Clinical and light microscopic studies of the conjunctival tissues of dogs with bilateral keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine C Izci1, İ Celik2, F Alkan1, M Erol1, E Sur2 1Departments
of Surgery and 2Histology and Embryology, Selcuk University, Veterinary Faculty, 40003, Konya, Turkey
Accepted February 26, 2014
Abstract We determined the concentrations of goblet and immune cells in conjunctival imprints and tissues of canines with keratoconjunctivitis sicca (KCS) before and after cyclosporine A (CsA) treatment. Twelve dogs with bilateral KCS were assigned to three groups: untreated, treatment group 1, and treatment group 2. The treatment groups were treated topically with 2% ophthalmic CsA solution for 45 days; CsA treatment group 2 was followed for an additional 30 days after discontinuation of the drug. Schirmer tear test (STT) scores were recorded prior to CsA treatment and on alternate days throughout the experiment. CsA treatment improved the STT scores, restored conjunctival histology, increased goblet and epithelial cell numbers, and decreased numbers of inflammatory cells. Although the STT scores regressed slightly at day 30 after discontinuing the treatment, the scores were higher than the baseline values. Topical CsA treatment resolved clinical signs of KCS, improved STT scores and restored normal conjunctival histology. Key words: canine, conjunctiva, cyclosporine A, keratoconjunctivitis sicca, mucosal immunity
Keratoconjunctivitis sicca (KCS) is an ophthalmic inflammatory disease of dogs that causes significant loss of the pre-corneal tear film (Morgan and Abrams 1991). In addition to the clinical symptoms, Schirmer tear test (STT) scores 10 mm/min indicate a dry eye condition (Izci et al. 2002). Kaswan and Salisbury (1990) suggested that autoimmune disorders of the third eyelid and lacrimal glands might be major causes of canine KCS. A healthy canine conjunctival epithelium is a stratified squamous epithelium with numerous goblet cells. The population of goblet cells in KCS is decreased significantly (Slatter 1990, Bounous et al. 1998). Histological and immunohistochemical studies have shown that multifocal chronic adenitis, lymphoid infiltrations, focal Correspondence: C İzci, Selcuk University, Veterinary Faculty, Department of Surgery, 40003, Konya, Turkey. Phone: 903322233579. E-mail: [email protected] © 2015 The Biological Stain Commission Biotechnic & Histochemistry 2015, 90(3): 223–230.
DOI: 10.3109/10520295.2014.930177
acinous atrophy and fibrous tissue are increased in the lacrimal glands of dogs with KCS (Kaswan and Salisbury 1990). Lymphocytic infiltrations are composed mainly of B lymphocytes and helper/inducer (CD4) T lymphocytes (Izci et al. 2002). Cyclosporine A (CsA) has been used widely since it was shown to be effective for interrupting immune-mediated reactions against the lacrimal glands. In addition, the drug has minimal side effects, because it specifically suppresses helper/ inducer (CD4) T lymphocyte activity (Scheiber and Crabtree 1992). Investigators have focused primarily on improving clinical signs and restoring histological structure, function and local immune elements of the lacrimal glands of CsA-treated animals (Bounous et al. 1995). Information is limited concerning the effects of ophthalmic 2% CsA treatment on the conjunctival mucosa of CsA-treated dogs with spontaneous KCS. We used the alpha-naphthyl acetate esterase (ANAE) reaction to assess canine T lymphocyte 223
levels in both peripheral blood smears and tissue sections, because it has been shown to be a cytochemical marker for canine T lymphocytes in an earlier study (Wulf et al. 1981). We investigated the effects of ophthalmic 2% CsA treatment on the clinical symptoms of KCS, STT scores, histology and immune cell populations in conjunctival imprints and tissues of dogs with KCS.
Material and methods Animal groups The local Animal Ethics Committee approved our investigation. Five female and seven 3–10-year-old male mixed breed dogs with bilateral KCS were selected based on STT scores 10 mm/min and clinical signs of KCS including hyperemia, mucoid discharge, chemosis and blepharospasm. The dogs were obtained from the Research Animal Facilities of Veterinary Faculty of Selcuk University. Complete blood counts and serum chemistry profiles were within normal ranges. Animals that had prior surgery involving the third eyelid gland or that were treated with topical or systemic drugs that affect tear production were excluded from the study. The dogs were assigned randomly to three groups of four: untreated, 2% CsA-treated for 45 days (CsA-1), and 2% CsA-treated for 45 days and followed-up for 30 days after stopping CsA treatment, because of the possibility of recurrence (CsA-2). CsA treatment and sampling Two percent CsA was prepared by diluting a commercial 10% CsA solution (Sandimmune; Sandoz,
Basel, Switzerland) with extra virgin olive oil for topical administration. The solution was filtered through a 0.45 μm Syrfil-MF® (Sigma-Aldrich, St. Louis, MO) filter and dispensed using a dropper bottle. One drop of 2% CsA was applied to both eyes of all dogs in the CsA-1 and CsA-2 groups, every 12 h for 45 days (Table 1). The untreated animals received no CsA. Conjunctival smears and biopsies of untreated animals were obtained at the beginning of experiment. The animals were returned to the Research Animal Facilities after the experiment. Histological results obtained from the untreated animals were used as baseline data and compared with the results of the CsA-treated groups. Dogs in the CsA-1 and CsA-2 groups received 2% ophthalmic solution of CsA for 45 days. Conjunctival imprints and tissue samples from the CsA-1 group were examined by histological, enzyme histochemical and immunohistochemical methods on day 45 of CsA administration. The dogs in the CsA-2 group were followed for 30 days after discontinuing CsA treatment and their tissue samples were evaluated on day 75 of the experiment. Clinical evaluations and STT scores Clinical signs of KCS in the animals of both treatment groups were monitored throughout the experiment by both direct and indirect ophthalmoscopy. STT scores were measured using tear test strips® (Clement Clarke, Harlow, UK) on alternate days throughout the experiment. STT scores for all groups, which were measured prior to CsA administration (day 0), were considered baseline values.
Table 1. Groups, treatments and sampling schedule Groups n ⴝ 4 Untreated
CsA-1
Treatments and samplings
Day 0 Day 45 Day 75
CsA treatment
none
STT Conjunctival impression slide Conjunctival biopsy
Day 0
Day 45 Day 75
45 days
Day 0
45 days and a following period without CsA treatment for 30 days (until day 75 of the experiment)
(), done; (), not done
224
CsA-2
Biotechnic & Histochemistry 2015, 90(3): 223–230
Day 45 Day 75
Collection and processing of conjunctival imprints and biopsies Conjunctival imprints and tissue samples were taken under general anesthesia from both conjunctivas of each dog. Conjunctival imprints were prepared from the centro-dorso-temporal region of palpebral conjunctiva (Bounous et al. 1995). The imprints were air dried, fixed in glutaraldehydeacetone, then stained with Pappenheim panoptic stain (Gabe 1961). Conjunctival biopsies, 2–3 mm, were collected from the ventral conjunctival fornices of both eyes. The biopsies of untreated animals were taken at the beginning of the study, and from the animals in the CsA-1 and CsA-2 groups on days 45 and 75, respectively (Table 1). Each biopsy specimen was divided into three pieces. One piece was fixed in formal-sucrose (pH 6.8), frozen sectioned and used for ANAE histochemistry. ANAE-positive T lymphocytes were detected using the method of Wulf et al. (1981). Lymphocytes with 2–5 reddish brown granules were classified as ANAE-positive T lymphocytes (Izci et al. 2002). The second piece of each biopsy was used for immunohistochemical detection of B lymphocytes, and CD4 and CD8 T lymphocytes (Izci et al. 2002). Immunostaining was performed by incubating frozen sections with one of the following primary monoclonal antibodies: anti-canine CD4 (MCA 1998S; Serotec, Kidlington, UK), anti-canine CD8 (MCA 1775S: Serotec) and anti-canine B lymphocyte (MCA 1781S; Serotec). A sensitive biotinstreptavidin detection system (STAR 2000®; Serotec) was used for visualization. Negative controls were prepared by incubating sections in PBS without primary antibody. Nuclei were stained with Mayer ’s hematoxylin. Positive cells displayed brownish black staining for each primary antibody. The third piece of each biopsy specimen was processed and embedded in paraffin using routine histological techniques. Sections 7 μm thick were cut and stained with Crossmon’s trichrome stain for general histology (Crossmon 1937), methyl greenpyronin for plasma cells, toluidine blue for mast cells and periodic acid-Schiff stain (PAS) for goblet cells (Stevens and Bancroft 1992). The specimens were viewed using a Nikon Eclipse E400 light microscope (Nikon, Tokyo, Japan). Digital images were processed using BAB BS 200 PRO image analysis software (BAB Microscope and Image Analysis Systems, Ankara, Turkey). Goblet cell density in conjunctival biopsy sections was defined as goblet cell number/50 epithelial cells. Lymphocyte types, plasma cells and mast cells were counted in a 2.25 105 μm2 area of the biopsy
specimens and the results were expressed as mean cell counts (mean cell number/unit area). Percentages of individual cell types were expressed as a percentage of the cells counted. Statistical analysis ANOVA test was used to evaluate STT results within and among the groups. The Wilcoxon-t test (MINITAB Release 12.1, MINITAB Inc.) was used to evaluate differences within the groups of peripheral blood, impression cytology and conjunctival biopsy results. Values for p 0.05 were considered statistically significant.
Results Clinical findings and STT results By day 45 of CsA treatment, clinical signs of KCS were resolved distinctly in both treatment groups (CsA-1 and CsA-2). Recurrences of clinical symptoms were not observed in the CsA-2 group 30 days after the CsA treatment was discontinued. No systemic or local adverse effects of the CsA treatment were observed during the experiment. The STT scores for the animals are given in Table 2. CsA application for 45 days improved STT scores significantly for both eyes for both CsA-treated groups. Although mean STT scores for the CsA-1 and CsA-2 groups were significantly lower than their scores at 45 days (p 0.05), the mean values were significantly higher than those for both the untreated animals and their own baseline levels (p 0.05) (Table 2). Conjunctival imprint and biopsy results Lymphocytes, neutrophils, monocytes and middle layer epithelial cells were abundant in conjunctival Table 2. STT scores (mm/min) STT Groups nⴝ4
Sampling periods
Right eye
Left eye
Untreated CsA-1
Baseline Baseline day 45 day 75 Baseline day 45 day 75
6.65 1.9c 6.50 2.3c 13.33 2.5a 10.00 2.3b 6.78 1.9c 11.33 5.7a 9.62 4.1b
6.73 1.4c 6.83 1.5c 13.50 4.2a 10.17 1.2b 6.82 1.9c 11.35 5.5a 9.79 3.9b
CsA-2
Means SD a,b,cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
Canine keratoconjunctivitis sicca histology 225
1.58 1.0b 11.86 2.6a 11.61 2.4a 1.50 0.9b 11.75 1.9a 11.53 1.7a 3.58 1.7a 1.11 0.8c 1.42 0.7b Means SD RC, right conjunctiva; LC, left conjunctiva. a, b, cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
3.86 1.6a 1.22 0.8c 1.50 0.7b 13.92 2.6a 1.06 0.9b 1.19 0.8b 15.36 3.0a 1.75 1.7b 1.92 1.6b 13.06 3.2a 1.97 0.9b 2.14 0.9b 68.78 4.4b 83.72 3.2a 83.75 2.9a 66.11 5.5b 83.31 3.0a 82.92 2.4a Untreated CsA-1 CsA-2
12.42 3.2a 1.97 0.9b 2.03 0.8b
LC RC LC RC LC RC RC LC RC
Groups (n ⴝ 4)
LC
Goblet cell number/50 epithelial cells Monocyte (%) Neutrophil (%) Lymphocyte (%) Epithelial cell (%)
Table 3. Percentages of cell types in conjunctival impression specimens
226
imprints of the untreated animals (Table 3, Fig. 1), whereas goblet cell densities were fewer. In both CsA-treated groups, the percentages of epithelial and goblet cell density increased significantly (p 0.05), whereas inflammatory cell ratios decreased significantly (p 0.05) in both conjunctivas of the animals on day 45 of CsA treatment (Fig. 2). Slight, but not statistically significant, decreases in the epithelial cell ratio and goblet cell density were observed, whereas a slight increase in inflammatory cell ratio of the CsA-2 group was observed on day 30 after discontinuing CsA treatment and the levels were lower than those of the untreated animals. Lymphocyte, neutrophil and monocyte ratios for the CsA-treated animals were significantly lower than for the untreated animals at days 30 and 75 (p 0.05, Table 3). In conjunctival biopsies, the conjunctival epithelium of untreated animals with KCS was mostly simple squamous with a few goblet cells and desquamated, eroded cellular debris frequently coated the surface of the epithelium (Fig. 3). Conjunctival histology improved in most animals in the CsA-1 (Fig. 4) and CsA-2 groups by day 45 of the CsA treatment. Lymphoid cell infiltration was not seen in the lamina propria of the animals in the CsA-2 group. Goblet cell density in the untreated group was very low; the density increased significantly in the CsA-1 group (p 0.05) and goblet cell density in the CsA-2 group decreased significantly (p 0.05) to a level close to that of the untreated animals (Table 4). The number of plasma cells was relatively low in the biopsies of untreated animals. The counts increased significantly in CsA-1 animals (p 0.05),
Fig. 1. Conjunctival impression of an untreated animal. Middle layer epithelial cells (1) and a neutrophil (2) are shown. Pappenheim panoptic stain. Bar 100 μm.
Biotechnic & Histochemistry 2015, 90(3): 223–230
Fig. 2. Conjunctival impression of an animal from the CsA-1 group on day 45 of treatment. Goblet cells (1, 2, 3) are clearly distinguished with mucous content, and flattened and dislocated nucleus. Basal layer (4) and superficial epithelial (5) cells are seen. Pappenheim panoptic stain. Bar 100 μm.
whereas the counts in CsA-2 animals were similar to those of untreated animals. The number of mast cells in the control animals was significantly higher (p 0.05) than for the CsA-treated groups. CsA treatment produced a significant decrease in mast cell numbers in the CsA-1 group on day 45 of the experiment (p 0.05), and did not change significantly afterward (Table 4). ANAE-positive lymphocyte counts were high in the conjunctival biopsies of untreated animals. The number of ANAE-positive lymphocytes declined gradually in both treatment groups. Although the decrease was not statistically significant in the
Fig. 3. Conjunctival biopsy of an untreated animal. Cellular debris mixed with fibrin (1) can be seen on the surface of the epithelium (2, 3). Crossmon’s trichrome. Bar 100 μm.
Fig. 4. Conjunctival biopsy of an animal from CsA-1 on day 45 of treatment. CsA-2 samples also displayed similar histology. Stratified epithelium (1, 2), desquamated epithelial cells (3) and lamina propria (4) are seen. Crossmon’s trichrome. Bar 100 μm.
CsA-1 group, the difference was significant for the right conjunctivas of the CsA-2 group (p 0.05). In the left conjunctiva, there were significant decreases in ANAE (p 0.05) T lymphocytes in the CsAtreated groups compared to the untreated group. A significant decrease (p 0.05) occurred in mean B lymphocyte count in the right conjunctivas of CsA-1 animals. Left conjunctivas of the treatment groups had significantly lower (p 0.05) B lymphocyte counts (Table 5). The number of CD4 T lymphocytes did not change after CsA treatment of the right conjunctiva and was decreased significantly (p 0.05) in the left conjunctiva of CsA-2 animals. The left conjunctivas of CsA-1 and CsA-2 animals were characterized by a slight decrease of CD4 T-lymphocytes on day 45 of treatment (p 0.05), whereas the decline was significant in the CsA-2 animals 30 days after stopping CsA treatment (p 0.05). The number of CD8positive T lymphocytes in both left and right conjunctivas of the untreated animals were relatively high. Changes in the CD8-positive T lymphocytes of the right conjunctiva were not statistically significant. The left conjunctivas of the CsA-1 group showed a significantly lower CD8-positive T lymphocyte count (p 0.05). Although the T lymphocyte count tended to decrease in the CsA-2 group animals 30 days after treatment, the difference was not statistically significant (Table 5).
Discussion KCS develops when the lacrimal and third eyelid glands are dysfunctional and it causes corneal Canine keratoconjunctivitis sicca histology 227
Table 4. Plasma and mast cell numbers/unit area (2.25 x 105 μm2) and goblet cell densities (goblet cells/50 epithelial cells) of conjunctival biopsies Groups (n ⴝ 4)
Plasma cell RC
Untreated 8.06 3.9b CsA-1 12.47 4.3a CsA-2 7.44 3.4b
Mast cell
LC
RC
Goblet cell LC
9.03 5.0ab 1.61 1.1a 9.50 3.7a 1.06 0.8b 7.64 2.8b 1.17 0.8ab
RC
2.00 1.0a 15.66 2.9b 1.39 1.1b 20.31 2.0a 1.11 0.8b 16.44 1.2b
LC 11.43 2.7b 16.80 1.7a 13.21 1.2b
RC, right conjunctiva; LC, left conjunctiva. a,bThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
symptoms in both CsA treated groups. Goblet cell density increased and inflammatory cell percentages decreased in conjunctival imprints of most animals in both CsA-treated groups by day 45. Baudouin et al. (1987) reported that topical CsA treatment reduced inflammatory infiltrates in conjunctival tissue owing to its immunosuppressive activity and it limited apoptosis in both the lacrimal glands and ocular surface. The significant decrease in apoptosis of the epithelial cells and the increase in lymphocytes of CsA-treated dogs provided additional evidence of the value of CsA in KCS therapy (Gao et al. 1998). In addition to its immunomodulatory activity, CsA also has lacrimo-stimulatory and lacrimo-mimetic effects by augmenting goblet cell restoration (Moore et al. 2001). We found that 2% ophthalmic CsA treatment restored the secretion of mucosubstance and goblet cell equilibrium, which is consistent with previous reports (Izci et al. 2002, Bolzan et al. 2005). Grevel and Kahan (1989) reported that clinical recovery due to CsA treatment might take several months in some animals because of individual differences in absorption, distribution and elimination of the topically administered drug. The delay between the start of CsA treatment and the onset of clinical recovery suggests that glandular restoration occurs during this time. We found that clinical signs of KCS in CsA-treated
and conjunctival changes. KCS typically occurs when the STT score of an affected eye declines to 10 mm/min (Helper 1976, Berdoulay et al. 2005). We selected dogs for CsA treatment based on both STT scores 10 mm/min and KCS-related clinical signs. Upon histological evaluation of the conjunctival tissue, the untreated animals exhibited severe epithelial erosion and loss of goblet cells. These findings were consistent with previous reports (Brignole et al. 2000, Argueso et al. 2002, Kunert et al. 2002, Ueta et al. 2005). Moore et al. (2001) reported that loss of goblet cells and mucosubstance were closely related to the severity of KCS. Although conjunctivitis is the main finding in more than 80% of KCS cases, little is known about specific changes in the conjunctival mucosa. Conjunctival imprints are useful for evaluating the course of KCS. The conjunctival epithelium of healthy dogs is stratified and consists of basal, middle and superficial cell layers (Lavach et al. 1977). Bolzan et al. (2005) observed leukocytes in 47% and mucoid substance in 53% of healthy imprints. We found that the conjunctival epithelium of the untreated animals with KCS frequently was desquamated and eroded. An abundant middle layer of epithelial cells and low numbers of goblet cells in imprint specimens also indicated a decrease in the number of goblet cells in KCS. By day 45, treatment with CsA produced recovery to normal STT levels and resolution of clinical
Table 5. Numbers of lymphocyte subtypes/unit area (2.25 x 105 μm2) of conjunctival biopsies Groups (n ⴝ 4) Untreated CsA-1 CsA-2
ANAEⴙ T lymphocyte
B lymphocyte
CD4 ⴙ T lymphocyte
CD8 ⴙT lymphocyte
RC
LC
RC
LC
RC
LC
RC
LC
3.39 1.2a 3.06 1.5ab 2.83 1.7b
3.81 1.2a 2.75 1.3b 2.00 0.9c
2.58 1.0a 2.44 1.1a 2.11 1.1b
2.69 0.9a 2.25 0.8b 1.47 0.9c
1.89 1.1a 2.14 1.3a 1.83 1.1a
2.11 0.9a 1.97 1.0a 1.47 0.9b
1.64 1.0a 1.65 1.1a 1.56 1.2a
1.92 1.1a 1.33 1.0b 0.97 1.0b
RC, right conjunctiva; LC, left conjunctiva. a,b,cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
228
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animals were resolved approximately 10 days earlier than suggested in an earlier report (Grevel and Kahan 1989). A prominent histological change in the conjunctival tissues of dogs with KCS is infiltration of lymphoid cells, i.e., mainly CD4- and CD8positive T lymphocytes (Izci et al. 2002); B cells are significantly fewer in number. We observed that the conjunctival epithelium and lamina propria of untreated animals were heavily populated with T and B lymphocytes and that their numbers were significantly greater than for both groups of CsA-treated animals. Slight differences between STT scores and conjunctival cell populations were found between the right and left eyes of both untreated and CsA-treated animals. Our experimental design did not permit us to explain the difference in response of right and left conjunctivas to CsA treatment. The high mast cell numbers in the conjunctival biopsies of the untreated animals may indicate that allergic reactions also affect the development of KCS. Because ocular tissues have numerous histamine receptors, allergic reactions may play a role in tissue destruction owing to excess IgE production, which triggers type I hypersensitivity by accelerating histamine release from mast cells (Munger 2001). CsA treatment decreased significantly the number of mast cells in the conjunctival samples of both the right and left eyes on day 45. Interestingly, the number of mast cells was not changed in the CsA-2 group on day 30 after stopping the treatment. Frequent recurrences of KCS have been reported after discontinuing ophthalmic CsA treatment (Olivero et al. 1991). Gilger et al. (1995) reported that the peripheral blood lymphocyte proliferation and cellular immune reactions also were suppressed in animals treated with 2% ophthalmic CsA. We observed no side effects attributable to CsA, however, and the symptoms of KCS were not observed in the animals on day 30 after stopping the treatment. We demonstrated that 2% topical CsA application for 45 days produced significant resolution of clinical signs of KCS, improvement in STT scores and restoration of conjunctival histology including increased goblet cell density; clinical symptoms of the disease did not recur. Although a slight regression in STT scores occurred 30 days after stopping CsA treatment, the scores were higher than the baseline levels of all groups. Scientific Research Projects (BAP) Coordinating Office of Selcuk University funded this study; Research Project Number 110/2003.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for its contents.
References Argueso P, Balaram M, Spurr-Michaud S, Keutmann HT, Reza DM, Gipson IK (2002) Decreased levels of the goblet cell mucin MUC5AC in tears of patients with Sjögren syndrome. Invest. Ophthalmol. Vis. Sci. 43: 1004–1011. Baudouin C, Brignole F, Becquet F, Pisella PJ, Goguel A (1997) Flow cytometry in impression cytology specimens. A new method for evaluation of conjunctival inflammation. Invest. Ophthalmol. Vis. Sci. 38: 1458–1464. Berdoulay A, English RV, Nadelstein B (2005) Effect of topical 0.02% tacrolimus aqueous suspension on tear production in dogs with keratoconjunctivitis sicca. Vet. Ophthalmol. 4: 225–232. Bolzan AA, Brunelli ADJ, Castro MB, Souza MA, Souza JL, Laus J (2005) Conjunctival impression cytology in dogs. Vet. Ophthalmol. 8: 401–405. Bounous DI, Carmichael KP, Kaswan RL, Hirsh S, Stiles J (1995) Effects of ophthalmic cyclosporine on lacrimal gland pathology and function in dogs with keratoconjunctivitis sicca. Vet. Comp. Ophthalmol. 1: 5–12. Bounous DI, Krenzer KL, Kaswan, RL, Hirsh SG (1998) Conjunctival impression cytology from dogs with keratoconjunctivitis sicca. Pre- and post-treatment with topical cyclosporine. Adv. Exp. Med. Biol. 438: 991–1000. Brignole F, Pisella PJ, Goldschild M, De Saint JM, Goguel A, Baudouin C (2000) Flow cytometric analysis of inflammatory markers in conjunctival epithelial cells of patients with dry eyes. Invest. Ophthalmol. Vis. Sci. 41: 1356–1363. Crossmon GA (1937) Modification of Mallory’s connective tissue stain with a discussion of the principles involved. Anat. Rec. 69: 33–38. Gabe M, Ed. (1961) Techniques Histologiques. 1st ed., Masson, Paris. pp. 784–785. Gao J, Schwalb TA, Addeo JV, Ghosn CR, Stern ME (1998) The role of apoptosis in the pathogenesis of canine keratoconjunctivitis sicca: the effect of topical cyclosporine A therapy. Cornea 17: 654–663. Gilger BC, Andrews J, Wilkie DA, Wyman M, Lairmore MD (1995) Cellular immunity in dogs with keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine. Vet. Immunol. Immunopathol. 3: 199–208. Grevel J, Kahan BD (1989) Pharmacokinetics of cyclosporine A. In: Thomson AW, Ed., Cylosporine: Mode of Action and Clinical Application, 1st ed., Kluwer Co., Dordrecht, The Netherlands. pp. 252–266. Helper LC (1976) Keratoconjunctivitis sicca in dogs. Trans. Am. Soc. Ophthalmol. Otolaryngol. 81: 624–628. Izci C, Çelik I, Alkan F, Oğurtan Z, Ceylan C, Sur E. Özkan Y (2002) Histologic characteristics and local cellular immunity of the gland of the third eyelid after topical ophthalmic administration of 2% cyclosporine for treatment of dogs with keratoconjunctivitis sicca. Am. J. Vet. Res. 63: 688–694.
Canine keratoconjunctivitis sicca histology 229
Kaswan RL, Salisbury MA (1990) A new perspective on canine keratoconjunctivitis sicca. Vet. Clin. N. Am-Small 20: 583–613. Kunert KS, Tisdule AS, Gipson IK (2002) Goblet cell numbers and epithelial proliferation in the conjunctiva of patients with dry eye syndrome treated with cyclosporine. Arch. Ophthalmol. 120: 330–337. Lavach JD, Thrall MA, Benjamin MM, Severin GA (1977) Cytology of normal and inflamed conjunctivas in dogs and cats. J. Am. Vet. Med. Assoc. 170: 722–727. Moore CP, McHugh JB, Thorne JG, Phillips TE (2001) Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model. Invest. Ophthalmol. Vis. Sci. 42: 653–659. Morgan RV, Abrams KL (1991) Topical administration of cyclosporine for treatment of keratoconjunctivitis sicca in dogs. J. Am. Vet. Med. Assoc. 199: 1043–1046. Munger R (2001) Diseases and immunity of the ocular surface. In: Proceedings of the World Small Animal Veterinary Association Congress, Vancouver, Canada.
230
Olivero DK, Davidson MG, English RV (1991) Clinical evaluation of 1% cyclosporine for topical treatment of keratoconjunctivitis sicca in dogs. J. Am. Vet. Med. Assoc. 199: 1039–1042. Schreiber SL, Crabtree GR (1992) The mechanism of action of cyclosporine and FK506. Immunol. Today 13: 136–141. Slatter DH (1990) Basic diagnostic techniques, conjunctiva and lacrimal system. In: Slatter DH, Ed., Fundamentals of Veterinary Ophthalmology, 4th ed. WB Saunders Co, Philadelphia. pp. 84–256. Stevens A, Bancroft JD (1992) Proteins and nucleic acids. In: Bancroft JD, Stevens A, Eds., The Theory and Practice of Histological Techniques, 3rd ed. The Bath Press, Avon. pp. 25, 143–153. Ueta M, Hamuro A, Yamamoto M, Kaseda K, Akira S, Kinoshita S (2005) Spontaneous ocular surface inflammation and goblet cell disappearance in 1 kappa B zeta gene-disrupted mice. Invest. Ophthalmol. Vis. Sci. 46: 579–588. Wulf JC, Sale GE, Deeg HJ, Storb R (1981) Nonspecific acid esterase activity as a marker for canine T-lymphocytes. Exp. Hematol. 9: 865–870.
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of Surgery and 2Histology and Embryology, Selcuk University, Veterinary Faculty, 40003, Konya, Turkey
Accepted February 26, 2014
Abstract We determined the concentrations of goblet and immune cells in conjunctival imprints and tissues of canines with keratoconjunctivitis sicca (KCS) before and after cyclosporine A (CsA) treatment. Twelve dogs with bilateral KCS were assigned to three groups: untreated, treatment group 1, and treatment group 2. The treatment groups were treated topically with 2% ophthalmic CsA solution for 45 days; CsA treatment group 2 was followed for an additional 30 days after discontinuation of the drug. Schirmer tear test (STT) scores were recorded prior to CsA treatment and on alternate days throughout the experiment. CsA treatment improved the STT scores, restored conjunctival histology, increased goblet and epithelial cell numbers, and decreased numbers of inflammatory cells. Although the STT scores regressed slightly at day 30 after discontinuing the treatment, the scores were higher than the baseline values. Topical CsA treatment resolved clinical signs of KCS, improved STT scores and restored normal conjunctival histology. Key words: canine, conjunctiva, cyclosporine A, keratoconjunctivitis sicca, mucosal immunity
Keratoconjunctivitis sicca (KCS) is an ophthalmic inflammatory disease of dogs that causes significant loss of the pre-corneal tear film (Morgan and Abrams 1991). In addition to the clinical symptoms, Schirmer tear test (STT) scores 10 mm/min indicate a dry eye condition (Izci et al. 2002). Kaswan and Salisbury (1990) suggested that autoimmune disorders of the third eyelid and lacrimal glands might be major causes of canine KCS. A healthy canine conjunctival epithelium is a stratified squamous epithelium with numerous goblet cells. The population of goblet cells in KCS is decreased significantly (Slatter 1990, Bounous et al. 1998). Histological and immunohistochemical studies have shown that multifocal chronic adenitis, lymphoid infiltrations, focal Correspondence: C İzci, Selcuk University, Veterinary Faculty, Department of Surgery, 40003, Konya, Turkey. Phone: 903322233579. E-mail: [email protected] © 2015 The Biological Stain Commission Biotechnic & Histochemistry 2015, 90(3): 223–230.
DOI: 10.3109/10520295.2014.930177
acinous atrophy and fibrous tissue are increased in the lacrimal glands of dogs with KCS (Kaswan and Salisbury 1990). Lymphocytic infiltrations are composed mainly of B lymphocytes and helper/inducer (CD4) T lymphocytes (Izci et al. 2002). Cyclosporine A (CsA) has been used widely since it was shown to be effective for interrupting immune-mediated reactions against the lacrimal glands. In addition, the drug has minimal side effects, because it specifically suppresses helper/ inducer (CD4) T lymphocyte activity (Scheiber and Crabtree 1992). Investigators have focused primarily on improving clinical signs and restoring histological structure, function and local immune elements of the lacrimal glands of CsA-treated animals (Bounous et al. 1995). Information is limited concerning the effects of ophthalmic 2% CsA treatment on the conjunctival mucosa of CsA-treated dogs with spontaneous KCS. We used the alpha-naphthyl acetate esterase (ANAE) reaction to assess canine T lymphocyte 223
levels in both peripheral blood smears and tissue sections, because it has been shown to be a cytochemical marker for canine T lymphocytes in an earlier study (Wulf et al. 1981). We investigated the effects of ophthalmic 2% CsA treatment on the clinical symptoms of KCS, STT scores, histology and immune cell populations in conjunctival imprints and tissues of dogs with KCS.
Material and methods Animal groups The local Animal Ethics Committee approved our investigation. Five female and seven 3–10-year-old male mixed breed dogs with bilateral KCS were selected based on STT scores 10 mm/min and clinical signs of KCS including hyperemia, mucoid discharge, chemosis and blepharospasm. The dogs were obtained from the Research Animal Facilities of Veterinary Faculty of Selcuk University. Complete blood counts and serum chemistry profiles were within normal ranges. Animals that had prior surgery involving the third eyelid gland or that were treated with topical or systemic drugs that affect tear production were excluded from the study. The dogs were assigned randomly to three groups of four: untreated, 2% CsA-treated for 45 days (CsA-1), and 2% CsA-treated for 45 days and followed-up for 30 days after stopping CsA treatment, because of the possibility of recurrence (CsA-2). CsA treatment and sampling Two percent CsA was prepared by diluting a commercial 10% CsA solution (Sandimmune; Sandoz,
Basel, Switzerland) with extra virgin olive oil for topical administration. The solution was filtered through a 0.45 μm Syrfil-MF® (Sigma-Aldrich, St. Louis, MO) filter and dispensed using a dropper bottle. One drop of 2% CsA was applied to both eyes of all dogs in the CsA-1 and CsA-2 groups, every 12 h for 45 days (Table 1). The untreated animals received no CsA. Conjunctival smears and biopsies of untreated animals were obtained at the beginning of experiment. The animals were returned to the Research Animal Facilities after the experiment. Histological results obtained from the untreated animals were used as baseline data and compared with the results of the CsA-treated groups. Dogs in the CsA-1 and CsA-2 groups received 2% ophthalmic solution of CsA for 45 days. Conjunctival imprints and tissue samples from the CsA-1 group were examined by histological, enzyme histochemical and immunohistochemical methods on day 45 of CsA administration. The dogs in the CsA-2 group were followed for 30 days after discontinuing CsA treatment and their tissue samples were evaluated on day 75 of the experiment. Clinical evaluations and STT scores Clinical signs of KCS in the animals of both treatment groups were monitored throughout the experiment by both direct and indirect ophthalmoscopy. STT scores were measured using tear test strips® (Clement Clarke, Harlow, UK) on alternate days throughout the experiment. STT scores for all groups, which were measured prior to CsA administration (day 0), were considered baseline values.
Table 1. Groups, treatments and sampling schedule Groups n ⴝ 4 Untreated
CsA-1
Treatments and samplings
Day 0 Day 45 Day 75
CsA treatment
none
STT Conjunctival impression slide Conjunctival biopsy
Day 0
Day 45 Day 75
45 days
Day 0
45 days and a following period without CsA treatment for 30 days (until day 75 of the experiment)
(), done; (), not done
224
CsA-2
Biotechnic & Histochemistry 2015, 90(3): 223–230
Day 45 Day 75
Collection and processing of conjunctival imprints and biopsies Conjunctival imprints and tissue samples were taken under general anesthesia from both conjunctivas of each dog. Conjunctival imprints were prepared from the centro-dorso-temporal region of palpebral conjunctiva (Bounous et al. 1995). The imprints were air dried, fixed in glutaraldehydeacetone, then stained with Pappenheim panoptic stain (Gabe 1961). Conjunctival biopsies, 2–3 mm, were collected from the ventral conjunctival fornices of both eyes. The biopsies of untreated animals were taken at the beginning of the study, and from the animals in the CsA-1 and CsA-2 groups on days 45 and 75, respectively (Table 1). Each biopsy specimen was divided into three pieces. One piece was fixed in formal-sucrose (pH 6.8), frozen sectioned and used for ANAE histochemistry. ANAE-positive T lymphocytes were detected using the method of Wulf et al. (1981). Lymphocytes with 2–5 reddish brown granules were classified as ANAE-positive T lymphocytes (Izci et al. 2002). The second piece of each biopsy was used for immunohistochemical detection of B lymphocytes, and CD4 and CD8 T lymphocytes (Izci et al. 2002). Immunostaining was performed by incubating frozen sections with one of the following primary monoclonal antibodies: anti-canine CD4 (MCA 1998S; Serotec, Kidlington, UK), anti-canine CD8 (MCA 1775S: Serotec) and anti-canine B lymphocyte (MCA 1781S; Serotec). A sensitive biotinstreptavidin detection system (STAR 2000®; Serotec) was used for visualization. Negative controls were prepared by incubating sections in PBS without primary antibody. Nuclei were stained with Mayer ’s hematoxylin. Positive cells displayed brownish black staining for each primary antibody. The third piece of each biopsy specimen was processed and embedded in paraffin using routine histological techniques. Sections 7 μm thick were cut and stained with Crossmon’s trichrome stain for general histology (Crossmon 1937), methyl greenpyronin for plasma cells, toluidine blue for mast cells and periodic acid-Schiff stain (PAS) for goblet cells (Stevens and Bancroft 1992). The specimens were viewed using a Nikon Eclipse E400 light microscope (Nikon, Tokyo, Japan). Digital images were processed using BAB BS 200 PRO image analysis software (BAB Microscope and Image Analysis Systems, Ankara, Turkey). Goblet cell density in conjunctival biopsy sections was defined as goblet cell number/50 epithelial cells. Lymphocyte types, plasma cells and mast cells were counted in a 2.25 105 μm2 area of the biopsy
specimens and the results were expressed as mean cell counts (mean cell number/unit area). Percentages of individual cell types were expressed as a percentage of the cells counted. Statistical analysis ANOVA test was used to evaluate STT results within and among the groups. The Wilcoxon-t test (MINITAB Release 12.1, MINITAB Inc.) was used to evaluate differences within the groups of peripheral blood, impression cytology and conjunctival biopsy results. Values for p 0.05 were considered statistically significant.
Results Clinical findings and STT results By day 45 of CsA treatment, clinical signs of KCS were resolved distinctly in both treatment groups (CsA-1 and CsA-2). Recurrences of clinical symptoms were not observed in the CsA-2 group 30 days after the CsA treatment was discontinued. No systemic or local adverse effects of the CsA treatment were observed during the experiment. The STT scores for the animals are given in Table 2. CsA application for 45 days improved STT scores significantly for both eyes for both CsA-treated groups. Although mean STT scores for the CsA-1 and CsA-2 groups were significantly lower than their scores at 45 days (p 0.05), the mean values were significantly higher than those for both the untreated animals and their own baseline levels (p 0.05) (Table 2). Conjunctival imprint and biopsy results Lymphocytes, neutrophils, monocytes and middle layer epithelial cells were abundant in conjunctival Table 2. STT scores (mm/min) STT Groups nⴝ4
Sampling periods
Right eye
Left eye
Untreated CsA-1
Baseline Baseline day 45 day 75 Baseline day 45 day 75
6.65 1.9c 6.50 2.3c 13.33 2.5a 10.00 2.3b 6.78 1.9c 11.33 5.7a 9.62 4.1b
6.73 1.4c 6.83 1.5c 13.50 4.2a 10.17 1.2b 6.82 1.9c 11.35 5.5a 9.79 3.9b
CsA-2
Means SD a,b,cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
Canine keratoconjunctivitis sicca histology 225
1.58 1.0b 11.86 2.6a 11.61 2.4a 1.50 0.9b 11.75 1.9a 11.53 1.7a 3.58 1.7a 1.11 0.8c 1.42 0.7b Means SD RC, right conjunctiva; LC, left conjunctiva. a, b, cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
3.86 1.6a 1.22 0.8c 1.50 0.7b 13.92 2.6a 1.06 0.9b 1.19 0.8b 15.36 3.0a 1.75 1.7b 1.92 1.6b 13.06 3.2a 1.97 0.9b 2.14 0.9b 68.78 4.4b 83.72 3.2a 83.75 2.9a 66.11 5.5b 83.31 3.0a 82.92 2.4a Untreated CsA-1 CsA-2
12.42 3.2a 1.97 0.9b 2.03 0.8b
LC RC LC RC LC RC RC LC RC
Groups (n ⴝ 4)
LC
Goblet cell number/50 epithelial cells Monocyte (%) Neutrophil (%) Lymphocyte (%) Epithelial cell (%)
Table 3. Percentages of cell types in conjunctival impression specimens
226
imprints of the untreated animals (Table 3, Fig. 1), whereas goblet cell densities were fewer. In both CsA-treated groups, the percentages of epithelial and goblet cell density increased significantly (p 0.05), whereas inflammatory cell ratios decreased significantly (p 0.05) in both conjunctivas of the animals on day 45 of CsA treatment (Fig. 2). Slight, but not statistically significant, decreases in the epithelial cell ratio and goblet cell density were observed, whereas a slight increase in inflammatory cell ratio of the CsA-2 group was observed on day 30 after discontinuing CsA treatment and the levels were lower than those of the untreated animals. Lymphocyte, neutrophil and monocyte ratios for the CsA-treated animals were significantly lower than for the untreated animals at days 30 and 75 (p 0.05, Table 3). In conjunctival biopsies, the conjunctival epithelium of untreated animals with KCS was mostly simple squamous with a few goblet cells and desquamated, eroded cellular debris frequently coated the surface of the epithelium (Fig. 3). Conjunctival histology improved in most animals in the CsA-1 (Fig. 4) and CsA-2 groups by day 45 of the CsA treatment. Lymphoid cell infiltration was not seen in the lamina propria of the animals in the CsA-2 group. Goblet cell density in the untreated group was very low; the density increased significantly in the CsA-1 group (p 0.05) and goblet cell density in the CsA-2 group decreased significantly (p 0.05) to a level close to that of the untreated animals (Table 4). The number of plasma cells was relatively low in the biopsies of untreated animals. The counts increased significantly in CsA-1 animals (p 0.05),
Fig. 1. Conjunctival impression of an untreated animal. Middle layer epithelial cells (1) and a neutrophil (2) are shown. Pappenheim panoptic stain. Bar 100 μm.
Biotechnic & Histochemistry 2015, 90(3): 223–230
Fig. 2. Conjunctival impression of an animal from the CsA-1 group on day 45 of treatment. Goblet cells (1, 2, 3) are clearly distinguished with mucous content, and flattened and dislocated nucleus. Basal layer (4) and superficial epithelial (5) cells are seen. Pappenheim panoptic stain. Bar 100 μm.
whereas the counts in CsA-2 animals were similar to those of untreated animals. The number of mast cells in the control animals was significantly higher (p 0.05) than for the CsA-treated groups. CsA treatment produced a significant decrease in mast cell numbers in the CsA-1 group on day 45 of the experiment (p 0.05), and did not change significantly afterward (Table 4). ANAE-positive lymphocyte counts were high in the conjunctival biopsies of untreated animals. The number of ANAE-positive lymphocytes declined gradually in both treatment groups. Although the decrease was not statistically significant in the
Fig. 3. Conjunctival biopsy of an untreated animal. Cellular debris mixed with fibrin (1) can be seen on the surface of the epithelium (2, 3). Crossmon’s trichrome. Bar 100 μm.
Fig. 4. Conjunctival biopsy of an animal from CsA-1 on day 45 of treatment. CsA-2 samples also displayed similar histology. Stratified epithelium (1, 2), desquamated epithelial cells (3) and lamina propria (4) are seen. Crossmon’s trichrome. Bar 100 μm.
CsA-1 group, the difference was significant for the right conjunctivas of the CsA-2 group (p 0.05). In the left conjunctiva, there were significant decreases in ANAE (p 0.05) T lymphocytes in the CsAtreated groups compared to the untreated group. A significant decrease (p 0.05) occurred in mean B lymphocyte count in the right conjunctivas of CsA-1 animals. Left conjunctivas of the treatment groups had significantly lower (p 0.05) B lymphocyte counts (Table 5). The number of CD4 T lymphocytes did not change after CsA treatment of the right conjunctiva and was decreased significantly (p 0.05) in the left conjunctiva of CsA-2 animals. The left conjunctivas of CsA-1 and CsA-2 animals were characterized by a slight decrease of CD4 T-lymphocytes on day 45 of treatment (p 0.05), whereas the decline was significant in the CsA-2 animals 30 days after stopping CsA treatment (p 0.05). The number of CD8positive T lymphocytes in both left and right conjunctivas of the untreated animals were relatively high. Changes in the CD8-positive T lymphocytes of the right conjunctiva were not statistically significant. The left conjunctivas of the CsA-1 group showed a significantly lower CD8-positive T lymphocyte count (p 0.05). Although the T lymphocyte count tended to decrease in the CsA-2 group animals 30 days after treatment, the difference was not statistically significant (Table 5).
Discussion KCS develops when the lacrimal and third eyelid glands are dysfunctional and it causes corneal Canine keratoconjunctivitis sicca histology 227
Table 4. Plasma and mast cell numbers/unit area (2.25 x 105 μm2) and goblet cell densities (goblet cells/50 epithelial cells) of conjunctival biopsies Groups (n ⴝ 4)
Plasma cell RC
Untreated 8.06 3.9b CsA-1 12.47 4.3a CsA-2 7.44 3.4b
Mast cell
LC
RC
Goblet cell LC
9.03 5.0ab 1.61 1.1a 9.50 3.7a 1.06 0.8b 7.64 2.8b 1.17 0.8ab
RC
2.00 1.0a 15.66 2.9b 1.39 1.1b 20.31 2.0a 1.11 0.8b 16.44 1.2b
LC 11.43 2.7b 16.80 1.7a 13.21 1.2b
RC, right conjunctiva; LC, left conjunctiva. a,bThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
symptoms in both CsA treated groups. Goblet cell density increased and inflammatory cell percentages decreased in conjunctival imprints of most animals in both CsA-treated groups by day 45. Baudouin et al. (1987) reported that topical CsA treatment reduced inflammatory infiltrates in conjunctival tissue owing to its immunosuppressive activity and it limited apoptosis in both the lacrimal glands and ocular surface. The significant decrease in apoptosis of the epithelial cells and the increase in lymphocytes of CsA-treated dogs provided additional evidence of the value of CsA in KCS therapy (Gao et al. 1998). In addition to its immunomodulatory activity, CsA also has lacrimo-stimulatory and lacrimo-mimetic effects by augmenting goblet cell restoration (Moore et al. 2001). We found that 2% ophthalmic CsA treatment restored the secretion of mucosubstance and goblet cell equilibrium, which is consistent with previous reports (Izci et al. 2002, Bolzan et al. 2005). Grevel and Kahan (1989) reported that clinical recovery due to CsA treatment might take several months in some animals because of individual differences in absorption, distribution and elimination of the topically administered drug. The delay between the start of CsA treatment and the onset of clinical recovery suggests that glandular restoration occurs during this time. We found that clinical signs of KCS in CsA-treated
and conjunctival changes. KCS typically occurs when the STT score of an affected eye declines to 10 mm/min (Helper 1976, Berdoulay et al. 2005). We selected dogs for CsA treatment based on both STT scores 10 mm/min and KCS-related clinical signs. Upon histological evaluation of the conjunctival tissue, the untreated animals exhibited severe epithelial erosion and loss of goblet cells. These findings were consistent with previous reports (Brignole et al. 2000, Argueso et al. 2002, Kunert et al. 2002, Ueta et al. 2005). Moore et al. (2001) reported that loss of goblet cells and mucosubstance were closely related to the severity of KCS. Although conjunctivitis is the main finding in more than 80% of KCS cases, little is known about specific changes in the conjunctival mucosa. Conjunctival imprints are useful for evaluating the course of KCS. The conjunctival epithelium of healthy dogs is stratified and consists of basal, middle and superficial cell layers (Lavach et al. 1977). Bolzan et al. (2005) observed leukocytes in 47% and mucoid substance in 53% of healthy imprints. We found that the conjunctival epithelium of the untreated animals with KCS frequently was desquamated and eroded. An abundant middle layer of epithelial cells and low numbers of goblet cells in imprint specimens also indicated a decrease in the number of goblet cells in KCS. By day 45, treatment with CsA produced recovery to normal STT levels and resolution of clinical
Table 5. Numbers of lymphocyte subtypes/unit area (2.25 x 105 μm2) of conjunctival biopsies Groups (n ⴝ 4) Untreated CsA-1 CsA-2
ANAEⴙ T lymphocyte
B lymphocyte
CD4 ⴙ T lymphocyte
CD8 ⴙT lymphocyte
RC
LC
RC
LC
RC
LC
RC
LC
3.39 1.2a 3.06 1.5ab 2.83 1.7b
3.81 1.2a 2.75 1.3b 2.00 0.9c
2.58 1.0a 2.44 1.1a 2.11 1.1b
2.69 0.9a 2.25 0.8b 1.47 0.9c
1.89 1.1a 2.14 1.3a 1.83 1.1a
2.11 0.9a 1.97 1.0a 1.47 0.9b
1.64 1.0a 1.65 1.1a 1.56 1.2a
1.92 1.1a 1.33 1.0b 0.97 1.0b
RC, right conjunctiva; LC, left conjunctiva. a,b,cThe difference between mean values in a column with different superscripts is statistically significant (p 0.05).
228
Biotechnic & Histochemistry 2015, 90(3): 223–230
animals were resolved approximately 10 days earlier than suggested in an earlier report (Grevel and Kahan 1989). A prominent histological change in the conjunctival tissues of dogs with KCS is infiltration of lymphoid cells, i.e., mainly CD4- and CD8positive T lymphocytes (Izci et al. 2002); B cells are significantly fewer in number. We observed that the conjunctival epithelium and lamina propria of untreated animals were heavily populated with T and B lymphocytes and that their numbers were significantly greater than for both groups of CsA-treated animals. Slight differences between STT scores and conjunctival cell populations were found between the right and left eyes of both untreated and CsA-treated animals. Our experimental design did not permit us to explain the difference in response of right and left conjunctivas to CsA treatment. The high mast cell numbers in the conjunctival biopsies of the untreated animals may indicate that allergic reactions also affect the development of KCS. Because ocular tissues have numerous histamine receptors, allergic reactions may play a role in tissue destruction owing to excess IgE production, which triggers type I hypersensitivity by accelerating histamine release from mast cells (Munger 2001). CsA treatment decreased significantly the number of mast cells in the conjunctival samples of both the right and left eyes on day 45. Interestingly, the number of mast cells was not changed in the CsA-2 group on day 30 after stopping the treatment. Frequent recurrences of KCS have been reported after discontinuing ophthalmic CsA treatment (Olivero et al. 1991). Gilger et al. (1995) reported that the peripheral blood lymphocyte proliferation and cellular immune reactions also were suppressed in animals treated with 2% ophthalmic CsA. We observed no side effects attributable to CsA, however, and the symptoms of KCS were not observed in the animals on day 30 after stopping the treatment. We demonstrated that 2% topical CsA application for 45 days produced significant resolution of clinical signs of KCS, improvement in STT scores and restoration of conjunctival histology including increased goblet cell density; clinical symptoms of the disease did not recur. Although a slight regression in STT scores occurred 30 days after stopping CsA treatment, the scores were higher than the baseline levels of all groups. Scientific Research Projects (BAP) Coordinating Office of Selcuk University funded this study; Research Project Number 110/2003.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for its contents.
References Argueso P, Balaram M, Spurr-Michaud S, Keutmann HT, Reza DM, Gipson IK (2002) Decreased levels of the goblet cell mucin MUC5AC in tears of patients with Sjögren syndrome. Invest. Ophthalmol. Vis. Sci. 43: 1004–1011. Baudouin C, Brignole F, Becquet F, Pisella PJ, Goguel A (1997) Flow cytometry in impression cytology specimens. A new method for evaluation of conjunctival inflammation. Invest. Ophthalmol. Vis. Sci. 38: 1458–1464. Berdoulay A, English RV, Nadelstein B (2005) Effect of topical 0.02% tacrolimus aqueous suspension on tear production in dogs with keratoconjunctivitis sicca. Vet. Ophthalmol. 4: 225–232. Bolzan AA, Brunelli ADJ, Castro MB, Souza MA, Souza JL, Laus J (2005) Conjunctival impression cytology in dogs. Vet. Ophthalmol. 8: 401–405. Bounous DI, Carmichael KP, Kaswan RL, Hirsh S, Stiles J (1995) Effects of ophthalmic cyclosporine on lacrimal gland pathology and function in dogs with keratoconjunctivitis sicca. Vet. Comp. Ophthalmol. 1: 5–12. Bounous DI, Krenzer KL, Kaswan, RL, Hirsh SG (1998) Conjunctival impression cytology from dogs with keratoconjunctivitis sicca. Pre- and post-treatment with topical cyclosporine. Adv. Exp. Med. Biol. 438: 991–1000. Brignole F, Pisella PJ, Goldschild M, De Saint JM, Goguel A, Baudouin C (2000) Flow cytometric analysis of inflammatory markers in conjunctival epithelial cells of patients with dry eyes. Invest. Ophthalmol. Vis. Sci. 41: 1356–1363. Crossmon GA (1937) Modification of Mallory’s connective tissue stain with a discussion of the principles involved. Anat. Rec. 69: 33–38. Gabe M, Ed. (1961) Techniques Histologiques. 1st ed., Masson, Paris. pp. 784–785. Gao J, Schwalb TA, Addeo JV, Ghosn CR, Stern ME (1998) The role of apoptosis in the pathogenesis of canine keratoconjunctivitis sicca: the effect of topical cyclosporine A therapy. Cornea 17: 654–663. Gilger BC, Andrews J, Wilkie DA, Wyman M, Lairmore MD (1995) Cellular immunity in dogs with keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine. Vet. Immunol. Immunopathol. 3: 199–208. Grevel J, Kahan BD (1989) Pharmacokinetics of cyclosporine A. In: Thomson AW, Ed., Cylosporine: Mode of Action and Clinical Application, 1st ed., Kluwer Co., Dordrecht, The Netherlands. pp. 252–266. Helper LC (1976) Keratoconjunctivitis sicca in dogs. Trans. Am. Soc. Ophthalmol. Otolaryngol. 81: 624–628. Izci C, Çelik I, Alkan F, Oğurtan Z, Ceylan C, Sur E. Özkan Y (2002) Histologic characteristics and local cellular immunity of the gland of the third eyelid after topical ophthalmic administration of 2% cyclosporine for treatment of dogs with keratoconjunctivitis sicca. Am. J. Vet. Res. 63: 688–694.
Canine keratoconjunctivitis sicca histology 229
Kaswan RL, Salisbury MA (1990) A new perspective on canine keratoconjunctivitis sicca. Vet. Clin. N. Am-Small 20: 583–613. Kunert KS, Tisdule AS, Gipson IK (2002) Goblet cell numbers and epithelial proliferation in the conjunctiva of patients with dry eye syndrome treated with cyclosporine. Arch. Ophthalmol. 120: 330–337. Lavach JD, Thrall MA, Benjamin MM, Severin GA (1977) Cytology of normal and inflamed conjunctivas in dogs and cats. J. Am. Vet. Med. Assoc. 170: 722–727. Moore CP, McHugh JB, Thorne JG, Phillips TE (2001) Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model. Invest. Ophthalmol. Vis. Sci. 42: 653–659. Morgan RV, Abrams KL (1991) Topical administration of cyclosporine for treatment of keratoconjunctivitis sicca in dogs. J. Am. Vet. Med. Assoc. 199: 1043–1046. Munger R (2001) Diseases and immunity of the ocular surface. In: Proceedings of the World Small Animal Veterinary Association Congress, Vancouver, Canada.
230
Olivero DK, Davidson MG, English RV (1991) Clinical evaluation of 1% cyclosporine for topical treatment of keratoconjunctivitis sicca in dogs. J. Am. Vet. Med. Assoc. 199: 1039–1042. Schreiber SL, Crabtree GR (1992) The mechanism of action of cyclosporine and FK506. Immunol. Today 13: 136–141. Slatter DH (1990) Basic diagnostic techniques, conjunctiva and lacrimal system. In: Slatter DH, Ed., Fundamentals of Veterinary Ophthalmology, 4th ed. WB Saunders Co, Philadelphia. pp. 84–256. Stevens A, Bancroft JD (1992) Proteins and nucleic acids. In: Bancroft JD, Stevens A, Eds., The Theory and Practice of Histological Techniques, 3rd ed. The Bath Press, Avon. pp. 25, 143–153. Ueta M, Hamuro A, Yamamoto M, Kaseda K, Akira S, Kinoshita S (2005) Spontaneous ocular surface inflammation and goblet cell disappearance in 1 kappa B zeta gene-disrupted mice. Invest. Ophthalmol. Vis. Sci. 46: 579–588. Wulf JC, Sale GE, Deeg HJ, Storb R (1981) Nonspecific acid esterase activity as a marker for canine T-lymphocytes. Exp. Hematol. 9: 865–870.
Biotechnic & Histochemistry 2015, 90(3): 223–230
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