of this treatment approach in nonhuman primate conjunctivitis. We suggest this approach to laboratory animal veterinarians as the treatment of choice for sustained or recurrent cases of bacterial conjunctivitis in nonhuman primate or other large animal species. TECHNIQUE Animals The three monkeys described previously were members of a colony of more than 100 macaques enrolled in research protocols approved by the IACUC of the University of South Florida (Tampa) for studies of aging, obesity, type 2 diabetes mellitus, and their associated complications11. The female cynomolgus macaque was 26 years old and weighed 4.3 kg. The two male rhesus macaques were 21.5 years old and 21 years old and weighed 18.5 kg and 14.8 kg, respectively. All three macaques were aged approximately the average macaque lifespan, and thus these monkeys were considered middle-aged or elderly12. The monkeys were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International in accordance with the Animal Welfare Act13 and the Guide for the Care and Use of Laboratory Animals14. The monkeys were singly housed in indoor enclosures. Their diabetic status required daily glucose checks, treatment with insulin, and complete measurement of food intake. Several macaques, singly caged with visual, auditory, olfactory and sometimes grooming (i.e., tactile) contact, were housed in large rooms and allowed for the optimal management of these monkeys at the time. They were provided Lab Diet 5038 (LabDiet, St. Louis, MO) with metabolizable energy distributed as 18.2% protein, 13.1% fat, and 68.7% carbohydrate. Food was always available to all monkeys under this ad libitum food intake regimen as was water via an autowatering system. Treat and non-food enrichment was provided to all on a rotating daily basis. Clinical diagnoses Upon presentation with clinical signs of conjunctivitis, we immobilized the monkeys with ketamine (Fort Dodge, Overland Park, KS) and carried out a general physical examination. We noted no abnormalities of the nasolacrimal duct, sinuses or eyelids. We examined the eyes with an ophthalmoscope (Welch Allyn Veterinary Diagnostic Set, 3.5 V; Welch Allyn, Skaneateles Falls, NY). All three monkeys presented several or all of the following common signs of bacterial conjunctivitis: epiphora, serous to mucopurulent ocular discharge, Volume 44, No. 3 | MARCH 2015 9 3
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chemosis, hyperemia, blepharospasm and periorbital swelling. Fluorescein dye testing (fluorescein sodium ophthalmic strips, Akorn, Lake Forest, IL) showed no corneal involvement in any of the monkeys. We collected samples from each monkey for cultures and sensitivity testing, which were carried out by an accredited commercial diagnostic laboratory. The cultures were found to be positive for Staphylococcus intermedius, S. aureus and Corynebacterium spp. The sensitivity testing confirmed susceptibility of these isolates to several antibiotics including gentamicin, a relatively broad-spectrum antibiotic that is widely used by ophthalmologists to treat severe and recalcitrant conjunctivitis in humans.
~30 ml, of which the eye occupies only one-fifth of the space17. A similar eyeball-to-orbit ratio can be expected in nonhuman primates; therefore, if an average rhesus macaque’s orbital volume is ~13 ml and one-fifth of it is occupied by the eyeball, the globe must occupy ~2.5 ml of the orbital volume. Thus, the maximum volume of drugs we considered safe for injection was 1 ml (0.75 ml gentamicin and 0.25 ml lidocaine). Gentamicin and lidocaine were mixed in one syringe (Becton Dickinson & Co, Franklin Lakes, NJ) just prior to injection. A 25-g, 1-in needle (Excel International, Los Angeles, CA) was attached to the syringe.
Antibiotic solution A solution of gentamicin (40 mg/ml; Hospira, Lake Forest, IL) and lidocaine (2%; Phoenix Pharmaceutical, St. Joseph, MO) was prepared for each monkey. Lidocaine was included to provide additional local anesthesia beyond the ketamine sedation to alleviate possible short-term discomfort after the injection. The amount of antibiotic solution to be injected was determined by the weights of the animals and the estimated corresponding orbital volumes. The anatomy of the orbit and surrounding structures of the eyeball of macaques is similar to those in other mammals and humans15. The eyeball (or globe) is located within and protected by a bony socket called the orbit. The size of the globe and the orbital volume increase in young rhesus monkeys before reaching a stable plateau in adulthood, between 5 years and 7 years of age16, and remain stable in adulthood independent of the monkey’s weight. The size of the globe (i.e., axial length) of an average adult rhesus macaque is about 19.4 ± 0.6 mm (ref. 12). De Rousseau and Bito16 report that a rhesus macaque’s orbital volume is 12.9 ± 1.7 ml on average, with males having an expectedly slightly larger orbit (13.9 ± 1.6 ml) than that of females (12.1 ± 1.4 ml). In comparison, the orbital volume of an adult human is
Injection procedure Human patients receiving deep subconjunctival injections commonly are not provided any form of anesthesia, can sit upright during the procedure and report that the pain associated with the procedure is momentary. Therefore, this injection could be administered to an awake but physically restrained animal. Sedation is advised, however, to reduce stress to the animal. Macaques in particular must be anesthetized for safety reasons. Therefore, we anesthetized the monkeys with 10–15 mg per kg body weight ketamine, with the dose depending on the monkey’s size and previous experience with anesthesia. We laid the monkey on a procedure table in a supine position. A technician secured the monkey’s head in a stable position to prevent any unexpected movements that may have resulted from the light level of anesthesia. Because of the sparse hair coat in the area surrounding the monkey’s eyes, shaving was not necessary. To prepare the injection site, we palpated the inferior orbital rim (Fig. 1) and disinfected the skin inferior to the eye with alcohol swabs. We gently pushed the eyeball upwards with a gloved finger, assessing the elasticity of the eyeball just prior to injection. While the eyeball was gently pushed upward, we inserted the needle through the skin, just above the inferior orbital rim and slightly medial to the lateral canthus of the eye (Fig. 2). We then
FIGURE 1 | Palpation of the inferior orbital rim to identify the landmarks for proper insertion of the needle.
FIGURE 2 | Syringe-mounted needle is inserted just above the inferior periorbital rim slightly medial to the lateral canthus of the eye.
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CONCLUSIONS We chose to administer antibiotics Superior rectus muscle via deep subconjunctival injection in Bulbar fascia macaques with conjunctivitis because Sclera Cilia Retina Cornea of the successful use of this approach Optic nerve (CNI I) Conjunctiva Bulbar in humans. The bioavailability of the Adipose tissue antibiotic is enhanced because it is Inferior rectus muscle administered directly to the affected Orbital septum tissue. The deep subconjunctival injection disperses the drug throughout the Inferior orbital rim tissues of the anterior segments of the FIGURE 3 | Illustration of a sagittal section of the human orbit (adapted from eye and results in the drug being diffused ref. 30), showing the needle position for the deep subconjunctival injection into the anterior orbit18,19, substanrelative to the inferior orbital rim. tially increasing drug concentrations at the treatment site compared with systemic antibiotic administration. slowly advanced the needle posteriorly along the orbital Therapeutic levels of kanamycin or penicillin adminisfloor ~10–15 mm deep and into the subconjunctival tered via the subconjunctival route were found in tears, space (Fig. 3). conjunctiva, cornea and orbital lacrimal glands20 or in conjunctival sac fluid21, respectively. Although it is Before injecting the drug solution, we attempted to aspirate the needle in order to avoid injecting into expected that a small fraction of the drug is distributed a blood vessel. Because the eyeball occupies a small systemically22, we did not observe any effects of such fraction of the orbital space, the risk of puncturing the systemic drug distribution in our animals. Baum8 investigated topical ocular administration globe during deep subconjunctival injection is small. Still, we determined whether the needle had pen- routes intensively and concluded that administering drugs by subconjunctival injection results in higher etrated the ocular sclera by slowly moving the needle to the left and to the right in a sweeping manner to see tissue concentrations than administering drugs via eye drops owing to the limited holding capacity of the whether the eyeball moved as well. If the sclera had been entered, the needle was withdrawn and moved eye and tear production dynamics that result in rapid away from the globe to ensure it was in the subcon- draining of the eye drop solution. To some extent, junctival space. Then, the drug solution was slowly ointments have prolonged contact time compared injected in three increments over a period of 1–2 min with eye drops, but they produce negative sensations to allow it to distribute. After withdrawing the needle, in humans, e.g., perceived increased intraocular pressure and blurriness of vision. A major advantage of we assessed the tension of the orbit and its contents. It is essential that the globe be normotensive; a hard the subconjunctival injection technique over topical globe indicates excessive intraocular pressure which approaches is the reduced administration frequency (three injections per animal at intervals of 48 h versus must be relieved, if necessary, by lateral canthotomy. This is done by applying a hemostat to the lateral can- several topical applications daily for days or weeks). thus and cutting the clamped area with scissors. Neither This lower frequency not only reduced the workload of the veterinary and animal care staff but, more sutures nor cautery are usually required. Chemosis importantly, improved the comfort and welfare of the and lower lid swelling are commonly seen after the deep subconjunctival injections and need no treatment. treated monkeys by reducing handling frequency, We carried out these injections in each eye three times preventing unpleasant sensations associated with instillation of eye drops or ointment, preventing the at 48-h intervals. anxiety associated with awake restraint methods and treatment and reducing the grogginess induced by OUTCOME Our goal was to achieve a positive treatment outcome, frequent anesthesia. As with almost any administration technique, there defined as cured conjunctivitis as well as prevented is the potential for complications associated with deep recurrence. This was achieved in all three monkeys. Two of the monkeys had no recurrent conjunctivitis subconjunctival injections. Complications reported with injections into peribulbar tissues include the risk for at least 12 months. One monkey had a relapse of conjunctivitis after 6.5 months and was treated again of perforation and penetration of the globe23, severe increase in intraocular pressure when large volumes with the same technique; no further conjunctivitis was noted during the follow-up period of 5 months. We are injected24 and acute orbital hemorrhage25. These did not observe any adverse events associated with complications are more common with retrobulbar (posterior sub-Tenon’s) injections, which also pose the the treatment.
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Levator palpebrae superioris muscle
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risk of direct optic nerve injury26 because the needle is advanced further into the orbit. If the injection is administered too fast, there is greater likelihood of significant subconjunctival hemorrhage22,27; vessel damage leading to retrobulbar hematoma; chemosis; swollen, partially closed or shut eyes; and transient congestion28. However, the latter outcome is far more common with superficial subconjunctival injections in the eyelid than with the technique we describe. We did not detect any of these problems in our monkeys. In our use of the technique described, the sclera was never entered. Although chemosis is sometimes induced after injections into the superficial subconjunctival area in humans, we did not experience this outcome. Anatomical variations such as a long eye, shallow orbit, staphyloma or enophthalmos, as well as the use of a too-long needle, are also risk factors for globe perforation29. One of the most important safety measures is familiarity with the anatomy of the eye and orbit to avoid damaging any structures within the eye socket (not only the eyeball itself, but also extraocular muscles, nerves, blood vessels and soft tissue). Deep subconjunctival injection is a valuable and simple technique for the treatment of conjunctivitis in macaques. This approach has improved treatment success in three monkeys in our colony. The technique is reliable and effective, can be learned easily, is safe for use in humans and can be safe for use in monkeys as well if basic precautions are followed. The technique presents a refinement of prior therapeutic approaches in that it reduces stress in the treated animals and improves the effectiveness of treatment, complementing the effort to improve animal welfare in research. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Received 16 June 2014; accepted 28 July 2014 Published online at http://www.labanimal.com/ 1. Kahn, C.M. & Line, S. (eds) The Merck Veterinary Manual 10th edn. (Merck & Co., Inc. Whitehouse Station, NJ, 2010). 2. Ormerod, L.D. Causes and management of bacterial keratitis in the elderly. Can. J. Ophthalmol. 24, 112–116 (1989). 3. Rubenstein, J.B. & Virach, V. in Ophthalmology 3rd edn. (Yanoff, M. &Duker, J.S., eds) 1528–1540 (Mosby, Edinburgh, Scotland, 2008). 4. Hansen, B.C. in The Metabolic Syndrome: Epidemiology, Clinical Treatment, and Underlying Mechanisms (Hansen, B.C. & Bray, G.A., eds.) 373–386 (Humana Press, Totawa, NJ, 2008). 5. Nakagawa, H. Treatment of chlamydial conjunctivitis. Ophthalmologica 211 (suppl. 1), 25–28 (1997). 6. Ward, D.A. & Clark, E.S. Ocular pharmacology. Vet. Clin. North Am. Food Anim. Pract. 7, 779–791 (1991). 7. Matsuura, K. Pharmacokinetics of subconjunctival injection of moxifloxacin in humans. Graefes. Arch. Clin. Exp. Ophthalmol. 251, 1019–1020 (2013). 8. Baum, J. Treatment of bacterial ulcers of the cornea in the rabbit: a comparison of administration by eye drops and subconjunctival injections. Trans. Am. Ophthalmol. Soc. 80, 369–390 (1982).
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9. Feldman-Billard, S., Du Pasquier-Fediaevsky, L. & Héron, E. Hyperglycemia after repeated periocular dexamethasone injections in patients with diabetes. Ophthalmology 113, 1720–1723 (2006). 10. Regnier, A., Toutain, P.L., Alvinerie, M., Perique, B. & Ruckebusch, Y. Adrenocortical function and plasma biochemical values in dogs after subconjunctival treatment with methylprednisolone acetate. Res. Vet. Sci. 32, 306–310 (1982). 11. Hansen, B.C. in Diabetes Mellitus: A Fundamental and Clinical Text (eds. LeRoith, D., Olefsky J. & Taylor, S.) 1060–1074 (Lippincott Williams and Wilkins, Philadelphia, PA 2004). 12. Bodkin, N.L., Alexander, T.M., Ortmeyer, H.K., Johnson, E. & Hansen, B.C. Mortality and morbidity in laboratory-maintained Rhesus monkeys and effects of long-term dietary restriction. J. Gerontol. A. Biol. Sci. Med. Sci. 58, 212–219 (2003). 13. Animal Welfare Act as Amended (7 USC 2131–2156). 14. Institute for Laboratory Animal Research, National Research Council. Guide for the Care and Use of Laboratory Animals 8th edn. (National Academy Press, Washington, DC, 2011). 15. Ankel-Simons, F. Primate Anatomy, Third Edition: An Introduction (Academic, New York NY, 2007). 16. De Rousseau, C. & Bito, L. Intraocular pressure of rhesus monkeys (Macaca mulatta). II. Juvenile ocular hypertension and its apparent relationship to ocular growth. Exp. Eye Res. 32, 407–417 (1981). 17. Riordan-Eva, P. in Vaughan & Asbury′s General Ophthalmology 18th edn. (Riordan-Eva, P. & Cunningham, E.T. Jr., eds.) Ch. 1 (McGraw-Hill, New York, NY, 2011). 18. Miller, T.R. Principles of therapeutics. Vet. Clin. North Am. Equine Pract. 8, 479–497 (1992). 19. Gordon, T.B. & Cunningham, R.D. Tobramycin levels in aqueous humor after subconjunctival injection in humans. Am. J. Ophthalmol. 93, 107–110 (1982). 20. George, L.W., Reina-Guerra, M., Baggot, J.D. & Mihalyi, J. Distribution of kanamycin in ocular tissues of calves. J. Vet. Pharmacol. Ther. 9, 183–191 (1986). 21. Abeynayake, P. & Cooper, B.S. The concentration of penicillin in bovine conjunctival sac fluid as it pertains to the treatment of Moraxella bovis infection. (I) Subconjunctival injection. J. Vet. Pharmacol. Ther. 12, 25–30 (1989). 22. Douglas, L.C., Yi, N.Y., Davis, J.L., Salmon, J.H. & Gilger, B.C. Ocular toxicity and distribution of subconjunctival and intravitreal rapamycin in horses. J. Vet. Pharmacol. Ther. 31, 511–516 (2008). 23. McLeod, S. in Ophthalmology 3rd edn. (Yanoff, M. & Duker, J.S., eds.) 262–270 (Mosby, Edinburgh, Scotland, 2008). 24. Senturk, S., Cetin, C., Temizel, M. & Ozel, E. Evaluation of the clinical efficacy of subconjunctival injection of clindamycin in the treatment of naturally occurring infectious bovine keratoconjunctivitis. Vet. Ophthalmol. 10, 186–189 (2007). 25. Azarbod, P., Mohammed, Q., Akram, I. & Moorman, C. Localised abscess following an injection of subtenon triamcinolone acitonide. Eye (Lond.) 21, 672–674 (2007). 26. Kim, S.K., Andreoli, C.M., Rizzo, J.F. III, Golden, M.A. & Bradbury, M.J. Optic neuropathy secondary to sub-tenon anesthetic injection in cataract surgery. Arch. Ophthalmol. 121, 907–909 (2003). 27. Kaderli, B. & Avci, R. Comparison of topical and subconjunctival anesthesia in intravitreal injection administrations. Eur. J. Ophthalmol. 16, 718–721 (2006). 28. Veneziale, R.W. et al. SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys. Cutan. Ocul. Toxicol. 26, 83–105 (2007). 29. Kumar, C.M. & Dodds, C. Ophthalmic regional block. Ann. Acad. Med. Singapore 35, 158–167 (2006). 30. Tank, P.W. & Gest, T.R. Lippincott Williams & Wilkins Atlas of Anatomy xv, 353 (Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia. PA, 2008).
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Deep subconjunctival injection of gentamicin for the treatment of bacterial conjunctivitis in macaques (Macaca mulatta and Macaca fascicularis)
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Sylvia I. Gografe, DVM, PhD1, Barbara C. Hansen, PhD2 & Kenneth D. Hansen, MD, JD3
Infectious conjunctivitis occurs in a number of domestic and laboratory animal species and is usually treated topically with eye drops or eye ointments, which must be administered several times a day and sometimes for a prolonged period of time. In aggressive nonhuman primates or other large laboratory animal species, this may require the use of anesthesia or physical restraint before each treatment, which can be stressful to the animals and demanding for personnel. The authors describe a technique for administering deep subconjunctival injections of an antibiotic to laboratory macaques for the treatment of conjunctivitis. Three cases of recurrent conjunctivitis in macaques that responded poorly to other treatment approaches were effectively treated using this technique. This approach is recommended for the treatment of conjunctivitis in macaques and other large animal species. Conjunctivitis and keratoconjunctivitis are relatively common in domestic and some laboratory animals (ref. 1; Orchard, E., Mannheimer Foundation, Homestead, FL, personal communication). In most cases, the pathogenesis of bacterial conjunctivitis is the disruption of host defense mechanisms. Ocular anomalies such as ectropion, misdirected cilia or keratoconjunctivitis sicca, as well as conditions like diabetes mellitus, can predispose an animal to recurring conjunctivitis2,3. In our colony of middle-aged to geriatric macaques, conjunctivitis, often relapsing, is an occasional veterinary medical problem. In a period of 18 months, a cynomolgus macaque (Macaca fascicularis) and two rhesus macaques (Macaca mulatta) in our colony were diagnosed with recurring conjunctivitis. The cynomolgus macaque had type 2 diabetes mellitus but had no prior history of conjunctivitis. In all four of its relapsing episodes, symptoms were restricted to the right eye. One of the rhesus macaques was prediabetic, with normal fasting glucose levels but impaired glucose tolerance, and had metabolic syndrome4, including obesity, elevated insulin levels and elevated
triglyceride levels. It too had no prior history of eye disease. Its first occurrence of conjunctivitis was unilateral, whereas the following two episodes involved both eyes. The second rhesus macaque was healthy upon arrival at our institution with a history of intermittent bilateral conjunctivitis. This monkey had four episodes of conjunctivitis affecting both eyes during the 18-month period. Conjunctivitis is typically treated with topical administration of antibiotic eye drops (four times per d) or ophthalmic ointment (two times per d) into the inferior conjunctival sac. This treatment must be administered multiple times daily and sometimes for a prolonged period of time in order to be effective1,5, requiring substantial technician time and facility resources6. Administering such treatment to nonhuman primate species requires anesthesia or physical restraint, because the monkeys can often be aggressive, posing a risk of injury to technicians and to themselves. Although conscious monkeys can be immobilized for the procedure using various restraint devices, these devices do not fully restrict the monkey’s movement,
1Florida Atlantic University, Boca Raton, FL. 2Departments of Internal Medicine and Pediatrics, College of Medicine, University of South Florida, Tampa, FL. 3International Health Foundation, McLean, VA. Correspondence should be addressed to S.I.G. ([email protected]).
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especially of the head. Furthermore, conscious monkeys tend to squeeze their eyes closed, making consistent administration of eye drops or ointment into the inferior conjunctival sac a challenge. Some individuals cannot tolerate intensive topical treatment3. Eye drops or ointment can elicit an uncomfortable foreign-body sensation in the eyes of the monkeys or can induce blurriness of vision, both of which can cause increased lacrimation that substantially dilutes the antibiotic solution. Thus, even if the animal is trained to facilitate therapeutic interventions under restraint, compliance with treatment may be compromised. We initially treated conjunctivitis in these macaques using the topical antibiotic or systemic antibiotic treatment approaches. While the monkeys were awake and restrained, we irrigated the inferior conjunctival sac with sterile saline (Baxter International, Deerfield, IL) or boric acid eye solution (Bausch and Lomb, Rochester, NY) 1–2 times per day to remove obvious exudates and applied antibiotic ophthalmic ointment (Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment; Akorn Animal Health, Lake Forest, IL or Vetropolycin; Dechra Veterinary Products, Overland Park, KS) to the eye 2–3 times per day. Two of the monkeys were treated with systemic antibiotics (ampicillin (Fort Dodge, Overland Park, KS) or enrofloxacin (Baytril; Bayer HealthCare, Shawnee Mission, KS)) for 6–8 days. These treatment approaches were unsuccessful, and all three monkeys had three or four relapses of conjunctivitis occurring from 8 days to 5 months apart. We sought new treatment options to improve therapeutic outcomes as well as to minimize the possible negative physical or psychological effects on the monkeys. We adapted a treatment approach frequently used in humans with severe or recalcitrant conjunctivitis that involves deep subconjunctival injection of an antibiotic solution for use in macaques. Subconjunctival injection of antibiotics has long been practiced in human medicine and is used by 16% of American and 77% of British practitioners7. It was also used in a previous study to effectively resolve a bacterial corneal ulcer in a rabbit8. Multiple terms are used for such injections, sometimes interchangeably but often without clear anatomical distinctions, including subconjunctival, periorbital or sub-Tenon’s. According to Baum8, whether the injection is subconjunctival or anterior sub-Tenon’s does not make a difference to therapeutic outcome. The treatment approach we used would most accurately be described as a deep subconjunctival injection. This approach was used to treat recurrent conjunctivitis in the same three macaques, and the technique that we used is described here. We found several advantages of this approach over oral or topical routes of antibiotic administration; the injections greatly increase drug concentrations at the treatment site, minimize the risk LAB ANIMAL
of systemic drug-induced side effects and reduce the frequency of dosing1,9,10. Furthermore, the procedure requires the same amount of anesthetic that would be used for instillation of topical ophthalmic medications. To our knowledge, this is the first reported use of this treatment approach in nonhuman primate conjunctivitis. We suggest this approach to laboratory animal veterinarians as the treatment of choice for sustained or recurrent cases of bacterial conjunctivitis in nonhuman primate or other large animal species. TECHNIQUE Animals The three monkeys described previously were members of a colony of more than 100 macaques enrolled in research protocols approved by the IACUC of the University of South Florida (Tampa) for studies of aging, obesity, type 2 diabetes mellitus, and their associated complications11. The female cynomolgus macaque was 26 years old and weighed 4.3 kg. The two male rhesus macaques were 21.5 years old and 21 years old and weighed 18.5 kg and 14.8 kg, respectively. All three macaques were aged approximately the average macaque lifespan, and thus these monkeys were considered middle-aged or elderly12. The monkeys were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International in accordance with the Animal Welfare Act13 and the Guide for the Care and Use of Laboratory Animals14. The monkeys were singly housed in indoor enclosures. Their diabetic status required daily glucose checks, treatment with insulin, and complete measurement of food intake. Several macaques, singly caged with visual, auditory, olfactory and sometimes grooming (i.e., tactile) contact, were housed in large rooms and allowed for the optimal management of these monkeys at the time. They were provided Lab Diet 5038 (LabDiet, St. Louis, MO) with metabolizable energy distributed as 18.2% protein, 13.1% fat, and 68.7% carbohydrate. Food was always available to all monkeys under this ad libitum food intake regimen as was water via an autowatering system. Treat and non-food enrichment was provided to all on a rotating daily basis. Clinical diagnoses Upon presentation with clinical signs of conjunctivitis, we immobilized the monkeys with ketamine (Fort Dodge, Overland Park, KS) and carried out a general physical examination. We noted no abnormalities of the nasolacrimal duct, sinuses or eyelids. We examined the eyes with an ophthalmoscope (Welch Allyn Veterinary Diagnostic Set, 3.5 V; Welch Allyn, Skaneateles Falls, NY). All three monkeys presented several or all of the following common signs of bacterial conjunctivitis: epiphora, serous to mucopurulent ocular discharge, Volume 44, No. 3 | MARCH 2015 9 3
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chemosis, hyperemia, blepharospasm and periorbital swelling. Fluorescein dye testing (fluorescein sodium ophthalmic strips, Akorn, Lake Forest, IL) showed no corneal involvement in any of the monkeys. We collected samples from each monkey for cultures and sensitivity testing, which were carried out by an accredited commercial diagnostic laboratory. The cultures were found to be positive for Staphylococcus intermedius, S. aureus and Corynebacterium spp. The sensitivity testing confirmed susceptibility of these isolates to several antibiotics including gentamicin, a relatively broad-spectrum antibiotic that is widely used by ophthalmologists to treat severe and recalcitrant conjunctivitis in humans.
~30 ml, of which the eye occupies only one-fifth of the space17. A similar eyeball-to-orbit ratio can be expected in nonhuman primates; therefore, if an average rhesus macaque’s orbital volume is ~13 ml and one-fifth of it is occupied by the eyeball, the globe must occupy ~2.5 ml of the orbital volume. Thus, the maximum volume of drugs we considered safe for injection was 1 ml (0.75 ml gentamicin and 0.25 ml lidocaine). Gentamicin and lidocaine were mixed in one syringe (Becton Dickinson & Co, Franklin Lakes, NJ) just prior to injection. A 25-g, 1-in needle (Excel International, Los Angeles, CA) was attached to the syringe.
Antibiotic solution A solution of gentamicin (40 mg/ml; Hospira, Lake Forest, IL) and lidocaine (2%; Phoenix Pharmaceutical, St. Joseph, MO) was prepared for each monkey. Lidocaine was included to provide additional local anesthesia beyond the ketamine sedation to alleviate possible short-term discomfort after the injection. The amount of antibiotic solution to be injected was determined by the weights of the animals and the estimated corresponding orbital volumes. The anatomy of the orbit and surrounding structures of the eyeball of macaques is similar to those in other mammals and humans15. The eyeball (or globe) is located within and protected by a bony socket called the orbit. The size of the globe and the orbital volume increase in young rhesus monkeys before reaching a stable plateau in adulthood, between 5 years and 7 years of age16, and remain stable in adulthood independent of the monkey’s weight. The size of the globe (i.e., axial length) of an average adult rhesus macaque is about 19.4 ± 0.6 mm (ref. 12). De Rousseau and Bito16 report that a rhesus macaque’s orbital volume is 12.9 ± 1.7 ml on average, with males having an expectedly slightly larger orbit (13.9 ± 1.6 ml) than that of females (12.1 ± 1.4 ml). In comparison, the orbital volume of an adult human is
Injection procedure Human patients receiving deep subconjunctival injections commonly are not provided any form of anesthesia, can sit upright during the procedure and report that the pain associated with the procedure is momentary. Therefore, this injection could be administered to an awake but physically restrained animal. Sedation is advised, however, to reduce stress to the animal. Macaques in particular must be anesthetized for safety reasons. Therefore, we anesthetized the monkeys with 10–15 mg per kg body weight ketamine, with the dose depending on the monkey’s size and previous experience with anesthesia. We laid the monkey on a procedure table in a supine position. A technician secured the monkey’s head in a stable position to prevent any unexpected movements that may have resulted from the light level of anesthesia. Because of the sparse hair coat in the area surrounding the monkey’s eyes, shaving was not necessary. To prepare the injection site, we palpated the inferior orbital rim (Fig. 1) and disinfected the skin inferior to the eye with alcohol swabs. We gently pushed the eyeball upwards with a gloved finger, assessing the elasticity of the eyeball just prior to injection. While the eyeball was gently pushed upward, we inserted the needle through the skin, just above the inferior orbital rim and slightly medial to the lateral canthus of the eye (Fig. 2). We then
FIGURE 1 | Palpation of the inferior orbital rim to identify the landmarks for proper insertion of the needle.
FIGURE 2 | Syringe-mounted needle is inserted just above the inferior periorbital rim slightly medial to the lateral canthus of the eye.
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CONCLUSIONS We chose to administer antibiotics Superior rectus muscle via deep subconjunctival injection in Bulbar fascia macaques with conjunctivitis because Sclera Cilia Retina Cornea of the successful use of this approach Optic nerve (CNI I) Conjunctiva Bulbar in humans. The bioavailability of the Adipose tissue antibiotic is enhanced because it is Inferior rectus muscle administered directly to the affected Orbital septum tissue. The deep subconjunctival injection disperses the drug throughout the Inferior orbital rim tissues of the anterior segments of the FIGURE 3 | Illustration of a sagittal section of the human orbit (adapted from eye and results in the drug being diffused ref. 30), showing the needle position for the deep subconjunctival injection into the anterior orbit18,19, substanrelative to the inferior orbital rim. tially increasing drug concentrations at the treatment site compared with systemic antibiotic administration. slowly advanced the needle posteriorly along the orbital Therapeutic levels of kanamycin or penicillin adminisfloor ~10–15 mm deep and into the subconjunctival tered via the subconjunctival route were found in tears, space (Fig. 3). conjunctiva, cornea and orbital lacrimal glands20 or in conjunctival sac fluid21, respectively. Although it is Before injecting the drug solution, we attempted to aspirate the needle in order to avoid injecting into expected that a small fraction of the drug is distributed a blood vessel. Because the eyeball occupies a small systemically22, we did not observe any effects of such fraction of the orbital space, the risk of puncturing the systemic drug distribution in our animals. Baum8 investigated topical ocular administration globe during deep subconjunctival injection is small. Still, we determined whether the needle had pen- routes intensively and concluded that administering drugs by subconjunctival injection results in higher etrated the ocular sclera by slowly moving the needle to the left and to the right in a sweeping manner to see tissue concentrations than administering drugs via eye drops owing to the limited holding capacity of the whether the eyeball moved as well. If the sclera had been entered, the needle was withdrawn and moved eye and tear production dynamics that result in rapid away from the globe to ensure it was in the subcon- draining of the eye drop solution. To some extent, junctival space. Then, the drug solution was slowly ointments have prolonged contact time compared injected in three increments over a period of 1–2 min with eye drops, but they produce negative sensations to allow it to distribute. After withdrawing the needle, in humans, e.g., perceived increased intraocular pressure and blurriness of vision. A major advantage of we assessed the tension of the orbit and its contents. It is essential that the globe be normotensive; a hard the subconjunctival injection technique over topical globe indicates excessive intraocular pressure which approaches is the reduced administration frequency (three injections per animal at intervals of 48 h versus must be relieved, if necessary, by lateral canthotomy. This is done by applying a hemostat to the lateral can- several topical applications daily for days or weeks). thus and cutting the clamped area with scissors. Neither This lower frequency not only reduced the workload of the veterinary and animal care staff but, more sutures nor cautery are usually required. Chemosis importantly, improved the comfort and welfare of the and lower lid swelling are commonly seen after the deep subconjunctival injections and need no treatment. treated monkeys by reducing handling frequency, We carried out these injections in each eye three times preventing unpleasant sensations associated with instillation of eye drops or ointment, preventing the at 48-h intervals. anxiety associated with awake restraint methods and treatment and reducing the grogginess induced by OUTCOME Our goal was to achieve a positive treatment outcome, frequent anesthesia. As with almost any administration technique, there defined as cured conjunctivitis as well as prevented is the potential for complications associated with deep recurrence. This was achieved in all three monkeys. Two of the monkeys had no recurrent conjunctivitis subconjunctival injections. Complications reported with injections into peribulbar tissues include the risk for at least 12 months. One monkey had a relapse of conjunctivitis after 6.5 months and was treated again of perforation and penetration of the globe23, severe increase in intraocular pressure when large volumes with the same technique; no further conjunctivitis was noted during the follow-up period of 5 months. We are injected24 and acute orbital hemorrhage25. These did not observe any adverse events associated with complications are more common with retrobulbar (posterior sub-Tenon’s) injections, which also pose the the treatment.
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Levator palpebrae superioris muscle
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risk of direct optic nerve injury26 because the needle is advanced further into the orbit. If the injection is administered too fast, there is greater likelihood of significant subconjunctival hemorrhage22,27; vessel damage leading to retrobulbar hematoma; chemosis; swollen, partially closed or shut eyes; and transient congestion28. However, the latter outcome is far more common with superficial subconjunctival injections in the eyelid than with the technique we describe. We did not detect any of these problems in our monkeys. In our use of the technique described, the sclera was never entered. Although chemosis is sometimes induced after injections into the superficial subconjunctival area in humans, we did not experience this outcome. Anatomical variations such as a long eye, shallow orbit, staphyloma or enophthalmos, as well as the use of a too-long needle, are also risk factors for globe perforation29. One of the most important safety measures is familiarity with the anatomy of the eye and orbit to avoid damaging any structures within the eye socket (not only the eyeball itself, but also extraocular muscles, nerves, blood vessels and soft tissue). Deep subconjunctival injection is a valuable and simple technique for the treatment of conjunctivitis in macaques. This approach has improved treatment success in three monkeys in our colony. The technique is reliable and effective, can be learned easily, is safe for use in humans and can be safe for use in monkeys as well if basic precautions are followed. The technique presents a refinement of prior therapeutic approaches in that it reduces stress in the treated animals and improves the effectiveness of treatment, complementing the effort to improve animal welfare in research. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Received 16 June 2014; accepted 28 July 2014 Published online at http://www.labanimal.com/ 1. Kahn, C.M. & Line, S. (eds) The Merck Veterinary Manual 10th edn. (Merck & Co., Inc. Whitehouse Station, NJ, 2010). 2. Ormerod, L.D. Causes and management of bacterial keratitis in the elderly. Can. J. Ophthalmol. 24, 112–116 (1989). 3. Rubenstein, J.B. & Virach, V. in Ophthalmology 3rd edn. (Yanoff, M. &Duker, J.S., eds) 1528–1540 (Mosby, Edinburgh, Scotland, 2008). 4. Hansen, B.C. in The Metabolic Syndrome: Epidemiology, Clinical Treatment, and Underlying Mechanisms (Hansen, B.C. & Bray, G.A., eds.) 373–386 (Humana Press, Totawa, NJ, 2008). 5. Nakagawa, H. Treatment of chlamydial conjunctivitis. Ophthalmologica 211 (suppl. 1), 25–28 (1997). 6. Ward, D.A. & Clark, E.S. Ocular pharmacology. Vet. Clin. North Am. Food Anim. Pract. 7, 779–791 (1991). 7. Matsuura, K. Pharmacokinetics of subconjunctival injection of moxifloxacin in humans. Graefes. Arch. Clin. Exp. Ophthalmol. 251, 1019–1020 (2013). 8. Baum, J. Treatment of bacterial ulcers of the cornea in the rabbit: a comparison of administration by eye drops and subconjunctival injections. Trans. Am. Ophthalmol. Soc. 80, 369–390 (1982).
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9. Feldman-Billard, S., Du Pasquier-Fediaevsky, L. & Héron, E. Hyperglycemia after repeated periocular dexamethasone injections in patients with diabetes. Ophthalmology 113, 1720–1723 (2006). 10. Regnier, A., Toutain, P.L., Alvinerie, M., Perique, B. & Ruckebusch, Y. Adrenocortical function and plasma biochemical values in dogs after subconjunctival treatment with methylprednisolone acetate. Res. Vet. Sci. 32, 306–310 (1982). 11. Hansen, B.C. in Diabetes Mellitus: A Fundamental and Clinical Text (eds. LeRoith, D., Olefsky J. & Taylor, S.) 1060–1074 (Lippincott Williams and Wilkins, Philadelphia, PA 2004). 12. Bodkin, N.L., Alexander, T.M., Ortmeyer, H.K., Johnson, E. & Hansen, B.C. Mortality and morbidity in laboratory-maintained Rhesus monkeys and effects of long-term dietary restriction. J. Gerontol. A. Biol. Sci. Med. Sci. 58, 212–219 (2003). 13. Animal Welfare Act as Amended (7 USC 2131–2156). 14. Institute for Laboratory Animal Research, National Research Council. Guide for the Care and Use of Laboratory Animals 8th edn. (National Academy Press, Washington, DC, 2011). 15. Ankel-Simons, F. Primate Anatomy, Third Edition: An Introduction (Academic, New York NY, 2007). 16. De Rousseau, C. & Bito, L. Intraocular pressure of rhesus monkeys (Macaca mulatta). II. Juvenile ocular hypertension and its apparent relationship to ocular growth. Exp. Eye Res. 32, 407–417 (1981). 17. Riordan-Eva, P. in Vaughan & Asbury′s General Ophthalmology 18th edn. (Riordan-Eva, P. & Cunningham, E.T. Jr., eds.) Ch. 1 (McGraw-Hill, New York, NY, 2011). 18. Miller, T.R. Principles of therapeutics. Vet. Clin. North Am. Equine Pract. 8, 479–497 (1992). 19. Gordon, T.B. & Cunningham, R.D. Tobramycin levels in aqueous humor after subconjunctival injection in humans. Am. J. Ophthalmol. 93, 107–110 (1982). 20. George, L.W., Reina-Guerra, M., Baggot, J.D. & Mihalyi, J. Distribution of kanamycin in ocular tissues of calves. J. Vet. Pharmacol. Ther. 9, 183–191 (1986). 21. Abeynayake, P. & Cooper, B.S. The concentration of penicillin in bovine conjunctival sac fluid as it pertains to the treatment of Moraxella bovis infection. (I) Subconjunctival injection. J. Vet. Pharmacol. Ther. 12, 25–30 (1989). 22. Douglas, L.C., Yi, N.Y., Davis, J.L., Salmon, J.H. & Gilger, B.C. Ocular toxicity and distribution of subconjunctival and intravitreal rapamycin in horses. J. Vet. Pharmacol. Ther. 31, 511–516 (2008). 23. McLeod, S. in Ophthalmology 3rd edn. (Yanoff, M. & Duker, J.S., eds.) 262–270 (Mosby, Edinburgh, Scotland, 2008). 24. Senturk, S., Cetin, C., Temizel, M. & Ozel, E. Evaluation of the clinical efficacy of subconjunctival injection of clindamycin in the treatment of naturally occurring infectious bovine keratoconjunctivitis. Vet. Ophthalmol. 10, 186–189 (2007). 25. Azarbod, P., Mohammed, Q., Akram, I. & Moorman, C. Localised abscess following an injection of subtenon triamcinolone acitonide. Eye (Lond.) 21, 672–674 (2007). 26. Kim, S.K., Andreoli, C.M., Rizzo, J.F. III, Golden, M.A. & Bradbury, M.J. Optic neuropathy secondary to sub-tenon anesthetic injection in cataract surgery. Arch. Ophthalmol. 121, 907–909 (2003). 27. Kaderli, B. & Avci, R. Comparison of topical and subconjunctival anesthesia in intravitreal injection administrations. Eur. J. Ophthalmol. 16, 718–721 (2006). 28. Veneziale, R.W. et al. SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys. Cutan. Ocul. Toxicol. 26, 83–105 (2007). 29. Kumar, C.M. & Dodds, C. Ophthalmic regional block. Ann. Acad. Med. Singapore 35, 158–167 (2006). 30. Tank, P.W. & Gest, T.R. Lippincott Williams & Wilkins Atlas of Anatomy xv, 353 (Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia. PA, 2008).
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