Veterinary Ophthalmology (2016) 19, 2, 110–116
DOI:10.1111/vop.12266
Clinical and histologic description of ocular anatomy in captive black-tailed prairie dogs (Cynomys ludovicianus) Jessica M. Meekins,* David Eshar,* Amy J. Rankin* and Jamie N. Henningson† *Department of Clinical Sciences, Kansas State University, Manhattan, KS, USA; and †Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA
Address communications to: J. Meekins Tel.: 785-532-5690 Fax: 785-532-4309 e-mail: [email protected]
Abstract Objective To describe the clinical and histologic ocular anatomy of the black-tailed prairie dog (PD). Animals studied Seventeen captive black-tailed PDs (11 males and six females), ranging in age from approximately 4 months to 4.5 years. Procedures Complete ocular examinations, including slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, were performed under isoflurane anesthesia. The globes (n = 2) of one black-tailed PD were harvested immediately after euthanasia and processed after formalin fixation. Staining with hematoxylin–eosin, cytokeratin AE1/AE3, glial fibrillary acidic protein, chromogranin A, claudin-5, smooth muscle actin, and vimentin was performed for light microscopic evaluation. Results A thick mucinous precorneal tear film was present on the ocular surface. A vestigial nictitating membrane was identified in the medial canthus area. The limbus was heavily pigmented, the iris was a dark homogenous brown, and the pupil was round. Funduscopically, there was no tapetum lucidum, the retinal vascular pattern was holangiotic, and a horizontally elongated optic disk was visualized. The most common ocular abnormalities were acquired eyelid margin defects, present in seven eyes of six black-tailed PDs (35.3%). On histologic examination, the retina was asymmetric, thicker below the optic disk and thinner above it. Conclusions The black-tailed PD fundus is atapetal with a holangiotic retinal vessel pattern and a horizontally elongated optic disk. Acquired lesions of the peri-ocular and eyelid region were the most common documented abnormality. Unique anatomic features of the globe and adnexa were confirmed with histologic and immunohistochemical analysis. Key Words: anatomy, Cynomys ludovicianus, eye, histology, ocular, prairie dog
INTRODUCTION
The black-tailed prairie dog (PD; Cynomys ludovicianus) is a member of the order Rodentia and the family Sciuridae (a family shared by squirrels and other small or mediumsized rodents).1 The PD is one of five keystone species, which is defined as a species that plays a critical role in maintaining the structure of its ecological community. They are found in the grasslands of North America, stretching from the United States–Canada border to the United States–Mexico border. Black-tailed PDs are the most common species found in zoological collections, research facilities, and private homes.1,2 However, descriptions of normal ocular anatomy in captive PDs are lacking.
To the authors’ knowledge, there are no publications describing normal ocular anatomy in this species. The purpose of this study was to describe the clinical and histologic anatomy of the globe and adnexa in captive PDs. MATERIALS AND METHODS
Seventeen PDs (C. ludovicianus) from two zoological collections (Manhattan, KS, and Milford, KS, USA) were admitted for an overall health examination. All examinations were performed at the Kansas State University Veterinary Health Center (KSU-VHC). The Kansas State University Institutional Animal Care and Use Committee approved this study. © 2015 American College of Veterinary Ophthalmologists
ocular anatomy in black-tailed prairie dogs 111
The PDs were transported to the KSU-VHC, then anesthetized for examination after arriving at the hospital. Anesthesia was achieved using a chamber induction with 5% isoflurane gas (IsoFlo; Abbott Laboratories, North Chicago, IL, USA) in 2 L/min of oxygen, followed by a small facemask and a Bain nonrebreathing anesthesia circuit. Anesthesia was maintained on 2.5% IsoFlo gas in 1.5 L/min of oxygen. Body temperature was monitored using a handheld rectal thermometer and maintained using a warm water blanket and heating packs. The heart rate was monitored by thoracic auscultation, and oxygen saturation was monitored by pulse oximetry (Nellcor Handheld Pulse Oximeter N20PA; Covidien, Dublin, Ireland). Respiratory rate and depth were monitored by visual observation. Following induction of anesthesia, each animal was weighed and a complete physical examination was performed. Complete blood count, serum biochemistry panel, and whole body radiographs were performed. A single board-certified ophthalmologist (JMM) performed all ocular examinations, immediately after anesthetic induction and as part of the physical examination. Examinations were conducted in dim lighting conditions and included slit-lamp biomicroscopy (SL-15; Kowa Co, Tokyo, Japan), indirect binocular ophthalmoscopy (Keeler Vantage Plus; Keeler Instruments, Inc., Broomall, PA, USA), and direct ophthalmoscopy (Welch Allyn direct ophthalmoscope, Skaneateles Falls, NY, USA). Pupil dilation was attempted using 1% tropicamide (Tropicamide Ophthalmic Solution; Akorn, Inc., Lake Forest, IL, USA); assessment of vision status was subjectively noted prior to induction of anesthesia by observing each animal’s ability to navigate within the confines of its transportation kennel. One PD was euthanized for reasons unrelated to the study (due to progressive systemic illness). The globes were harvested immediately after death. The right eye was enucleated by a subconjunctival approach, and the left eye was enucleated by a transpalpebral approach (to allow for analysis of adnexal structures). Histologic analysis was performed after fixation of the tissues in 10% neutral buffered formalin. Globes were sectioned in the dorsoventral plane, and paraffin wax-embedded tissues were sectioned and stained with hematoxylin–eosin (H&E). Hematoxylin– eosin were scanned at 9200 by a 3D Histotech Midi digital scanner, and then measurements of ocular structures were acquired using 3D HISTOTECH CASEVIEWER software (Budapest, Hungary). Additionally, immunohistochemical (IHC) staining with cytokeratin AE1/AE3 (pancytokeratin, ready to use [RTU]); Leica Biosystems (Buffalo Grove, IL, USA), glial fibrillary acidic protein (GFAP, 1:500; Dako, Carpinteria, CA, USA), claudin-5 (1:100, clone 4C3C2; Invitrogen Grand Island, NY, USA), smooth muscle actin (SMA, RTU, clone alpha sm-1; Leica), chromogranin A (1:500; Immunostar, Hudson, WI, USA), and vimentin (RTU, srl-33; Leica) was performed. Antigen retrieval for cytokeratin AE1/AE3, vimentin, and claudin5 consisted of EDTA pH 9.0 for 20 min at 100 °C; for
chromogranin A, proteinase K for 10 min at ambient temperature; and for SMA, citrate pH 6.0 for 10 min at 100 °C. Glial fibrillary acidic protein did not require antigen retrieval. Cytokeratin AE1/AE3, vimentin, SMA, and claudin-5 used the polymer-PowerVision-AP-a-mouse IgG and GFAP used the polymer AP-rabbit IgG. Cytokeratin, vimentin, SMA, GFAP, and claudin-5 were incubated for 15 min at ambient temperature and then developed using an alkaline phosphatase kit, the Bond Polymer Refine Red Detection Kit, on the automated Leica Bond. RESULTS
Black-tailed PDs ranged in age from approximately 4 months to 4.5 years, and body weights ranged from 582 to 1200 g for the 11 males and 759 to 1073 g for the six females. All but one PD was deemed healthy based on results of general physical examination, clinical pathology results, and diagnostic imaging. Clinical pathology testing and diagnostic imaging were performed as part of a concurrent study. The one unhealthy PD euthanized due to progressive systemic illness had a recent history of lower incisor overgrowth into the hard palate, which was the hypothesized reason for weight loss due to decreased food intake. Necropsy results were inconclusive in determining any other cause of illness. Because all animals required general anesthesia for handling and examination, menace response and other neuro-ophthalmic parameters influenced by anesthesia could not be assessed. The normal appearance of the PD eye was characterized (Fig. 1). A single row of cilia lined the dorsal and ventral eyelids. Lacrimal puncta were not identified. A thick, mucinous precorneal tear film coated the ocular surface. A vestigial, poorly developed nictitating membrane was located in the medial canthus. The limbus was heavily pigmented, the iris was a dark homogenous brown with no distinct zones of coloration, and the pupil was round. The pupil was poorly responsive to pharmacologic dilation in all animals. Funduscopically, there was no tapetum lucidum, the retinal vascular pattern was holangiotic, and a horizontally elongated optic disc was identified (Fig. 2). On gross examination during postmortem enucleation, a complete bony orbit was confirmed. A prominent
Figure 1. The normal right eye of a black-tailed prairie dog.
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Figure 2. Clinical image of the black-tailed prairie dog (PD) fundus (right eye) taken with the RetCam 3. The black-tailed PD fundus is atapetal with a holangiotic retinal vessel pattern and a horizontally elongated optic disc. There is a slight shadow artifact from the iris, as the image was obtained through a nondilated pupil.
cartilaginous shelf protruded from the dorsal orbital rim. Additionally, a large gland was present in the ventromedial anterior orbit, which was loosely adhered to the globe (Fig. 3). The most common ocular abnormalities were acquired eyelid margin and peri-ocular area defects (Fig. 4) in 6/17 (35.3%) of examined PDs. These abnormalities ranged from recent puncture wounds and superficial abrasions in the peri-ocular region to more chronic cicatrices of the eyelid margin and were presumed to be traumatic in origin. Five of six PDs were unilaterally affected with eyelid margin and peri-ocular area defects; one PD exhibited bilateral abnormalities, with a fresh puncture wound approximately 8 mm from the ventral eyelid margin of the right eye and a chronic eyelid margin defect occupying one-third of the eyelid length at the ventromedial lid of the left eye. Other abnormalities were uncommon and included an incipient anterior cortical cataract in one eye of one 4-year-old male
Figure 3. Photograph of a black-tailed prairie dog globe that was enucleated by the transpalpebral technique. A large orbital gland was present in the ventromedial anterior orbit and was loosely adherent to the globe. Histologically, the gland consisted of mucous-producing acinar cells. This gland is presumed to have a lacrimal function in secreting the prominent mucin component of the black-tailed prairie dog tear film.
Figure 4. An example of the most commonly documented ocular abnormality in black-tailed prairie dogs. An acquired eyelid margin defect was present and involving approximately 30% of the ventromedial lower left eyelid.
PD and an iris-to-iris persistent pupillary membrane in one eye of one 3-year-old female PD. Distinguishing features identified upon histologic examination of the globe and adnexa (Fig. 5) included many prominent meibomian glands within the eyelids (Fig. 5c) and a large orbital gland (Fig. 5d). In the conjunctiva, goblet cells were not observed in the bulbar conjunctiva but were dense in the fornices of the conjunctival pseudostratified epithelium (Fig. 5e,f). Within the conjunctival fornices, density of goblet cells varied; two highly goblet cell dense areas had eight to 10 goblet cells present per 100 lm, while less dense areas had three to four goblet cells per 100 lm. Histologically, the large orbital gland consisted diffusely of mucous acinar cells. The basal layer of the peripheral corneal epithelium contained pigment within the cytoplasm of cells. The cornea measured 382.8 lm in thickness. The ciliary body was composed of a pars plicata anteriorly and a pars plana posteriorly. An asymmetric retina, thicker ventrally below the horizontally elongated optic disc, was identified (Fig. 6); the retina measured 326.4 lm in thickness ventrally and 162.4 lm in thickness dorsally. Comparing the thinner retina to the thicker retina, there was a reduction in thickness of all cell layers that appeared to be proportional in the thinner segment of retina. The variation was due to relative thinning of all retinal cell layers and was not attributable to a specific cell layer. The ganglion cell density ranged from two to five large neuronal perikarya per 100 lm in all areas of the retina (thinner and thicker segments). Myelinated axon bundles from the horizontally elongated optic disc were observed to extend into the retina. No tapetum lucidum was observed. Immunohistochemistry for SMA demonstrated iridal sphincter and dilator muscles and the ciliary muscle at the periphery of the ciliary body, which attached to the
© 2015 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 19, 110–116
ocular anatomy in black-tailed prairie dogs 113
Figure 5. Representative histologic images of the black-tailed prairie dog globe and adnexa. (a) Photomicrograph of a globe (hematoxylin–eosin [H&E] stain, 910). (b) Photomicrograph of the anterior segment, illustrating the cornea, iris, and lens (H&E stain, 930). (c) Photomicrograph of prominent and numerous meibomian glands (arrows) in the eyelid (H&E stain, 9200). (d) Photomicrograph of the large orbital gland (H&E stain, 9200). (e) Photomicrograph of the conjunctival epithelium with numerous goblet cells (arrows; H&E stain, 9400). (f) Photomicrograph of numerous goblet cells stained bright pink (arrow) in the conjunctival epithelium (periodic acid–Schiff stain, 9400).
(a)
(b)
(c)
(d)
(e)
(f)
corneal stroma, sclera, and trabecular meshwork of the iridocorneal angle. The only areas of corneal epithelium positive for pancytokeratin were the peripheral pigmented corneal epithelium and the basal layer of epithelium. Claudin-5, a vascular marker, highlighted vessels in many parts of the eye, including the iris, choroid, and retina; vessels were not present in the cornea or lens. Complete results of IHC staining of the globe and adnexal tissues are reported in Table 1. DISCUSSION
This is the first study to describe normal ocular examination findings and the complete histologic anatomy of the PD eye. Menace response and other neuro-ophthalmic parameters influenced by anesthesia could not be directly assessed due to the necessity of general anesthesia to handle and examine the animals. However, evaluation of navigation within the transportation kennel indicated functional vision in each animal, and no ocular abnormalities expected to cause vision impairment were identified in any animal.
Specific features of PD ocular morphology were identified and are represented in Fig. 1. The tear film was noticeably thick as it coated the ocular surface. One hypothesis for the function of this tear film adaptation relates to the natural behavior of PDs as burrowing rodents; the act of digging through soil, grit, and other debris to create burrows as habitats poses a challenge to ocular surface protection. A more viscous tear film could protect the cornea from inadvertent damage. The presence of prominent meibomian glands supports the concept of a robust lipid component to the PD tear film. In addition, the large anterior orbital gland consisted entirely of mucous-producing cells in the areas examined, indicating an additional source of abundant mucin levels within the thick PD tear film. A vestigial nictitating membrane was identified at the medial canthus. Other rodents, such as the Canadian beaver,3 the capybara,4 and the chinchilla,5,6 have also been described to possess a vestigial nictitating membrane. Lacrimal puncta, the proximal openings to the mammalian nasolacrimal drainage apparatus, were not identified in this group of PDs.
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(a)
(b)
(c)
Figure 6. Representative histologic images of the black-tailed prairie dog ocular fundus. (a) Photomicrograph showing the asymmetric retina, thicker ventral to the horizontally elongated optic disc (hematoxylin–eosin [H&E] stain, 930). H&E stain. (b) Extensions of the horizontally elongated optic disc (arrows) extend into the retina (H&E stain, 9100). (c) Higher magnification of (b) illustrating the extensions from the optic nerve and the thick segment of the retina (H&E stain, 9200).
Another unique clinical feature of the PD eye was the heavy pigmentation present at the limbus. A pigmented basal corneal epithelial cell layer at the peripheral cornea supported this finding histologically. Peripheral corneal pigmentation was also noted clinically upon examination of a group of Canadian beavers3 and one each of a capybara4,7 and a PD.7 Pigment serves a protective role against ultraviolet light exposure, and it is possible that the heavy limbal corneal pigmentation noted clinically and histologically in these PDs could be an evolutionary adaptation to increased UV light exposure in the natural environment. Bowman’s layer, the basement membrane of the corneal epithelium that is observed in some species, was not readily apparent in histologically examined sections.
As complete evaluation of the posterior segment of the eye is greatly enhanced by pupil dilation, pharmacologic dilation was attempted in this group of PDs. The pupils of all 34 eyes failed to appropriately dilate with the use of topically applied 1% tropicamide. Tropicamide is a cholinergic antagonist drug that exerts its pupil dilating effect by paralysis of the iris sphincter muscle. It is most commonly used in a clinical examination setting for its relatively rapid onset and short duration of effect, to facilitate examination of the lens and posterior segment. Tropicamide targets cholinergic receptors in smooth muscle, and histologically, the PD iris possessed an iris sphincter composed of smooth muscle. Differences in iris musculature composition were eliminated as a possible reason for failure to respond to tropicamide. Rather, we suspect that the poor response of the PD pupil to pharmacologic dilation may have occurred as a result of melanin binding within the heavily pigmented iris tissue. Accumulation of topically administered drugs in the pigmented uveal tissue decreases free drug available for binding to target tissues.8 Melanin acts as a site of loss for the drug, and smaller initial ocular effects are seen due to decreased drug concentration at the target tissue. The mydriatic response of eyes with variably pigmented uveal tissue has been studied. Atropine is a cholinergic antagonist with a slower onset, longer duration of action, and increased cycloplegic effect when compared to tropicamide. Drug binding to melanin containing tissues explains the smaller initial response of more heavily pigmented eyes to the mydriatic effects of a cholinergic antagonist such as atropine.9 Given the heavy pigmentation of the PD iris, we suspect this phenomenon is responsible for failure of pharmacologic pupil dilation. The animals were observed for approximately 1 h after application of tropicamide, and no real mydriatic response was appreciated. However, quantification of the exact pupil diameter at baseline and at different time points after tropicamide application was not attempted, nor were the animals observed for longer than 1 h to determine delayed onset of action. Despite poor pupil dilation in all animals, funduscopic examination was attempted. The fundus lacked a tapetum, the retinal vessel pattern was holangiotic, and the optic disc was horizontally elongated in a bright streak. Histologically, the retina displayed an asymmetric morphology, with an increased thickness below the optic disc compared to above it. Measurements of histologic sections revealed that the ventral retinal thickness was approximately twice that of the dorsal retina. This retinal asymmetry has been previously described by Rodriguez-Ramos Fernandez and Dubielzig.7 We observed that the variation in retinal thickness was due to relative thinning of all retinal cell layers and was not attributable to a specific cell layer. Our study is the first to report extensions of the horizontally elongated optic disc that extend into the retina. Interestingly, these extensions are present throughout the entire course of the horizontal optic nerve head in the PD. In
© 2015 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 19, 110–116
ocular anatomy in black-tailed prairie dogs 115 Table 1. Summary of immunohistochemical results
Cornea Epithelium Stroma Endothelium Iris Stroma Muscles Epithelium Ciliary body Epithelium Stroma Vessels Muscles Lens Choroid Retina RPE Rods and cones Plexiform layers Ganglion layer Sclera Optic nerve Conjunctiva Epithelium Submucosa Vessels
Pancytokeratin
GFAP
Vimentin
Pos* Neg Neg
Neg Neg Neg
Neg Neg Pos
Neg Neg Neg
Neg Neg Neg
Pos Pos Pos
Neg Neg Neg Neg
Pos Pos Pos Pos
Neg
Neg Neg Neg Neg Neg Neg
Pos Neg Neg Neg Neg Neg
Neg Neg Neg Pos Neg Neg
Pos Neg Neg
Neg Neg Neg
Claudin-5
Chromogranin A
SMA
Neg Neg Neg
Neg Pos Pos Pos Pos Pos
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Pos Pos
Neg Neg Neg
Pos
Pos
Neg Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos
GFAP = Glial fibrillary acidic protein; SMA = smooth muscle actin. *Weak positive staining of basal epithelium.
comparison, the extensions in dogs, cats, and humans converge in one location at the optic nerve head. Acquired eyelid lesions were the predominant ocular abnormality in this group of captive PDs. Five of six animals were unilaterally affected with varying degrees of eyelid margin cicatrization or excoriation. Capturerelated trauma may explain the acute lesions (e.g. excoriations) that were observed, as the PDs were captured in nets prior to transportation to the KSU-VHC. However, we hypothesize that interanimal aggression was a likely cause of the eyelid lesions, especially the chronic lesions (e.g. scars). Fights and territorial disputes have been documented in wild PDs; these aggressive behaviors are considered one of the costs of coloniality in a species that lives in a densely populated area with competition for limited resources such as food and mates.10 While competition for resources is essentially eliminated in captivity, it is likely that PDs may still exhibit behaviors that mimic those observed in the wild. The PD euthanized due to general and progressive ill thrift was the only animal with bilateral eyelid involvement, and his eyelid lesions appeared to be the most recently acquired of those affected in the group. Interestingly, ocular abnormalities in a group of Canadian beavers were also primarily attributable to trauma of the eyelids and peri-orbital area.3 Other ocular abnormalities in our group of animals were rare and included an incipient
anterior cortical cataract in one eye of one PD and an iris-to-iris persistent pupillary membrane in one eye of one PD. Immunohistochemical stains are commonly used to diagnose anaplastic tumors in all species. The IHC stains pancytokeratin, GFAP, vimentin, claudin-5, chromogranin A, and SMA are commonly used to identify the origin of tumors in many species. As IHC analysis has never been reported in the eye of the PD, these stains were performed to establish a baseline for tumor diagnosis by providing information on how normal ocular structures label with particular IHC stains. Immunohistochemical positive staining in the PD globe occurred in anatomic locations similar to domestic species.11 Based on the results of this study, peri-ocular abnormalities were relatively common, while the prevalence of intraocular abnormalities appears to be low in captive PDs. Our description of normal clinical and histologic ocular and adnexal anatomy in this animal provides valuable information for the diagnosis and management of ocular diseases in this species. ACKNOWLEDGMENTS
This study was supported by a research grant from the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University. The authors would
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like to thank the Sunset Zoo, Manhattan, KS, and the Milford Nature Center, Junction City, KS, for the use of their animals. We also thank Mal Rooks Hoover, CMI, for her assistance with the preparation of figures. REFERENCES 1. Hoogland JL, James DA, Watson L. Nutrition, care, and behavior of captive prairie dogs. The Veterinary Clinics of North America. Exotic Animal Practice 2009; 12: 255–266. 2. Lennox AM. Emergency and critical care procedures in sugar gliders (Petaurus breviceps), African hedgehogs (Atelerix albiventris), and prairie dogs (Cynomys spp). The Veterinary Clinics of North America. Exotic Animal Practice 2007; 10: 533–555. 3. Cullen CL. Normal ocular features, conjunctival microflora and intraocular pressure in the Canadian beaver (Castor canadensis). Veterinary Ophthalmology 2003; 6: 279–284. 4. Montiani-Ferreira F, Truppel J, Tramontin MH et al. The capybara eye: clinical tests, anatomic and biometric features. Veterinary Ophthalmology 2008; 11: 386–394.
5. Peiffer RL, Johnson PT. Clinical ocular findings in a colony of chinchillas (Chinchilla laniger). Laboratory Animals 1980; 14: 331– 335. 6. Voigt S, Fuchs-Baumgartinger A, Egerbacher M et al. Investigations on the conjunctival goblet cells and the characteristics of the glands associated with the eye in chinchillas (Chinchilla laniger). Veterinary Ophthalmology 2012; 15: 333–344. 7. Rodriguez-Ramos Fernandez J, Dubielzig RR. Ocular comparative anatomy of the family Rodentia. Veterinary Ophthalmology 2013; 16(Suppl 1): 94–99. 8. Sasaki H, Yamamura K, Nishida K et al. Delivery of drugs to the eye by topical application. Progress in Retinal and Eye Research 1996; 15: 583–620. 9. Salazar M, Shimada K, Patil PN. Iris pigmentation and atropine mydriasis. Journal of Pharmacology and Experimental Therapeutics 1976; 197: 79–88. 10. Hoogland JL. Aggression, ecto-parasitism, and other possible costs of prairie dog (Sciuridae, Cynomys spp) coloniality. Behaviour 1979; 69: 1–35. 11. Labelle P, Reilly CM, Naydan DK et al. Immunohistochemical characteristics of normal canine eyes. Veterinary Pathology 2012; 49: 860–869.
© 2015 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 19, 110–116
DOI:10.1111/vop.12266
Clinical and histologic description of ocular anatomy in captive black-tailed prairie dogs (Cynomys ludovicianus) Jessica M. Meekins,* David Eshar,* Amy J. Rankin* and Jamie N. Henningson† *Department of Clinical Sciences, Kansas State University, Manhattan, KS, USA; and †Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA
Address communications to: J. Meekins Tel.: 785-532-5690 Fax: 785-532-4309 e-mail: [email protected]
Abstract Objective To describe the clinical and histologic ocular anatomy of the black-tailed prairie dog (PD). Animals studied Seventeen captive black-tailed PDs (11 males and six females), ranging in age from approximately 4 months to 4.5 years. Procedures Complete ocular examinations, including slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, were performed under isoflurane anesthesia. The globes (n = 2) of one black-tailed PD were harvested immediately after euthanasia and processed after formalin fixation. Staining with hematoxylin–eosin, cytokeratin AE1/AE3, glial fibrillary acidic protein, chromogranin A, claudin-5, smooth muscle actin, and vimentin was performed for light microscopic evaluation. Results A thick mucinous precorneal tear film was present on the ocular surface. A vestigial nictitating membrane was identified in the medial canthus area. The limbus was heavily pigmented, the iris was a dark homogenous brown, and the pupil was round. Funduscopically, there was no tapetum lucidum, the retinal vascular pattern was holangiotic, and a horizontally elongated optic disk was visualized. The most common ocular abnormalities were acquired eyelid margin defects, present in seven eyes of six black-tailed PDs (35.3%). On histologic examination, the retina was asymmetric, thicker below the optic disk and thinner above it. Conclusions The black-tailed PD fundus is atapetal with a holangiotic retinal vessel pattern and a horizontally elongated optic disk. Acquired lesions of the peri-ocular and eyelid region were the most common documented abnormality. Unique anatomic features of the globe and adnexa were confirmed with histologic and immunohistochemical analysis. Key Words: anatomy, Cynomys ludovicianus, eye, histology, ocular, prairie dog
INTRODUCTION
The black-tailed prairie dog (PD; Cynomys ludovicianus) is a member of the order Rodentia and the family Sciuridae (a family shared by squirrels and other small or mediumsized rodents).1 The PD is one of five keystone species, which is defined as a species that plays a critical role in maintaining the structure of its ecological community. They are found in the grasslands of North America, stretching from the United States–Canada border to the United States–Mexico border. Black-tailed PDs are the most common species found in zoological collections, research facilities, and private homes.1,2 However, descriptions of normal ocular anatomy in captive PDs are lacking.
To the authors’ knowledge, there are no publications describing normal ocular anatomy in this species. The purpose of this study was to describe the clinical and histologic anatomy of the globe and adnexa in captive PDs. MATERIALS AND METHODS
Seventeen PDs (C. ludovicianus) from two zoological collections (Manhattan, KS, and Milford, KS, USA) were admitted for an overall health examination. All examinations were performed at the Kansas State University Veterinary Health Center (KSU-VHC). The Kansas State University Institutional Animal Care and Use Committee approved this study. © 2015 American College of Veterinary Ophthalmologists
ocular anatomy in black-tailed prairie dogs 111
The PDs were transported to the KSU-VHC, then anesthetized for examination after arriving at the hospital. Anesthesia was achieved using a chamber induction with 5% isoflurane gas (IsoFlo; Abbott Laboratories, North Chicago, IL, USA) in 2 L/min of oxygen, followed by a small facemask and a Bain nonrebreathing anesthesia circuit. Anesthesia was maintained on 2.5% IsoFlo gas in 1.5 L/min of oxygen. Body temperature was monitored using a handheld rectal thermometer and maintained using a warm water blanket and heating packs. The heart rate was monitored by thoracic auscultation, and oxygen saturation was monitored by pulse oximetry (Nellcor Handheld Pulse Oximeter N20PA; Covidien, Dublin, Ireland). Respiratory rate and depth were monitored by visual observation. Following induction of anesthesia, each animal was weighed and a complete physical examination was performed. Complete blood count, serum biochemistry panel, and whole body radiographs were performed. A single board-certified ophthalmologist (JMM) performed all ocular examinations, immediately after anesthetic induction and as part of the physical examination. Examinations were conducted in dim lighting conditions and included slit-lamp biomicroscopy (SL-15; Kowa Co, Tokyo, Japan), indirect binocular ophthalmoscopy (Keeler Vantage Plus; Keeler Instruments, Inc., Broomall, PA, USA), and direct ophthalmoscopy (Welch Allyn direct ophthalmoscope, Skaneateles Falls, NY, USA). Pupil dilation was attempted using 1% tropicamide (Tropicamide Ophthalmic Solution; Akorn, Inc., Lake Forest, IL, USA); assessment of vision status was subjectively noted prior to induction of anesthesia by observing each animal’s ability to navigate within the confines of its transportation kennel. One PD was euthanized for reasons unrelated to the study (due to progressive systemic illness). The globes were harvested immediately after death. The right eye was enucleated by a subconjunctival approach, and the left eye was enucleated by a transpalpebral approach (to allow for analysis of adnexal structures). Histologic analysis was performed after fixation of the tissues in 10% neutral buffered formalin. Globes were sectioned in the dorsoventral plane, and paraffin wax-embedded tissues were sectioned and stained with hematoxylin–eosin (H&E). Hematoxylin– eosin were scanned at 9200 by a 3D Histotech Midi digital scanner, and then measurements of ocular structures were acquired using 3D HISTOTECH CASEVIEWER software (Budapest, Hungary). Additionally, immunohistochemical (IHC) staining with cytokeratin AE1/AE3 (pancytokeratin, ready to use [RTU]); Leica Biosystems (Buffalo Grove, IL, USA), glial fibrillary acidic protein (GFAP, 1:500; Dako, Carpinteria, CA, USA), claudin-5 (1:100, clone 4C3C2; Invitrogen Grand Island, NY, USA), smooth muscle actin (SMA, RTU, clone alpha sm-1; Leica), chromogranin A (1:500; Immunostar, Hudson, WI, USA), and vimentin (RTU, srl-33; Leica) was performed. Antigen retrieval for cytokeratin AE1/AE3, vimentin, and claudin5 consisted of EDTA pH 9.0 for 20 min at 100 °C; for
chromogranin A, proteinase K for 10 min at ambient temperature; and for SMA, citrate pH 6.0 for 10 min at 100 °C. Glial fibrillary acidic protein did not require antigen retrieval. Cytokeratin AE1/AE3, vimentin, SMA, and claudin-5 used the polymer-PowerVision-AP-a-mouse IgG and GFAP used the polymer AP-rabbit IgG. Cytokeratin, vimentin, SMA, GFAP, and claudin-5 were incubated for 15 min at ambient temperature and then developed using an alkaline phosphatase kit, the Bond Polymer Refine Red Detection Kit, on the automated Leica Bond. RESULTS
Black-tailed PDs ranged in age from approximately 4 months to 4.5 years, and body weights ranged from 582 to 1200 g for the 11 males and 759 to 1073 g for the six females. All but one PD was deemed healthy based on results of general physical examination, clinical pathology results, and diagnostic imaging. Clinical pathology testing and diagnostic imaging were performed as part of a concurrent study. The one unhealthy PD euthanized due to progressive systemic illness had a recent history of lower incisor overgrowth into the hard palate, which was the hypothesized reason for weight loss due to decreased food intake. Necropsy results were inconclusive in determining any other cause of illness. Because all animals required general anesthesia for handling and examination, menace response and other neuro-ophthalmic parameters influenced by anesthesia could not be assessed. The normal appearance of the PD eye was characterized (Fig. 1). A single row of cilia lined the dorsal and ventral eyelids. Lacrimal puncta were not identified. A thick, mucinous precorneal tear film coated the ocular surface. A vestigial, poorly developed nictitating membrane was located in the medial canthus. The limbus was heavily pigmented, the iris was a dark homogenous brown with no distinct zones of coloration, and the pupil was round. The pupil was poorly responsive to pharmacologic dilation in all animals. Funduscopically, there was no tapetum lucidum, the retinal vascular pattern was holangiotic, and a horizontally elongated optic disc was identified (Fig. 2). On gross examination during postmortem enucleation, a complete bony orbit was confirmed. A prominent
Figure 1. The normal right eye of a black-tailed prairie dog.
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Figure 2. Clinical image of the black-tailed prairie dog (PD) fundus (right eye) taken with the RetCam 3. The black-tailed PD fundus is atapetal with a holangiotic retinal vessel pattern and a horizontally elongated optic disc. There is a slight shadow artifact from the iris, as the image was obtained through a nondilated pupil.
cartilaginous shelf protruded from the dorsal orbital rim. Additionally, a large gland was present in the ventromedial anterior orbit, which was loosely adhered to the globe (Fig. 3). The most common ocular abnormalities were acquired eyelid margin and peri-ocular area defects (Fig. 4) in 6/17 (35.3%) of examined PDs. These abnormalities ranged from recent puncture wounds and superficial abrasions in the peri-ocular region to more chronic cicatrices of the eyelid margin and were presumed to be traumatic in origin. Five of six PDs were unilaterally affected with eyelid margin and peri-ocular area defects; one PD exhibited bilateral abnormalities, with a fresh puncture wound approximately 8 mm from the ventral eyelid margin of the right eye and a chronic eyelid margin defect occupying one-third of the eyelid length at the ventromedial lid of the left eye. Other abnormalities were uncommon and included an incipient anterior cortical cataract in one eye of one 4-year-old male
Figure 3. Photograph of a black-tailed prairie dog globe that was enucleated by the transpalpebral technique. A large orbital gland was present in the ventromedial anterior orbit and was loosely adherent to the globe. Histologically, the gland consisted of mucous-producing acinar cells. This gland is presumed to have a lacrimal function in secreting the prominent mucin component of the black-tailed prairie dog tear film.
Figure 4. An example of the most commonly documented ocular abnormality in black-tailed prairie dogs. An acquired eyelid margin defect was present and involving approximately 30% of the ventromedial lower left eyelid.
PD and an iris-to-iris persistent pupillary membrane in one eye of one 3-year-old female PD. Distinguishing features identified upon histologic examination of the globe and adnexa (Fig. 5) included many prominent meibomian glands within the eyelids (Fig. 5c) and a large orbital gland (Fig. 5d). In the conjunctiva, goblet cells were not observed in the bulbar conjunctiva but were dense in the fornices of the conjunctival pseudostratified epithelium (Fig. 5e,f). Within the conjunctival fornices, density of goblet cells varied; two highly goblet cell dense areas had eight to 10 goblet cells present per 100 lm, while less dense areas had three to four goblet cells per 100 lm. Histologically, the large orbital gland consisted diffusely of mucous acinar cells. The basal layer of the peripheral corneal epithelium contained pigment within the cytoplasm of cells. The cornea measured 382.8 lm in thickness. The ciliary body was composed of a pars plicata anteriorly and a pars plana posteriorly. An asymmetric retina, thicker ventrally below the horizontally elongated optic disc, was identified (Fig. 6); the retina measured 326.4 lm in thickness ventrally and 162.4 lm in thickness dorsally. Comparing the thinner retina to the thicker retina, there was a reduction in thickness of all cell layers that appeared to be proportional in the thinner segment of retina. The variation was due to relative thinning of all retinal cell layers and was not attributable to a specific cell layer. The ganglion cell density ranged from two to five large neuronal perikarya per 100 lm in all areas of the retina (thinner and thicker segments). Myelinated axon bundles from the horizontally elongated optic disc were observed to extend into the retina. No tapetum lucidum was observed. Immunohistochemistry for SMA demonstrated iridal sphincter and dilator muscles and the ciliary muscle at the periphery of the ciliary body, which attached to the
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ocular anatomy in black-tailed prairie dogs 113
Figure 5. Representative histologic images of the black-tailed prairie dog globe and adnexa. (a) Photomicrograph of a globe (hematoxylin–eosin [H&E] stain, 910). (b) Photomicrograph of the anterior segment, illustrating the cornea, iris, and lens (H&E stain, 930). (c) Photomicrograph of prominent and numerous meibomian glands (arrows) in the eyelid (H&E stain, 9200). (d) Photomicrograph of the large orbital gland (H&E stain, 9200). (e) Photomicrograph of the conjunctival epithelium with numerous goblet cells (arrows; H&E stain, 9400). (f) Photomicrograph of numerous goblet cells stained bright pink (arrow) in the conjunctival epithelium (periodic acid–Schiff stain, 9400).
(a)
(b)
(c)
(d)
(e)
(f)
corneal stroma, sclera, and trabecular meshwork of the iridocorneal angle. The only areas of corneal epithelium positive for pancytokeratin were the peripheral pigmented corneal epithelium and the basal layer of epithelium. Claudin-5, a vascular marker, highlighted vessels in many parts of the eye, including the iris, choroid, and retina; vessels were not present in the cornea or lens. Complete results of IHC staining of the globe and adnexal tissues are reported in Table 1. DISCUSSION
This is the first study to describe normal ocular examination findings and the complete histologic anatomy of the PD eye. Menace response and other neuro-ophthalmic parameters influenced by anesthesia could not be directly assessed due to the necessity of general anesthesia to handle and examine the animals. However, evaluation of navigation within the transportation kennel indicated functional vision in each animal, and no ocular abnormalities expected to cause vision impairment were identified in any animal.
Specific features of PD ocular morphology were identified and are represented in Fig. 1. The tear film was noticeably thick as it coated the ocular surface. One hypothesis for the function of this tear film adaptation relates to the natural behavior of PDs as burrowing rodents; the act of digging through soil, grit, and other debris to create burrows as habitats poses a challenge to ocular surface protection. A more viscous tear film could protect the cornea from inadvertent damage. The presence of prominent meibomian glands supports the concept of a robust lipid component to the PD tear film. In addition, the large anterior orbital gland consisted entirely of mucous-producing cells in the areas examined, indicating an additional source of abundant mucin levels within the thick PD tear film. A vestigial nictitating membrane was identified at the medial canthus. Other rodents, such as the Canadian beaver,3 the capybara,4 and the chinchilla,5,6 have also been described to possess a vestigial nictitating membrane. Lacrimal puncta, the proximal openings to the mammalian nasolacrimal drainage apparatus, were not identified in this group of PDs.
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(a)
(b)
(c)
Figure 6. Representative histologic images of the black-tailed prairie dog ocular fundus. (a) Photomicrograph showing the asymmetric retina, thicker ventral to the horizontally elongated optic disc (hematoxylin–eosin [H&E] stain, 930). H&E stain. (b) Extensions of the horizontally elongated optic disc (arrows) extend into the retina (H&E stain, 9100). (c) Higher magnification of (b) illustrating the extensions from the optic nerve and the thick segment of the retina (H&E stain, 9200).
Another unique clinical feature of the PD eye was the heavy pigmentation present at the limbus. A pigmented basal corneal epithelial cell layer at the peripheral cornea supported this finding histologically. Peripheral corneal pigmentation was also noted clinically upon examination of a group of Canadian beavers3 and one each of a capybara4,7 and a PD.7 Pigment serves a protective role against ultraviolet light exposure, and it is possible that the heavy limbal corneal pigmentation noted clinically and histologically in these PDs could be an evolutionary adaptation to increased UV light exposure in the natural environment. Bowman’s layer, the basement membrane of the corneal epithelium that is observed in some species, was not readily apparent in histologically examined sections.
As complete evaluation of the posterior segment of the eye is greatly enhanced by pupil dilation, pharmacologic dilation was attempted in this group of PDs. The pupils of all 34 eyes failed to appropriately dilate with the use of topically applied 1% tropicamide. Tropicamide is a cholinergic antagonist drug that exerts its pupil dilating effect by paralysis of the iris sphincter muscle. It is most commonly used in a clinical examination setting for its relatively rapid onset and short duration of effect, to facilitate examination of the lens and posterior segment. Tropicamide targets cholinergic receptors in smooth muscle, and histologically, the PD iris possessed an iris sphincter composed of smooth muscle. Differences in iris musculature composition were eliminated as a possible reason for failure to respond to tropicamide. Rather, we suspect that the poor response of the PD pupil to pharmacologic dilation may have occurred as a result of melanin binding within the heavily pigmented iris tissue. Accumulation of topically administered drugs in the pigmented uveal tissue decreases free drug available for binding to target tissues.8 Melanin acts as a site of loss for the drug, and smaller initial ocular effects are seen due to decreased drug concentration at the target tissue. The mydriatic response of eyes with variably pigmented uveal tissue has been studied. Atropine is a cholinergic antagonist with a slower onset, longer duration of action, and increased cycloplegic effect when compared to tropicamide. Drug binding to melanin containing tissues explains the smaller initial response of more heavily pigmented eyes to the mydriatic effects of a cholinergic antagonist such as atropine.9 Given the heavy pigmentation of the PD iris, we suspect this phenomenon is responsible for failure of pharmacologic pupil dilation. The animals were observed for approximately 1 h after application of tropicamide, and no real mydriatic response was appreciated. However, quantification of the exact pupil diameter at baseline and at different time points after tropicamide application was not attempted, nor were the animals observed for longer than 1 h to determine delayed onset of action. Despite poor pupil dilation in all animals, funduscopic examination was attempted. The fundus lacked a tapetum, the retinal vessel pattern was holangiotic, and the optic disc was horizontally elongated in a bright streak. Histologically, the retina displayed an asymmetric morphology, with an increased thickness below the optic disc compared to above it. Measurements of histologic sections revealed that the ventral retinal thickness was approximately twice that of the dorsal retina. This retinal asymmetry has been previously described by Rodriguez-Ramos Fernandez and Dubielzig.7 We observed that the variation in retinal thickness was due to relative thinning of all retinal cell layers and was not attributable to a specific cell layer. Our study is the first to report extensions of the horizontally elongated optic disc that extend into the retina. Interestingly, these extensions are present throughout the entire course of the horizontal optic nerve head in the PD. In
© 2015 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 19, 110–116
ocular anatomy in black-tailed prairie dogs 115 Table 1. Summary of immunohistochemical results
Cornea Epithelium Stroma Endothelium Iris Stroma Muscles Epithelium Ciliary body Epithelium Stroma Vessels Muscles Lens Choroid Retina RPE Rods and cones Plexiform layers Ganglion layer Sclera Optic nerve Conjunctiva Epithelium Submucosa Vessels
Pancytokeratin
GFAP
Vimentin
Pos* Neg Neg
Neg Neg Neg
Neg Neg Pos
Neg Neg Neg
Neg Neg Neg
Pos Pos Pos
Neg Neg Neg Neg
Pos Pos Pos Pos
Neg
Neg Neg Neg Neg Neg Neg
Pos Neg Neg Neg Neg Neg
Neg Neg Neg Pos Neg Neg
Pos Neg Neg
Neg Neg Neg
Claudin-5
Chromogranin A
SMA
Neg Neg Neg
Neg Pos Pos Pos Pos Pos
Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Pos Pos
Neg Neg Neg
Pos
Pos
Neg Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos
GFAP = Glial fibrillary acidic protein; SMA = smooth muscle actin. *Weak positive staining of basal epithelium.
comparison, the extensions in dogs, cats, and humans converge in one location at the optic nerve head. Acquired eyelid lesions were the predominant ocular abnormality in this group of captive PDs. Five of six animals were unilaterally affected with varying degrees of eyelid margin cicatrization or excoriation. Capturerelated trauma may explain the acute lesions (e.g. excoriations) that were observed, as the PDs were captured in nets prior to transportation to the KSU-VHC. However, we hypothesize that interanimal aggression was a likely cause of the eyelid lesions, especially the chronic lesions (e.g. scars). Fights and territorial disputes have been documented in wild PDs; these aggressive behaviors are considered one of the costs of coloniality in a species that lives in a densely populated area with competition for limited resources such as food and mates.10 While competition for resources is essentially eliminated in captivity, it is likely that PDs may still exhibit behaviors that mimic those observed in the wild. The PD euthanized due to general and progressive ill thrift was the only animal with bilateral eyelid involvement, and his eyelid lesions appeared to be the most recently acquired of those affected in the group. Interestingly, ocular abnormalities in a group of Canadian beavers were also primarily attributable to trauma of the eyelids and peri-orbital area.3 Other ocular abnormalities in our group of animals were rare and included an incipient
anterior cortical cataract in one eye of one PD and an iris-to-iris persistent pupillary membrane in one eye of one PD. Immunohistochemical stains are commonly used to diagnose anaplastic tumors in all species. The IHC stains pancytokeratin, GFAP, vimentin, claudin-5, chromogranin A, and SMA are commonly used to identify the origin of tumors in many species. As IHC analysis has never been reported in the eye of the PD, these stains were performed to establish a baseline for tumor diagnosis by providing information on how normal ocular structures label with particular IHC stains. Immunohistochemical positive staining in the PD globe occurred in anatomic locations similar to domestic species.11 Based on the results of this study, peri-ocular abnormalities were relatively common, while the prevalence of intraocular abnormalities appears to be low in captive PDs. Our description of normal clinical and histologic ocular and adnexal anatomy in this animal provides valuable information for the diagnosis and management of ocular diseases in this species. ACKNOWLEDGMENTS
This study was supported by a research grant from the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University. The authors would
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like to thank the Sunset Zoo, Manhattan, KS, and the Milford Nature Center, Junction City, KS, for the use of their animals. We also thank Mal Rooks Hoover, CMI, for her assistance with the preparation of figures. REFERENCES 1. Hoogland JL, James DA, Watson L. Nutrition, care, and behavior of captive prairie dogs. The Veterinary Clinics of North America. Exotic Animal Practice 2009; 12: 255–266. 2. Lennox AM. Emergency and critical care procedures in sugar gliders (Petaurus breviceps), African hedgehogs (Atelerix albiventris), and prairie dogs (Cynomys spp). The Veterinary Clinics of North America. Exotic Animal Practice 2007; 10: 533–555. 3. Cullen CL. Normal ocular features, conjunctival microflora and intraocular pressure in the Canadian beaver (Castor canadensis). Veterinary Ophthalmology 2003; 6: 279–284. 4. Montiani-Ferreira F, Truppel J, Tramontin MH et al. The capybara eye: clinical tests, anatomic and biometric features. Veterinary Ophthalmology 2008; 11: 386–394.
5. Peiffer RL, Johnson PT. Clinical ocular findings in a colony of chinchillas (Chinchilla laniger). Laboratory Animals 1980; 14: 331– 335. 6. Voigt S, Fuchs-Baumgartinger A, Egerbacher M et al. Investigations on the conjunctival goblet cells and the characteristics of the glands associated with the eye in chinchillas (Chinchilla laniger). Veterinary Ophthalmology 2012; 15: 333–344. 7. Rodriguez-Ramos Fernandez J, Dubielzig RR. Ocular comparative anatomy of the family Rodentia. Veterinary Ophthalmology 2013; 16(Suppl 1): 94–99. 8. Sasaki H, Yamamura K, Nishida K et al. Delivery of drugs to the eye by topical application. Progress in Retinal and Eye Research 1996; 15: 583–620. 9. Salazar M, Shimada K, Patil PN. Iris pigmentation and atropine mydriasis. Journal of Pharmacology and Experimental Therapeutics 1976; 197: 79–88. 10. Hoogland JL. Aggression, ecto-parasitism, and other possible costs of prairie dog (Sciuridae, Cynomys spp) coloniality. Behaviour 1979; 69: 1–35. 11. Labelle P, Reilly CM, Naydan DK et al. Immunohistochemical characteristics of normal canine eyes. Veterinary Pathology 2012; 49: 860–869.
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