BASIC INVESTIGATION
A New Nonhuman Primate Model of Severe Dry Eye Yi Qin, MD,* Xiaobo Tan, MD, PhD,* Yingnan Zhang, MD, PhD,* Ying Jie, MD, PhD,* Antoine Labbe, MD, PhD,*†‡ and Zhiqiang Pan, MD, PhD*
Purpose: The aim of this study was to establish a new rhesus monkey model of severe dry eye.
Key Words: dry eye, animal model, rhesus monkey, impression cytology, lacrimal gland (Cornea 2014;33:510–517)
Methods: A total of 8 rhesus monkeys were used for the study. Four monkeys had their main lacrimal gland and nictitating membrane surgically removed (group 1). Another 4 monkeys had a similar surgery with further application of 50% trichloroacetic acid on the bulbar conjunctiva (group 2). The ocular surface was evaluated before and after the surgery (1, 4, 8, 12, and 24 weeks) using Schirmer-1 test, corneal fluorescein staining, and the lissamine green test. Conjunctival impression cytology was also performed before and 24 weeks after the surgery. Finally, the cornea and the conjunctiva were evaluated using light microscopy.
Results: A significant decrease in tear secretion was observed in all operated eyes. Schirmer test data measured were #4 mm in all the operated eyes. Slit-lamp examination also revealed abnormal staining in all the operated eyes that remained stable until the end of the experiment. In group 2, corneal fluorescein staining and lissamine green test values were always $5 (max 12) and $4 (max 9), respectively. Impression cytology specimens of both the treated groups showed conjunctival squamous metaplasia and a decreased number of goblet cells. Under light microscopy, the corneal epithelium appeared irregular with edematous basal epithelial cells. The conjunctiva showed a decreased goblet cell density with infiltration of inflammatory cells. Conclusions: Complete removal of the principal lacrimal gland and nictitating membrane associated with the application of 50% trichloroacetic acid on the conjunctiva could induce severe dry eye in rhesus monkeys.
Received for publication November 14, 2013; revision received January 5, 2014; accepted January 7, 2014. Published online ahead of print February 26, 2014. From the *Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Lab, Beijing, China; †Department of Ophthalmology, Quinze-Vingts National Ophthalmology Hospital, Paris and Versailles Saint-Quentin-enYvelines University, Versailles, France; and ‡INSERM, U968, UPMC Univ Paris 06, UMR S 968, Institut de la Vision, CNRS, UMR 7210, Paris, France. Supported by the Beijing Health Systems High-level Health and Technical Talent Training Plan (2009-2-05) and the National Natural Science Foundation of China, project No. 30801264. The authors have no conflicts of interest to disclose. Reprints: Zhiqiang Pan, Beijing Tongren Eye Center, Beijing Tongren Hospital, 1# Dong Jiao Min Xiang, Beijing 100730, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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ry eye is one of the most common ocular diseases and may affect from 5% to .30% of people aged $50 years.1 According to the 2007 Dry Eye Workshop, dry eye is a multifactorial disease of tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability.2 Severe dry eye may also lead to corneal ulceration, opacification, and even blindness.3 Despite recent advances in the treatment of dry eye with a new antiinflammatory drug such as cyclosporine A,4 the management of patients with severe dry eye remains difficult and is often unsatisfactory.5 In patients with severe pathology despite medical therapy, a new surgical approach, transplantation of salivary glands, has been proposed.6 In particular, the transplantation of minor salivary glands could be an effective treatment option for severe dry eyes, because it is a simple procedure and has minimal surgical risks.7 Promising results have thus been reported in patients with severe dry eye because of chemical burns or Stevens–Johnson syndrome.7–10 Nevertheless, with very few published studies available, little is known about the survival of the glands, the characteristics of the salivary tear film, and the benefit for the ocular surface.7–10 Consequently, animal models of severe dry eye that allow one to study the mechanisms and to evaluate the results of new surgical strategies, such as salivary gland transplantation, are needed.11 Numerous animal models, including monkey, dog, rabbit, rat, and mouse have been developed to mimic different pathophysiologic mechanisms involved in dry eye.12 The choice of an animal model plays an important role, because there are notable differences in the anatomical, biochemical, physiological, and morphological characteristics of the ocular surface between these different animal species and humans. Among dry eye models, the monkey eye model was found to be the most similar to human eye models.13 Monkeys also have one main lacrimal gland with an anatomical structure similar to that observed in humans. In a previous study, Maitchouk et al14 demonstrated that the removal of the lacrimal gland decreased tear secretion in monkeys. However, no reproducible ocular surface damage was observed in that model,14 and to date, no severe dry eye model has been successfully developed in monkeys. Considering the need for animal models to evaluate new surgical therapies for dry eye, the aim of our study was to create a new monkey model of severe dry eye by removing the main lacrimal gland and nictitating membrane and applying 50% trichloroacetic acid on the palpebral and bulbar conjunctiva. Cornea Volume 33, Number 5, May 2014
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MATERIALS AND METHODS A total of 8 male rhesus monkeys (age, 3–5 years; weight, 5.5–6.0 kg) were used for the study. All the monkeys were purchased from the Academy of Military Medical Sciences (Beijing, China). Animal housing and handling were done by the Capital Medical University Laboratory Animal Center (Beijing, China). This study was performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Animal Experimental Committee of Capital Medical University. Slit-lamp examinations were performed before surgery. Only animals with a normal anterior segment and ocular surface were included in the study. All the monkeys underwent the surgery in their right eye. The left eyes remained untreated and were used as intact controls. The monkeys were first anesthetized by giving them an intramuscular injection of 0.2 mL/kg of ketamine hydrochloride (Shanghai No 1 Biochemical and Pharmaceutical Co Ltd, Shanghai, China). The skin around the right eye was shaved and wiped with 2% iodine tincture (Shuang Qiao Pharmaceutical Company, Beijing, China). Lidocaine hydrochloride 2% (Yi Min Pharmaceutical Co Ltd, Beijing, China) was injected subcutaneously to provide local anesthesia to the skin. A horizontal skin incision (15 mm) was made above the lateral canthus and 2 mm above the eyelashes of the right eye, through the skin and the underlying fascia. The major lacrimal gland was identified in each animal and removed together with its duct, vessels, and nerves. Then, the incision was sutured with 6-0 silk sutures (Unik Surgical Sutures, Taipei, Taiwan). The nictitating membrane was removed from its base with curved scissors without sutures. Tobramycin and dexamethasone eye ointment (Tobradex, Alcon, Fort Worth, TX) was applied into the conjunctival cul-de-sac of operated eyes. The sutures were removed 7 days after the surgery. Healing was uneventful in all the animals. The monkeys were divided into 2 groups. In group 1 (n = 4), rhesus monkeys underwent only the surgical procedure. In group 2 (n = 4), in addition to the surgery, the bulbar conjunctiva was swabbed with 50% trichloroacetic acid for 60 seconds with the aim to further destroy goblet cells and the accessory glands of the conjunctiva.11
Ocular Surface Tests Ocular surface tests were performed on both eyes during the same examination time—before and 1, 4, 8, 12, and 24 weeks after the surgery–in the following order: fluorescein test, lissamine green staining, and Schirmer-1 test. For fluorescein corneal staining, a fluorescein strip (Tianjin Jingming New Technology Development Co Ltd, Tianjin, China) was wetted with isotonic sodium chloride solution and gently applied to the lower conjunctiva. After the animals blinked several times, the slit-lamp examination was performed using a cobalt blue filter to visualize the areas of fluorescein staining. The cornea was divided into 4 quadrants, and the intensity of the staining was graded as follows: 0, no staining; 1, few separate punctate staining; 2, many separate punctate staining; and 3, confluent punctate staining or presence of filament.11 Then, a lissamine green 2014 Lippincott Williams & Wilkins
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strip (HUB Pharmaceuticals, LLC) was wetted with isotonic sodium chloride solution and gently applied to the lower conjunctiva. The intensity of the staining was analyzed in the cornea and the conjunctiva using the Van Bijsterveld scoring system.15,16 After a 3-minute washout period, the Schirmer test strip (Tianjin Jingming New Technology Development Co Ltd) was inserted into the lower conjunctival fornix at the junction of the middle and lateral third of the lower eyelid margin. The extent of wetting was measured after 5 minutes.
Conjunctival Impression Cytology Conjunctival impression cytology specimens were collected using round 5-mm cellulose acetate filters (Toyo, Roshi Kaisha, Ltd, Japan). To evaluate the normal distribution of goblet cells in rhesus monkeys, impression cytology specimens were first collected in 3 superior bulbar conjunctival areas: 2 mm from the limbus, in the conjunctival fornix, and between these 2 areas (Fig. 5). After the experimental procedures were performed, only the area adjacent to the limbus was evaluated and quantified. After 5 seconds, the filters were gently removed to collect a homogeneous population of superficial conjunctival cells and were then fixed with 95% alcohol. Then, specimens were stained with periodic acid–Schiff.17 After staining, the number of goblet cells was counted under a light microscope (Olympus CH-2, Japan) with a ·40 objective high-power field, and the morphology of the conjunctival epithelium was graded according to the Nelson classification.17 Ten randomly chosen areas (220 · 162 mm, 0.036 mm2) around the center of the filter were evaluated; the results of the goblet cell count and grading represented the mean value of these 10 areas.
Histopathologic Examination The animals were killed at the end of the experiment (24 weeks). The bulbar conjunctiva and the cornea were obtained from both the operated and the control eyes. Tissue samples, including the nictitating membrane and lacrimal gland excised during the surgery, were fixed in 10% formalin. After dehydration, the specimens were embedded in paraffin, cross-sectioned, and stained with hematoxylin–eosin. The sections were observed under an optical light microscope (Olympus CH-2).
Statistical Analysis Schirmer test data were analyzed preoperatively and postoperatively using repeated analysis of variance. A Kruskal–Wallis test was used postoperatively to compare the ocular surface staining scores for both the operated and control eyes in the 2 groups at each time point. The statistical significance of the goblet cell density was evaluated between both the operated and control eyes using a 1-way analysis of variance. Statistical analysis was performed on a computer (SPSS ver 18.0; SPSS Inc, Chicago, IL). A value of P , 0.05 was considered as significant. www.corneajrnl.com |
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FIGURE 1. Comparison of preoperative and postoperative Schirmer test results in both the groups. A, Group 1, preoperative and postoperative Schirmer test results in both the eyes. B, Group 2 (with trichloroacetic acid) preoperative and postoperative Schirmer test results in both the eyes. Tear secretion from the operated eyes decreased significantly after the surgery (P , 0.001). C, Tear secretion from the operated eyes was significantly lower in group 2 than in group 1 before 12 weeks, postoperatively (P , 0.05).
RESULTS Before the surgery, slit-lamp examination, Schirmer test, and ocular surface staining procedures showed no abnormality in any of the eyes. Healing of the surgical wounds was uneventful. Slit-lamp examination revealed no eyelid abnormalities after the surgery. Some secretions were observed in the operated eyes of group 2 during the first week.
Ocular Surface Tests In both groups, a significant decrease in tear secretion (;80%) was observed in the operated eyes when compared with that in the contralateral untreated eyes at all time points (P , 0.001) (Fig. 1). Schirmer tests showed that aqueous tear secretion decreased in the operated eyes as soon as 1 week after the surgery from 28.25 6 2.06 to 6.25 6 1.71 mm and from 27.5 6 1.91 to 3.75 6 0.96 mm in groups 1 and 2, respectively. A further slight decrease in aqueous tear secretion was also observed in both groups during the follow-up. From 8 weeks after the surgery, Schirmer test data measured were #4 mm in all the operated eyes. Tear secretion in the eyes of group 2 was significantly lower than that observed postoperatively for the eyes of group 1 until 12 weeks (P = 0.03), but no significant difference was observed after this time (Fig. 1).
During the 24 weeks of follow-up after the surgery, slitlamp examination revealed abnormal fluorescein and lissamine green staining in all the operated eyes. In contrast, no abnormal staining was observed in the control eyes. Moreover, ocular surface staining test results remained stable in both groups until the end of the experiment (Figs. 2, 3). After the surgical procedure, fluorescein and lissamine green scores were higher in group 2 than in group 1 at all time points (P , 0.05) (Fig. 4). In group 2, corneal fluorescein staining and lissamine green test values were always $5 (max. 12) and $4 (max. 9), respectively.
Conjunctival Impression Cytology In untreated rhesus monkey eyes, impression cytology specimens showed numerous round and small conjunctival epithelial cells, and fewer large and oval goblet cells (Fig. 5). Epithelial cells had a large nucleus with a nucleocytoplasmic ratio of approximately 1:1, and goblet cells had an intensely periodic acid–Schiff–positive cytoplasm (Fig. 5). The number of goblet cells decreased from the conjunctival fornix to the limbus. Before the experimental procedures were performed, there was no difference in the goblet cell density between the 2 monkey groups (437.85 6 189.6–451.19 6 199.88 cells per square millimeter). After 24 weeks, impression cytology specimens of both treated groups showed conjunctival squamous
FIGURE 2. Slip-lamp photographs of the fluorescein staining test. A, In the control eyes, no abnormal fluorescein staining was detected. B, In the operated eyes, a diffuse fluorescein staining was observed.
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FIGURE 3. Slip-lamp photographs of the lissamine green test. A, In the control eyes, the findings of the lissamine green test were normal. B, In the operated eyes, a diffuse staining was observed.
metaplasia and a decreased number of goblet cells. The grading score of all the operated eyes reached Nelson classification grade 3. By comparison, all control eyes had a grading of 0 to 1. Moreover, the number of pycnotic cells and the nucleocytoplasmic ratio were further increased in group 2 as compared with that in group 1. The number of goblet cells in both treated groups (group 1: 82.01 6 57.27 cells per square millimeter; group 2: 9.73 6 18.07 cells per square millimeter) was significantly lower than in the control untreated eyes (429.77 6 201.63 cells per square millimeter, P , 0.001 for both comparisons). Moreover, in group 2, the number of goblet cells was significantly lower than in group 1 (P = 0.003) (Fig. 6).
Histopathological Examination Under light microscopy, the cornea of rhesus monkeys was similar to that observed in humans. The corneal epithelium was a well-stratified squamous nonkeratinized epithelium with 4 to 5 cell layers. Basal cells appeared as a single layer of columnar cells (Fig. 7A). In all the operated eyes, after inducing dry eye, the corneal epithelium was found to be irregular with a decreased number of epithelial cell layers and basal cell edema (Fig. 7B). The conjunctiva of the control eyes consisted of a stratified multilayer of cuboidal epithelial cells with homogeneous staining. Goblet cells were randomly distributed within the conjunctival epithelium (Fig. 7C). In contrast, the conjunctival epithelium in the operated eyes showed squamous metaplasia with an irregular epithelium thickness, loss of stratification, less homogeneous cell size and staining, and an obvious loss of goblet cells with infiltration of inflammatory cells. Further, vasodilation of conjunctival blood vessels was
observed (Fig. 7D). No significant difference was observed between the cornea and the conjunctiva of the 2 operated groups. Finally, the nictitating membrane contained numerous goblet cells, accessory lacrimal glands, and some cartilage tissue and muscles (Fig. 8).
DISCUSSION Because dry eye is a complex disease involving several mechanisms and having different treatment modalities, a large variety of animal models have been developed.13 However, to our knowledge, no previous study has reported the development of a simple, long-standing, and severe dry eye model in monkeys. In this study, after removing the main lacrimal gland and the nictitating membrane of rhesus monkeys, an important decrease in the quantity of tears was associated with dry eye ocular surface tissue alterations during at least 24 weeks. The surgical removal of the main lacrimal gland is an approach that has already been used in several species to obtain aqueous deficient dry eye models.12 Associated with the removal of the nictitating membrane, this model was able to induce clinical signs of dry eye in dogs.18 However, in other species such as mice or rabbits, the removal of the main lacrimal glands did not necessarily cause ocular surface disease.12,19,20 In squirrel monkeys that have ocular surface structures more similar to those of humans, although the removal of the main lacrimal gland caused an important decrease in aqueous tear secretion (basal and reflex tears), no clinical or histological change suggesting dry eye was observed.14 Maitchouk et al14 reported that as in humans, nonhuman primates do not have a nictitating
FIGURE 4. Comparison of staining scores between the 2 groups. A, Corneal fluorescein staining scores. B, Lissamine green staining scores. Group 1: without trichloroacetic acid application; group 2: with trichloroacetic acid application. *P , 0.05. 2014 Lippincott Williams & Wilkins
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FIGURE 5. Conjunctival impression cytology specimens showing the distribution of goblet cells in the bulbar conjunctiva of normal rhesus monkeys. Epithelial cells appear small and round. Nuclei are large and intensely stained with a nucleocytoplasmic ratio of 1:1. Goblet cells are plump and oval. They had an intensely periodic acid Schiff–positive cytoplasm (·400). A, Specimen collection location. Impression cytology taken from the fornix (B), above the limbus (C), and in the middle of the bulbar conjunctiva (D). The number of goblet cells decreased from the conjunctival fornix to the limbus.
membrane. Similarly, in 2 studies on rhesus monkey ocular glands, Stephens et al21,22 did not mention the presence of a nictitating membrane. However, in this study, we found a nictitating membrane in all rhesus monkey eyes as a narrow, highly vascular crescent-shaped fold of the conjunctiva located in the medial canthus. This nictitating membrane could extend from a crescent-shaped fold to a membrane of a size of
about 15 · 15 mm (Fig. 8). Histologically, this membrane was found to contain a large number of goblet cells and accessory lacrimal glands. Therefore, this ocular surface tissue may contribute largely to the tear film aqueous and mucous secretion and may explain the absence of dry eye in monkeys after removal of only their main lacrimal gland.14 The plica semilunaris, a vestigial structure located in the medial canthus in
FIGURE 6. Results of conjunctival impression cytology. In the control eyes (A), many goblet cells randomly distributed were observed. In group 1 (B) and group 2 (treated with trichloroacetic acid) (C), few goblet cells were observed. The densities of the goblet cells were significantly different among the control (429.77 6 201.63 cells per square millimeter), group 1 (82.01 6 57.27 cells per square millimeter), and group 2 (9.73 6 18.07 cells per square millimeter). *P , 0.01.
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FIGURE 7. Histologic examination of the cornea and conjunctiva (hematoxylin–eosin, ·400). A, Normal cornea in the control eyes. B, The corneal epithelium was irregular with a decreased number of epithelial cell layers and basal cells edema in all the operated eyes. C, Normal bulbar conjunctiva in the control eyes showing numerous goblet cells randomly distributed. D, In all the operated eyes, the conjunctival epithelium showed squamous metaplasia with an irregular epithelium thickness, loss of stratification, less homogeneous cell size and staining, and an obvious loss of goblet cells with infiltration of inflammatory cells.
humans, may also correspond to this membrane observed in monkeys or to the nictitating membrane or third eyelid of some other animal species.23 In addition to the removal of the main lacrimal gland and nictitating membrane, Zhu et al24 swabbed the palpebral and bulbar conjunctiva with 30% trichloroacetic acid to induce dry eye in rabbits. These authors observed a decrease in the goblet cell density in the conjunctiva with a moderate or severe infiltration of inflammatory cells in the cornea and
conjunctiva. However, Chen et al,11 in a similar rabbit model using 50% trichloroacetic acid, failed to show a difference in terms of clinical ocular surface tests or goblet cell density between the eyes with or without application of this weak acid. Similarly, before this study, we evaluated the effects of 50% trichloroacetic acid application on the rhesus monkey conjunctiva, and no abnormal clinical sign or ocular surface test results were observed after 1 month (data not shown). These results suggested that swabbing the conjunctiva with
FIGURE 8. The nictitating membrane of the rhesus monkey. A, Slit-lamp photograph of the nictitating membrane in the medial canthus. B, The nictitating membrane extended to an approximately 15- · 15-mm-sized membrane after traction. C, Numerous goblet cells were observed in this tissue (hematoxylin–eosin, ·200). D, Accessory lacrimal glands and cartilage tissue were also detected (hematoxylin–eosin, ·100). 2014 Lippincott Williams & Wilkins
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50% trichloroacetic acid did not induce extensive damage to the ocular surface as was observed in alkali burn models.25 Accordingly, to create a more severe dry eye model than obtained only by removing the main lacrimal gland and nictitating membrane, we also applied 50% trichloroacetic acid on the bulbar and palpebral conjunctiva. Unlike in rabbits, the use of this weak acid was associated with more severe dry eye clinical tests and lower conjunctival goblet cells as compared with that in monkeys that underwent only the surgical procedure. Moreover, no destruction of the ocular surface tissue architecture was observed. According to the 2007 Dry Eye Workshop, dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and ocular surface inflammation.2 Although subjective symptoms and osmolarity were not evaluated in this study, the association of decreased tear secretion and ocular surface histological changes—decreased goblet cell density and an infiltration of inflammatory cells—confirmed the development of a severe dry eye model mimicking dry eye tissue alterations. The investigation techniques available to assess the functional or histological changes induced by the disease are also important parameters to determine the choice of an animal model. In particular for dry eye disease, the precise in vivo evaluation of ocular surface changes and the severity of damage remains a challenge for both ophthalmologist clinicians and researchers. Besides the classic evaluation of clinical parameters, such as the Schirmer test values or fluorescein and rose Bengal staining, we performed conjunctival impression cytology to evaluate in vivo the tissue changes induced on this monkey model. Impression cytology, by applying filter membranes, is a collection technique for the most superficial layers of the conjunctiva, and some authors consider it as an equivalent of conjunctival biopsy.26 Interestingly, impression cytology specimens of rhesus monkeys were similar to those observed in humans, particularly in terms of goblet cell density and distribution over the conjunctiva.24,27–29 Moreover, the results were correlated to the histological tissue analyses that we eventually performed. The loss of goblet cells is considered as a sensible indicator of ocular surface disease.30,31 Accordingly, in this study, the severity of dry eye clinical tests was correlated to the number of goblet cells observed within the conjunctiva. To our knowledge, this is the first study using impression cytology to evaluate the rhesus monkey conjunctiva, and this technique seems to be extremely useful in monitoring in vivo ocular surface changes in that animal model. Although the quantitative evaluation of impression cytology in this study has some limitations, a more precise quantification using flow cytometry, already used in humans,32 might be used in future studies. The therapeutic intervention plan is also a major determining factor in the animal model choice of dry eye. Although monkey models have several negative aspects including the ethical considerations, the costs, and the need of special facilities, they are very similar to human models both anatomically and physiologically.21,22 Stephens
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et al21,22,33 described and illustrated the overall anatomy of the eyelid and of the various ocular surface glands and salivary glands in rhesus monkeys, and concluded that they were highly similar to those of humans in terms of location and gross and microscopic features. Because new therapeutic strategies for severe dry eye need to be evaluated, and in particular, surgical strategies such as autologous minor salivary gland transfer, our aim was to develop a new monkey model of severe dry eye. Combined with the use of impression cytology, this new monkey model may be an interesting tool for the development of new surgical approaches for the treatment of severe dry eye disease.
ACKNOWLEDGMENTS The authors thank Dr Chunyan He, Dr Xiaolin Xu, and Prof Bin Li, Department of Histopathology, Beijing Tongren Hospital and Beijing Institute of Ophthalmology, Capital University of Medical Science, for help with the histology. REFERENCES 1. The epidemiology of dry eye disease: report of the Epidemiology Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007;5:93–107. 2. The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007;5:75–92. 3. Luo SR, Zou LH, Yan C, et al. Transplantation of autologous labial salivary glands for severe dry eye [in Chinese]. Zhonghua Yan Ke Za Zhi. 2013;49:22–26. 4. Tsubota K, Kawashima M, Inaba T, et al. The antiaging approach for the treatment of dry eye. Cornea. 2012;31:S3–S8. 5. Lemp MA. Advances in understanding and managing dry eye disease. Am J Ophthalmol. 2008;146:350–356. 6. Murube-del-Castillo J. Transplantation of salivary gland to the lacrimal basin. Scand J Rheumatol Suppl. 1986;61:264–267. 7. Geerling G, Raus P, Murube J. Minor salivary gland transplantation. Dev Ophthalmol. 2008;41:243–254. 8. Sant’ Anna AE, Hazarbassanov RM, de Freitas D, et al. Estela A, Minor salivary glands and labial mucous membrane graft in the treatment of severe symblepharon and dry eye in patients with Stevens–Johnson syndrome. Br J Ophthalmol. 2012;96:234–239. 9. Marinho DR, Burmann TG, Kwitko S. Labial salivary gland transplantation for severe dry eye due to chemical burns and Stevens–Johnson syndrome. Ophthal Plast Reconstr Surg. 2010;26:182–184. 10. Murube J, Manyari A, Chen-Zhuo L, et al. Labial salivary gland transplantation in severe dry eye. Oper Tech Oculoplast Orbit Reconstr Surg. 1998;1:104–110. 11. Chen ZY, Liang QF, Yu GY. Establishment of a rabbit model for keratoconjunctivitis sicca. Cornea. 2011;30:1024–1029. 12. Barabino S, Dana MR. Animal models of dry eye: a critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci. 2004;45:1641–1646. 13. Schrader S, Mircheff AK, Geerling G. Animal models of dry eye. Dev Ophthalmol. 2008;41:298–312. 14. Maitchouk DY, Beuerman RW, Ohta T, et al. Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys. Arch Ophthalmol. 2000;118:246–252. 15. van Bijsterveld OP. Diagnostic tests in the sicca syndrome. Arch Ophthalmol. 1969;82:10–14. 16. Yoon KC, Im SK, Kim HG, et al. Usefulness of double vital staining with 1% fluorescein and 1% lissamine green in patients with dry eye syndrome. Cornea. 2011;30:972–976. 17. Nelson JD. Impression cytology. Cornea. 1988;7:71–81. 18. Helper LC, Magrane WG, Koehm J, et al. Surgical induction of keratoconjunctivitis sicca in the dog. J Am Vet Med Assoc. 1974;165:172–174.
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19. Jester JV, Nicolaides N, Kiss-Palvolgyi I, et al. Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD. Invest Ophthalmol Vis Sci. 1989;30:936–945. 20. Maudgal PC. The epithelial response in keratitis sicca and keratitis herpetica (an experimental and clinical study). Doc Ophthalmol. 1978;45: 223–227. 21. Stephens LC, Schultheiss TE, Ang KK, et al. Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta). J Med Primatol. 1987;16:407–419. 22. Stephens LC, Schultheiss TE, Vargas KJ, et al. Glands of the eyelids of rhesus monkeys (Macaca mulatta). J Med Primatol. 1989;18: 383–396. 23. Arends G, Schramm U. The structure of the human semilunar plica at different stages of its development—a morphological and morphometric study. Ann Anat. 2004;186:195–207. 24. Zhu ZH, Yu GY, Zou LH, et al. Autologous submandibular gland transfer for the management of xerophthalmia: an experimental study. J Mod Stomatol (Chin). 2001;15:179–181. 25. Ueno M, Lyons LB, et al. Accelerated wound healing of alkali-burned corneas in mrl mice is associated with a reduced inflammatory signature. Invest Ophthalmol Vis Sci. 2005;46:4097–4106.
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26. Calonge M, Diebold Y, Sáez V, et al. Impression cytology of the ocular surface: a review. Exp Eye Res. 2004;78:457–472. 27. Rivas L, Oroza MA, Perez-Esteban A, et al. Topographical distribution of ocular surface cells by the use of impression cytology. Acta Ophthalmol (Copenh). 1991;65:371–376. 28. Rivas L, Alvarez MI, Rodriguez JJ, et al. Ophthalmological tests in patients with keratoconjunctivitis sicca with and without association of primary Sjögren’s syndrome. Ger J Ophthalmol. 1995;4:306–310. 29. Vujković V, Mikac G, Kozomara R. Distribution and density of conjunctival goblet cells. Med Pregl. 2002;55:195–200. 30. Kinoshita S, Kiorpes TC, Friend J, et al. Goblet cell density in ocular surface disease. A better indicator than tear mucin. Arch Ophthalmol. 1983;101:1284–1287. 31. Adams GG, Dilly PN, Kirkness CM. Monitoring ocular disease by impression cytology. Eye (Lond). 1988;2:506–516. 32. Brignole-Baudouin F, Ott AC, Warnet JM, et al. Flow cytometry in conjunctival impression cytology: a new tool for exploring ocular surface pathologies. Exp Eye Res. 2004;78:473–481. 33. Stephens LC, King GK, Ang KK, et al. Surgical and microscopic anatomy of parotid and submandibular salivary glands of rhesus monkeys (Macaca mulatta). J Med Primatol. 1986;15:105–119.
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A New Nonhuman Primate Model of Severe Dry Eye Yi Qin, MD,* Xiaobo Tan, MD, PhD,* Yingnan Zhang, MD, PhD,* Ying Jie, MD, PhD,* Antoine Labbe, MD, PhD,*†‡ and Zhiqiang Pan, MD, PhD*
Purpose: The aim of this study was to establish a new rhesus monkey model of severe dry eye.
Key Words: dry eye, animal model, rhesus monkey, impression cytology, lacrimal gland (Cornea 2014;33:510–517)
Methods: A total of 8 rhesus monkeys were used for the study. Four monkeys had their main lacrimal gland and nictitating membrane surgically removed (group 1). Another 4 monkeys had a similar surgery with further application of 50% trichloroacetic acid on the bulbar conjunctiva (group 2). The ocular surface was evaluated before and after the surgery (1, 4, 8, 12, and 24 weeks) using Schirmer-1 test, corneal fluorescein staining, and the lissamine green test. Conjunctival impression cytology was also performed before and 24 weeks after the surgery. Finally, the cornea and the conjunctiva were evaluated using light microscopy.
Results: A significant decrease in tear secretion was observed in all operated eyes. Schirmer test data measured were #4 mm in all the operated eyes. Slit-lamp examination also revealed abnormal staining in all the operated eyes that remained stable until the end of the experiment. In group 2, corneal fluorescein staining and lissamine green test values were always $5 (max 12) and $4 (max 9), respectively. Impression cytology specimens of both the treated groups showed conjunctival squamous metaplasia and a decreased number of goblet cells. Under light microscopy, the corneal epithelium appeared irregular with edematous basal epithelial cells. The conjunctiva showed a decreased goblet cell density with infiltration of inflammatory cells. Conclusions: Complete removal of the principal lacrimal gland and nictitating membrane associated with the application of 50% trichloroacetic acid on the conjunctiva could induce severe dry eye in rhesus monkeys.
Received for publication November 14, 2013; revision received January 5, 2014; accepted January 7, 2014. Published online ahead of print February 26, 2014. From the *Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Lab, Beijing, China; †Department of Ophthalmology, Quinze-Vingts National Ophthalmology Hospital, Paris and Versailles Saint-Quentin-enYvelines University, Versailles, France; and ‡INSERM, U968, UPMC Univ Paris 06, UMR S 968, Institut de la Vision, CNRS, UMR 7210, Paris, France. Supported by the Beijing Health Systems High-level Health and Technical Talent Training Plan (2009-2-05) and the National Natural Science Foundation of China, project No. 30801264. The authors have no conflicts of interest to disclose. Reprints: Zhiqiang Pan, Beijing Tongren Eye Center, Beijing Tongren Hospital, 1# Dong Jiao Min Xiang, Beijing 100730, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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ry eye is one of the most common ocular diseases and may affect from 5% to .30% of people aged $50 years.1 According to the 2007 Dry Eye Workshop, dry eye is a multifactorial disease of tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability.2 Severe dry eye may also lead to corneal ulceration, opacification, and even blindness.3 Despite recent advances in the treatment of dry eye with a new antiinflammatory drug such as cyclosporine A,4 the management of patients with severe dry eye remains difficult and is often unsatisfactory.5 In patients with severe pathology despite medical therapy, a new surgical approach, transplantation of salivary glands, has been proposed.6 In particular, the transplantation of minor salivary glands could be an effective treatment option for severe dry eyes, because it is a simple procedure and has minimal surgical risks.7 Promising results have thus been reported in patients with severe dry eye because of chemical burns or Stevens–Johnson syndrome.7–10 Nevertheless, with very few published studies available, little is known about the survival of the glands, the characteristics of the salivary tear film, and the benefit for the ocular surface.7–10 Consequently, animal models of severe dry eye that allow one to study the mechanisms and to evaluate the results of new surgical strategies, such as salivary gland transplantation, are needed.11 Numerous animal models, including monkey, dog, rabbit, rat, and mouse have been developed to mimic different pathophysiologic mechanisms involved in dry eye.12 The choice of an animal model plays an important role, because there are notable differences in the anatomical, biochemical, physiological, and morphological characteristics of the ocular surface between these different animal species and humans. Among dry eye models, the monkey eye model was found to be the most similar to human eye models.13 Monkeys also have one main lacrimal gland with an anatomical structure similar to that observed in humans. In a previous study, Maitchouk et al14 demonstrated that the removal of the lacrimal gland decreased tear secretion in monkeys. However, no reproducible ocular surface damage was observed in that model,14 and to date, no severe dry eye model has been successfully developed in monkeys. Considering the need for animal models to evaluate new surgical therapies for dry eye, the aim of our study was to create a new monkey model of severe dry eye by removing the main lacrimal gland and nictitating membrane and applying 50% trichloroacetic acid on the palpebral and bulbar conjunctiva. Cornea Volume 33, Number 5, May 2014
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MATERIALS AND METHODS A total of 8 male rhesus monkeys (age, 3–5 years; weight, 5.5–6.0 kg) were used for the study. All the monkeys were purchased from the Academy of Military Medical Sciences (Beijing, China). Animal housing and handling were done by the Capital Medical University Laboratory Animal Center (Beijing, China). This study was performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Animal Experimental Committee of Capital Medical University. Slit-lamp examinations were performed before surgery. Only animals with a normal anterior segment and ocular surface were included in the study. All the monkeys underwent the surgery in their right eye. The left eyes remained untreated and were used as intact controls. The monkeys were first anesthetized by giving them an intramuscular injection of 0.2 mL/kg of ketamine hydrochloride (Shanghai No 1 Biochemical and Pharmaceutical Co Ltd, Shanghai, China). The skin around the right eye was shaved and wiped with 2% iodine tincture (Shuang Qiao Pharmaceutical Company, Beijing, China). Lidocaine hydrochloride 2% (Yi Min Pharmaceutical Co Ltd, Beijing, China) was injected subcutaneously to provide local anesthesia to the skin. A horizontal skin incision (15 mm) was made above the lateral canthus and 2 mm above the eyelashes of the right eye, through the skin and the underlying fascia. The major lacrimal gland was identified in each animal and removed together with its duct, vessels, and nerves. Then, the incision was sutured with 6-0 silk sutures (Unik Surgical Sutures, Taipei, Taiwan). The nictitating membrane was removed from its base with curved scissors without sutures. Tobramycin and dexamethasone eye ointment (Tobradex, Alcon, Fort Worth, TX) was applied into the conjunctival cul-de-sac of operated eyes. The sutures were removed 7 days after the surgery. Healing was uneventful in all the animals. The monkeys were divided into 2 groups. In group 1 (n = 4), rhesus monkeys underwent only the surgical procedure. In group 2 (n = 4), in addition to the surgery, the bulbar conjunctiva was swabbed with 50% trichloroacetic acid for 60 seconds with the aim to further destroy goblet cells and the accessory glands of the conjunctiva.11
Ocular Surface Tests Ocular surface tests were performed on both eyes during the same examination time—before and 1, 4, 8, 12, and 24 weeks after the surgery–in the following order: fluorescein test, lissamine green staining, and Schirmer-1 test. For fluorescein corneal staining, a fluorescein strip (Tianjin Jingming New Technology Development Co Ltd, Tianjin, China) was wetted with isotonic sodium chloride solution and gently applied to the lower conjunctiva. After the animals blinked several times, the slit-lamp examination was performed using a cobalt blue filter to visualize the areas of fluorescein staining. The cornea was divided into 4 quadrants, and the intensity of the staining was graded as follows: 0, no staining; 1, few separate punctate staining; 2, many separate punctate staining; and 3, confluent punctate staining or presence of filament.11 Then, a lissamine green 2014 Lippincott Williams & Wilkins
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strip (HUB Pharmaceuticals, LLC) was wetted with isotonic sodium chloride solution and gently applied to the lower conjunctiva. The intensity of the staining was analyzed in the cornea and the conjunctiva using the Van Bijsterveld scoring system.15,16 After a 3-minute washout period, the Schirmer test strip (Tianjin Jingming New Technology Development Co Ltd) was inserted into the lower conjunctival fornix at the junction of the middle and lateral third of the lower eyelid margin. The extent of wetting was measured after 5 minutes.
Conjunctival Impression Cytology Conjunctival impression cytology specimens were collected using round 5-mm cellulose acetate filters (Toyo, Roshi Kaisha, Ltd, Japan). To evaluate the normal distribution of goblet cells in rhesus monkeys, impression cytology specimens were first collected in 3 superior bulbar conjunctival areas: 2 mm from the limbus, in the conjunctival fornix, and between these 2 areas (Fig. 5). After the experimental procedures were performed, only the area adjacent to the limbus was evaluated and quantified. After 5 seconds, the filters were gently removed to collect a homogeneous population of superficial conjunctival cells and were then fixed with 95% alcohol. Then, specimens were stained with periodic acid–Schiff.17 After staining, the number of goblet cells was counted under a light microscope (Olympus CH-2, Japan) with a ·40 objective high-power field, and the morphology of the conjunctival epithelium was graded according to the Nelson classification.17 Ten randomly chosen areas (220 · 162 mm, 0.036 mm2) around the center of the filter were evaluated; the results of the goblet cell count and grading represented the mean value of these 10 areas.
Histopathologic Examination The animals were killed at the end of the experiment (24 weeks). The bulbar conjunctiva and the cornea were obtained from both the operated and the control eyes. Tissue samples, including the nictitating membrane and lacrimal gland excised during the surgery, were fixed in 10% formalin. After dehydration, the specimens were embedded in paraffin, cross-sectioned, and stained with hematoxylin–eosin. The sections were observed under an optical light microscope (Olympus CH-2).
Statistical Analysis Schirmer test data were analyzed preoperatively and postoperatively using repeated analysis of variance. A Kruskal–Wallis test was used postoperatively to compare the ocular surface staining scores for both the operated and control eyes in the 2 groups at each time point. The statistical significance of the goblet cell density was evaluated between both the operated and control eyes using a 1-way analysis of variance. Statistical analysis was performed on a computer (SPSS ver 18.0; SPSS Inc, Chicago, IL). A value of P , 0.05 was considered as significant. www.corneajrnl.com |
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FIGURE 1. Comparison of preoperative and postoperative Schirmer test results in both the groups. A, Group 1, preoperative and postoperative Schirmer test results in both the eyes. B, Group 2 (with trichloroacetic acid) preoperative and postoperative Schirmer test results in both the eyes. Tear secretion from the operated eyes decreased significantly after the surgery (P , 0.001). C, Tear secretion from the operated eyes was significantly lower in group 2 than in group 1 before 12 weeks, postoperatively (P , 0.05).
RESULTS Before the surgery, slit-lamp examination, Schirmer test, and ocular surface staining procedures showed no abnormality in any of the eyes. Healing of the surgical wounds was uneventful. Slit-lamp examination revealed no eyelid abnormalities after the surgery. Some secretions were observed in the operated eyes of group 2 during the first week.
Ocular Surface Tests In both groups, a significant decrease in tear secretion (;80%) was observed in the operated eyes when compared with that in the contralateral untreated eyes at all time points (P , 0.001) (Fig. 1). Schirmer tests showed that aqueous tear secretion decreased in the operated eyes as soon as 1 week after the surgery from 28.25 6 2.06 to 6.25 6 1.71 mm and from 27.5 6 1.91 to 3.75 6 0.96 mm in groups 1 and 2, respectively. A further slight decrease in aqueous tear secretion was also observed in both groups during the follow-up. From 8 weeks after the surgery, Schirmer test data measured were #4 mm in all the operated eyes. Tear secretion in the eyes of group 2 was significantly lower than that observed postoperatively for the eyes of group 1 until 12 weeks (P = 0.03), but no significant difference was observed after this time (Fig. 1).
During the 24 weeks of follow-up after the surgery, slitlamp examination revealed abnormal fluorescein and lissamine green staining in all the operated eyes. In contrast, no abnormal staining was observed in the control eyes. Moreover, ocular surface staining test results remained stable in both groups until the end of the experiment (Figs. 2, 3). After the surgical procedure, fluorescein and lissamine green scores were higher in group 2 than in group 1 at all time points (P , 0.05) (Fig. 4). In group 2, corneal fluorescein staining and lissamine green test values were always $5 (max. 12) and $4 (max. 9), respectively.
Conjunctival Impression Cytology In untreated rhesus monkey eyes, impression cytology specimens showed numerous round and small conjunctival epithelial cells, and fewer large and oval goblet cells (Fig. 5). Epithelial cells had a large nucleus with a nucleocytoplasmic ratio of approximately 1:1, and goblet cells had an intensely periodic acid–Schiff–positive cytoplasm (Fig. 5). The number of goblet cells decreased from the conjunctival fornix to the limbus. Before the experimental procedures were performed, there was no difference in the goblet cell density between the 2 monkey groups (437.85 6 189.6–451.19 6 199.88 cells per square millimeter). After 24 weeks, impression cytology specimens of both treated groups showed conjunctival squamous
FIGURE 2. Slip-lamp photographs of the fluorescein staining test. A, In the control eyes, no abnormal fluorescein staining was detected. B, In the operated eyes, a diffuse fluorescein staining was observed.
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FIGURE 3. Slip-lamp photographs of the lissamine green test. A, In the control eyes, the findings of the lissamine green test were normal. B, In the operated eyes, a diffuse staining was observed.
metaplasia and a decreased number of goblet cells. The grading score of all the operated eyes reached Nelson classification grade 3. By comparison, all control eyes had a grading of 0 to 1. Moreover, the number of pycnotic cells and the nucleocytoplasmic ratio were further increased in group 2 as compared with that in group 1. The number of goblet cells in both treated groups (group 1: 82.01 6 57.27 cells per square millimeter; group 2: 9.73 6 18.07 cells per square millimeter) was significantly lower than in the control untreated eyes (429.77 6 201.63 cells per square millimeter, P , 0.001 for both comparisons). Moreover, in group 2, the number of goblet cells was significantly lower than in group 1 (P = 0.003) (Fig. 6).
Histopathological Examination Under light microscopy, the cornea of rhesus monkeys was similar to that observed in humans. The corneal epithelium was a well-stratified squamous nonkeratinized epithelium with 4 to 5 cell layers. Basal cells appeared as a single layer of columnar cells (Fig. 7A). In all the operated eyes, after inducing dry eye, the corneal epithelium was found to be irregular with a decreased number of epithelial cell layers and basal cell edema (Fig. 7B). The conjunctiva of the control eyes consisted of a stratified multilayer of cuboidal epithelial cells with homogeneous staining. Goblet cells were randomly distributed within the conjunctival epithelium (Fig. 7C). In contrast, the conjunctival epithelium in the operated eyes showed squamous metaplasia with an irregular epithelium thickness, loss of stratification, less homogeneous cell size and staining, and an obvious loss of goblet cells with infiltration of inflammatory cells. Further, vasodilation of conjunctival blood vessels was
observed (Fig. 7D). No significant difference was observed between the cornea and the conjunctiva of the 2 operated groups. Finally, the nictitating membrane contained numerous goblet cells, accessory lacrimal glands, and some cartilage tissue and muscles (Fig. 8).
DISCUSSION Because dry eye is a complex disease involving several mechanisms and having different treatment modalities, a large variety of animal models have been developed.13 However, to our knowledge, no previous study has reported the development of a simple, long-standing, and severe dry eye model in monkeys. In this study, after removing the main lacrimal gland and the nictitating membrane of rhesus monkeys, an important decrease in the quantity of tears was associated with dry eye ocular surface tissue alterations during at least 24 weeks. The surgical removal of the main lacrimal gland is an approach that has already been used in several species to obtain aqueous deficient dry eye models.12 Associated with the removal of the nictitating membrane, this model was able to induce clinical signs of dry eye in dogs.18 However, in other species such as mice or rabbits, the removal of the main lacrimal glands did not necessarily cause ocular surface disease.12,19,20 In squirrel monkeys that have ocular surface structures more similar to those of humans, although the removal of the main lacrimal gland caused an important decrease in aqueous tear secretion (basal and reflex tears), no clinical or histological change suggesting dry eye was observed.14 Maitchouk et al14 reported that as in humans, nonhuman primates do not have a nictitating
FIGURE 4. Comparison of staining scores between the 2 groups. A, Corneal fluorescein staining scores. B, Lissamine green staining scores. Group 1: without trichloroacetic acid application; group 2: with trichloroacetic acid application. *P , 0.05. 2014 Lippincott Williams & Wilkins
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FIGURE 5. Conjunctival impression cytology specimens showing the distribution of goblet cells in the bulbar conjunctiva of normal rhesus monkeys. Epithelial cells appear small and round. Nuclei are large and intensely stained with a nucleocytoplasmic ratio of 1:1. Goblet cells are plump and oval. They had an intensely periodic acid Schiff–positive cytoplasm (·400). A, Specimen collection location. Impression cytology taken from the fornix (B), above the limbus (C), and in the middle of the bulbar conjunctiva (D). The number of goblet cells decreased from the conjunctival fornix to the limbus.
membrane. Similarly, in 2 studies on rhesus monkey ocular glands, Stephens et al21,22 did not mention the presence of a nictitating membrane. However, in this study, we found a nictitating membrane in all rhesus monkey eyes as a narrow, highly vascular crescent-shaped fold of the conjunctiva located in the medial canthus. This nictitating membrane could extend from a crescent-shaped fold to a membrane of a size of
about 15 · 15 mm (Fig. 8). Histologically, this membrane was found to contain a large number of goblet cells and accessory lacrimal glands. Therefore, this ocular surface tissue may contribute largely to the tear film aqueous and mucous secretion and may explain the absence of dry eye in monkeys after removal of only their main lacrimal gland.14 The plica semilunaris, a vestigial structure located in the medial canthus in
FIGURE 6. Results of conjunctival impression cytology. In the control eyes (A), many goblet cells randomly distributed were observed. In group 1 (B) and group 2 (treated with trichloroacetic acid) (C), few goblet cells were observed. The densities of the goblet cells were significantly different among the control (429.77 6 201.63 cells per square millimeter), group 1 (82.01 6 57.27 cells per square millimeter), and group 2 (9.73 6 18.07 cells per square millimeter). *P , 0.01.
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FIGURE 7. Histologic examination of the cornea and conjunctiva (hematoxylin–eosin, ·400). A, Normal cornea in the control eyes. B, The corneal epithelium was irregular with a decreased number of epithelial cell layers and basal cells edema in all the operated eyes. C, Normal bulbar conjunctiva in the control eyes showing numerous goblet cells randomly distributed. D, In all the operated eyes, the conjunctival epithelium showed squamous metaplasia with an irregular epithelium thickness, loss of stratification, less homogeneous cell size and staining, and an obvious loss of goblet cells with infiltration of inflammatory cells.
humans, may also correspond to this membrane observed in monkeys or to the nictitating membrane or third eyelid of some other animal species.23 In addition to the removal of the main lacrimal gland and nictitating membrane, Zhu et al24 swabbed the palpebral and bulbar conjunctiva with 30% trichloroacetic acid to induce dry eye in rabbits. These authors observed a decrease in the goblet cell density in the conjunctiva with a moderate or severe infiltration of inflammatory cells in the cornea and
conjunctiva. However, Chen et al,11 in a similar rabbit model using 50% trichloroacetic acid, failed to show a difference in terms of clinical ocular surface tests or goblet cell density between the eyes with or without application of this weak acid. Similarly, before this study, we evaluated the effects of 50% trichloroacetic acid application on the rhesus monkey conjunctiva, and no abnormal clinical sign or ocular surface test results were observed after 1 month (data not shown). These results suggested that swabbing the conjunctiva with
FIGURE 8. The nictitating membrane of the rhesus monkey. A, Slit-lamp photograph of the nictitating membrane in the medial canthus. B, The nictitating membrane extended to an approximately 15- · 15-mm-sized membrane after traction. C, Numerous goblet cells were observed in this tissue (hematoxylin–eosin, ·200). D, Accessory lacrimal glands and cartilage tissue were also detected (hematoxylin–eosin, ·100). 2014 Lippincott Williams & Wilkins
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50% trichloroacetic acid did not induce extensive damage to the ocular surface as was observed in alkali burn models.25 Accordingly, to create a more severe dry eye model than obtained only by removing the main lacrimal gland and nictitating membrane, we also applied 50% trichloroacetic acid on the bulbar and palpebral conjunctiva. Unlike in rabbits, the use of this weak acid was associated with more severe dry eye clinical tests and lower conjunctival goblet cells as compared with that in monkeys that underwent only the surgical procedure. Moreover, no destruction of the ocular surface tissue architecture was observed. According to the 2007 Dry Eye Workshop, dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and ocular surface inflammation.2 Although subjective symptoms and osmolarity were not evaluated in this study, the association of decreased tear secretion and ocular surface histological changes—decreased goblet cell density and an infiltration of inflammatory cells—confirmed the development of a severe dry eye model mimicking dry eye tissue alterations. The investigation techniques available to assess the functional or histological changes induced by the disease are also important parameters to determine the choice of an animal model. In particular for dry eye disease, the precise in vivo evaluation of ocular surface changes and the severity of damage remains a challenge for both ophthalmologist clinicians and researchers. Besides the classic evaluation of clinical parameters, such as the Schirmer test values or fluorescein and rose Bengal staining, we performed conjunctival impression cytology to evaluate in vivo the tissue changes induced on this monkey model. Impression cytology, by applying filter membranes, is a collection technique for the most superficial layers of the conjunctiva, and some authors consider it as an equivalent of conjunctival biopsy.26 Interestingly, impression cytology specimens of rhesus monkeys were similar to those observed in humans, particularly in terms of goblet cell density and distribution over the conjunctiva.24,27–29 Moreover, the results were correlated to the histological tissue analyses that we eventually performed. The loss of goblet cells is considered as a sensible indicator of ocular surface disease.30,31 Accordingly, in this study, the severity of dry eye clinical tests was correlated to the number of goblet cells observed within the conjunctiva. To our knowledge, this is the first study using impression cytology to evaluate the rhesus monkey conjunctiva, and this technique seems to be extremely useful in monitoring in vivo ocular surface changes in that animal model. Although the quantitative evaluation of impression cytology in this study has some limitations, a more precise quantification using flow cytometry, already used in humans,32 might be used in future studies. The therapeutic intervention plan is also a major determining factor in the animal model choice of dry eye. Although monkey models have several negative aspects including the ethical considerations, the costs, and the need of special facilities, they are very similar to human models both anatomically and physiologically.21,22 Stephens
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et al21,22,33 described and illustrated the overall anatomy of the eyelid and of the various ocular surface glands and salivary glands in rhesus monkeys, and concluded that they were highly similar to those of humans in terms of location and gross and microscopic features. Because new therapeutic strategies for severe dry eye need to be evaluated, and in particular, surgical strategies such as autologous minor salivary gland transfer, our aim was to develop a new monkey model of severe dry eye. Combined with the use of impression cytology, this new monkey model may be an interesting tool for the development of new surgical approaches for the treatment of severe dry eye disease.
ACKNOWLEDGMENTS The authors thank Dr Chunyan He, Dr Xiaolin Xu, and Prof Bin Li, Department of Histopathology, Beijing Tongren Hospital and Beijing Institute of Ophthalmology, Capital University of Medical Science, for help with the histology. REFERENCES 1. The epidemiology of dry eye disease: report of the Epidemiology Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007;5:93–107. 2. The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007;5:75–92. 3. Luo SR, Zou LH, Yan C, et al. Transplantation of autologous labial salivary glands for severe dry eye [in Chinese]. Zhonghua Yan Ke Za Zhi. 2013;49:22–26. 4. Tsubota K, Kawashima M, Inaba T, et al. The antiaging approach for the treatment of dry eye. Cornea. 2012;31:S3–S8. 5. Lemp MA. Advances in understanding and managing dry eye disease. Am J Ophthalmol. 2008;146:350–356. 6. Murube-del-Castillo J. Transplantation of salivary gland to the lacrimal basin. Scand J Rheumatol Suppl. 1986;61:264–267. 7. Geerling G, Raus P, Murube J. Minor salivary gland transplantation. Dev Ophthalmol. 2008;41:243–254. 8. Sant’ Anna AE, Hazarbassanov RM, de Freitas D, et al. Estela A, Minor salivary glands and labial mucous membrane graft in the treatment of severe symblepharon and dry eye in patients with Stevens–Johnson syndrome. Br J Ophthalmol. 2012;96:234–239. 9. Marinho DR, Burmann TG, Kwitko S. Labial salivary gland transplantation for severe dry eye due to chemical burns and Stevens–Johnson syndrome. Ophthal Plast Reconstr Surg. 2010;26:182–184. 10. Murube J, Manyari A, Chen-Zhuo L, et al. Labial salivary gland transplantation in severe dry eye. Oper Tech Oculoplast Orbit Reconstr Surg. 1998;1:104–110. 11. Chen ZY, Liang QF, Yu GY. Establishment of a rabbit model for keratoconjunctivitis sicca. Cornea. 2011;30:1024–1029. 12. Barabino S, Dana MR. Animal models of dry eye: a critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci. 2004;45:1641–1646. 13. Schrader S, Mircheff AK, Geerling G. Animal models of dry eye. Dev Ophthalmol. 2008;41:298–312. 14. Maitchouk DY, Beuerman RW, Ohta T, et al. Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys. Arch Ophthalmol. 2000;118:246–252. 15. van Bijsterveld OP. Diagnostic tests in the sicca syndrome. Arch Ophthalmol. 1969;82:10–14. 16. Yoon KC, Im SK, Kim HG, et al. Usefulness of double vital staining with 1% fluorescein and 1% lissamine green in patients with dry eye syndrome. Cornea. 2011;30:972–976. 17. Nelson JD. Impression cytology. Cornea. 1988;7:71–81. 18. Helper LC, Magrane WG, Koehm J, et al. Surgical induction of keratoconjunctivitis sicca in the dog. J Am Vet Med Assoc. 1974;165:172–174.
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19. Jester JV, Nicolaides N, Kiss-Palvolgyi I, et al. Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD. Invest Ophthalmol Vis Sci. 1989;30:936–945. 20. Maudgal PC. The epithelial response in keratitis sicca and keratitis herpetica (an experimental and clinical study). Doc Ophthalmol. 1978;45: 223–227. 21. Stephens LC, Schultheiss TE, Ang KK, et al. Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta). J Med Primatol. 1987;16:407–419. 22. Stephens LC, Schultheiss TE, Vargas KJ, et al. Glands of the eyelids of rhesus monkeys (Macaca mulatta). J Med Primatol. 1989;18: 383–396. 23. Arends G, Schramm U. The structure of the human semilunar plica at different stages of its development—a morphological and morphometric study. Ann Anat. 2004;186:195–207. 24. Zhu ZH, Yu GY, Zou LH, et al. Autologous submandibular gland transfer for the management of xerophthalmia: an experimental study. J Mod Stomatol (Chin). 2001;15:179–181. 25. Ueno M, Lyons LB, et al. Accelerated wound healing of alkali-burned corneas in mrl mice is associated with a reduced inflammatory signature. Invest Ophthalmol Vis Sci. 2005;46:4097–4106.
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26. Calonge M, Diebold Y, Sáez V, et al. Impression cytology of the ocular surface: a review. Exp Eye Res. 2004;78:457–472. 27. Rivas L, Oroza MA, Perez-Esteban A, et al. Topographical distribution of ocular surface cells by the use of impression cytology. Acta Ophthalmol (Copenh). 1991;65:371–376. 28. Rivas L, Alvarez MI, Rodriguez JJ, et al. Ophthalmological tests in patients with keratoconjunctivitis sicca with and without association of primary Sjögren’s syndrome. Ger J Ophthalmol. 1995;4:306–310. 29. Vujković V, Mikac G, Kozomara R. Distribution and density of conjunctival goblet cells. Med Pregl. 2002;55:195–200. 30. Kinoshita S, Kiorpes TC, Friend J, et al. Goblet cell density in ocular surface disease. A better indicator than tear mucin. Arch Ophthalmol. 1983;101:1284–1287. 31. Adams GG, Dilly PN, Kirkness CM. Monitoring ocular disease by impression cytology. Eye (Lond). 1988;2:506–516. 32. Brignole-Baudouin F, Ott AC, Warnet JM, et al. Flow cytometry in conjunctival impression cytology: a new tool for exploring ocular surface pathologies. Exp Eye Res. 2004;78:473–481. 33. Stephens LC, King GK, Ang KK, et al. Surgical and microscopic anatomy of parotid and submandibular salivary glands of rhesus monkeys (Macaca mulatta). J Med Primatol. 1986;15:105–119.
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