Comparison of topical dry eye medications for the treatment of keratoconjunctivitis sicca in a botulinum toxin B-induced mouse model

PURPOSE: To compare the effects of topical dry eye medications including anti-inflammatory agents and lubricant eyedrops for the treatment of keratoconjunctivitis sicca (KCS) in a botulinum toxin B (BTX-B)-induced mouse model. METHODS: CBA mice were randomized into 10 groups. The first 5 groups received a transconjunctival injection of saline into the lacrimal gland, and the remaining groups were injected with 0.05 mL of 20 mU BTX-B. Each group received treatment with 0.1% fluorometholone (FML), 0.05% cyclosporine A (CsA), a 50:50 combination of FML and CsA, artificial tears, or saline 3 days after injections. Tear production, corneal staining, and blink rate were compared in each of the 10 groups. RESULTS: Tear production in BTX-B-injected CsA-treated, FML-treated, and combined-treated groups started to return to baseline level within 2 weeks of treatment, whereas those treated with saline or artificial tears still exhibited reduction of lacrimation up to 4 weeks after injection. Topical FML significantly reversed the staining score within 1 week of treatment. The improvement of corneal staining in BTX-B-challenged combined-treated and CsA-treated groups occurred later within 2 weeks after treatment. No significant improvement in corneal staining was observed for the BTX-B-injected mice treated with artificial tears or saline. No significant changes were noted in blink frequency between the control and study groups undergoing the various dry eye therapies. CONCLUSION: The therapeutic effects of dry eye medications in a BTX-B-induced mouse model of KCS are similar to the human response.     

Effects of topical anti-inflammatory agents in a botulinum toxin B-induced mouse model of keratoconjunctivitis sicca.

PURPOSE: The aim of this study was to compare the effects of topical nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroid, doxycycline, and artificial tears for the treatment of ocular surface damage in the Botulinum toxin B (BTX-B)-induced mouse model of dry eye. METHODS: CBA/J mice were randomized into 2 experimental groups of 35 animals each. The control group received a transconjunctival injection of 0.05 mL of saline into the left lacrimal gland, and another group was injected with 0.05 mL of 20 milliunits BTX-B solution (SPSS, Inc., Chicago, IL). Three (3) days after intralacrimal gland injections, each group was equally randomized into 7 subgroups (n=5 each) to receive treatment unilaterally into their left eyes with topical artificial tears (0.5% carboxymethylcellulose sodium), 0.1% fluorometholone, 0.1% nepafenac, 0.4% ketorolac, 0.09% bromfenac, 0.1% diclofenac, or 0.025% doxycycline. Tear volume, ocular surface changes, and spontaneous blink rate were evaluated in each of the 14 experimental subgroups. RESULTS: Topical fluorometholone, nepafenac, and doxycycline significantly improved corneal surface staining in the BTX-B-injected mice within 2 weeks of treatment. Topical ketorolac, diclofenac, and bromfenac, applied twice-daily, partially reduce corneal staining, and did so more slowly by the 4-week time point. In comparison, topical artificial tear-treated mice did not demonstrate significant improvement of the corneal surface at any time point. Aqueous tear production in the BTX-B-injected fluorometholone-treated group started to return to baseline level within 2 weeks, although not significantly. Meanwhile, BTX-B-injected mice treated with artificial tears, topical NSAIDs, and doxycycline still exhibited a reduction in tear production up to 4 weeks. No significant differences in blink rate between the control and study groups undergoing the various treatments were noted at all time points. CONCLUSIONS: This study suggests the potential usefulness of topical NSAIDs, corticosteroid, and doxycycline for the clinical treatment of ocular surface epithelial disorders associated with dry eye.     

肉毒杆菌B诱导大鼠干眼模型中泪腺炎症因子表达研究

目的: 建立肉毒杆菌B诱导大鼠炎症干眼模型,通过观察泪腺炎症因子表达变化为干眼病实验研究提供实验依据。方法: 健康8周龄雌性SD大鼠,30只,随机分为2组,20只为实验组,10只为对照组。右侧泪腺注射肉毒杆菌B0.1mL(20mU),生理盐水0.1mL。分别于实验前1d及实验后第3d;1,4,6wk,观察基础泪液分泌量、角膜荧光染色,并且酶联免疫吸附试验(ELISA)检测泪腺组织中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和巨噬细胞移动抑制因子(MIF)变化。结果: 术后实验组从第3d时出现泪液分泌减少,4wk达到高峰,6wk后恢复;角膜荧光染色第3d时出现,持续6wk;泪腺组织IL-1β从第3d开始出现增加,持续6wk;TNF-α表达水平未增加,MIF检测从第4wk开始表达增加,持续6wk。结论: 泪腺注射肉毒杆菌B可以成功诱导大鼠炎症干眼模型,炎症因子的表达变化具有一定的特征性,为干眼病实验提供较理想的动物模型。     

Botulinum toxin B-induced mouse model of keratoconjunctivitis sicca

PURPOSE: To develop a mouse model of human chronic dry eye (keratoconjunctivitis sicca [KCS]). METHODS: Under direct visualization with an operating microscope, CBA/J mice received a transconjunctival injection of saline or 1.25, 5, or 20 milliunits (mU) of botulinum toxin B (BTX-B) into the lacrimal gland. The mice were either left unstressed or were subjected to an air blower for 5 h/d, 5 d/wk in fixed temperature and humidity conditions. Tear production and corneal fluorescein staining were evaluated in all groups before injection and at several time points after. Tear production was measured with phenol red-impregnated cotton threads. Corneal fluorescein staining was photographed under cobalt blue light with a digital camera fitted with a macro lens. RESULTS: BTX-B-injected mice displayed significantly decreased tear production until the 4-week time point. Throughout all time points, the addition of environmental blower stress did not appear to alter tear production significantly. Linear regression models, used to evaluate the effects of various doses of BTX-B on tear production, showed that doses higher than 1.25 mU did not provide significantly different outcomes. After 3 days, saline-injected mice showed no corneal staining, whereas BTX-B-injected mice displayed various amounts of staining. At the early time point (day 3), there did not appear to be an additional effect of the blower on corneal fluorescein staining. However, at 1, 2, and 4 weeks, the blower stress appeared to increase the amount of corneal fluorescein staining at each BTX-B dose, although not significantly. Furthermore, at 8 to 10 weeks, in the BTX B-injected groups, corneas had persistent staining, even though tear production had already returned to normal levels. Histopathologic analyses revealed no inflammatory cell infiltration of the stroma or acini of the lacrimal glands and conjunctivae of both saline-injected and BTX-B-injected animals. CONCLUSIONS: Intralacrimal gland injection of BTX-B resulted in persistent corneal fluorescein staining within 3 days, and a significant decrease in aqueous tear production that persisted for 1 month. Intralacrimal gland injection of BTX-B suppressed lacrimation, thereby establishing a dry eye state. This animal model could be a useful tool for investigating the pathogenesis of the chronic condition KCS in humans.     

Effect of botulinum toxin injections into rabbit eye

The aim of this study was to investigate the adverse effects of the intraocular injection of botulinum toxin in rabbits. Intravitreal injections of botulinum toxin A in five doses, 1.25, 2.5, 5, 10, 25 units, were given into five rabbit eyes. The same volume of saline was injected into the second eye of the rabbit as a control. External examination, ophthalmoscopy, visual evoked potentials and electroretinography were done before injection and repeated at the first and second weeks after the injection. There were no significant differences in retinal function between toxin- and saline-injected eyes, neither ophthalmoscopically nor electrophysiologically. Ipsilateral mydriasis developed in the eyes injected with botulinum toxin. This study suggests that botulinum toxin has no harmful effect on retinal function.     

Effects of botulinum toxin type A on the treatment of dry eye disease and tear cytokines

Graefe's Archive for Clinical and Experimental Ophthalmology - To determine the effects of botulinum toxin type A (BTX-A) injection on dry eye signs, symptoms, and tear cytokine levels in...     

摘除泪腺和泪腺注射肉毒杆菌毒素A制备大鼠干眼模型的比较

目的：分别采用摘除泪腺法和泪腺注射肉毒杆菌毒素A法诱导大鼠干眼症模型,通过对比两种干眼症模型的临床表现、病理学特点和细胞因子变化,探讨其优缺点和适用范围。

方法：健康8周龄雄性Brown Norway大鼠30只,随机分为3组：A组左眼为空白组,B组摘除大鼠左侧主泪腺,C组左侧泪腺注射肉毒杆菌毒素A。比较实验前1d,实验后3、7、14、28、42d的泪液分泌量(SⅠt)、泪膜破裂时间(BUT)、角膜荧光素钠染色评分的变化。42d观察结膜、角膜和泪腺组织病理学改变; 通过实时荧光定量PCR对白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)和上皮生长因子(EGF)表达量进行检测。

结果：实验后第3d开始,B、C组均出现泪液分泌量持续性减少,与A组均有差异(P<0.05); 实验后第7d开始,B、C组均出现BUT持续性缩短以及角膜上皮染色明显增多,与A组比较有差异(P<0.05); 以上临床数据B、C组间比较均无差异(P>0.05)。A组角膜上皮细胞形态正常,B组及C组角膜上皮均存在不同程度表层细胞丝状分离,结膜杯状细胞数目明显减少,C组泪腺组织明显萎缩。B、C组结膜和角膜组织的EGF、TNF-α和IL-6表达量均显著增高,与A组比较均有差异(P<0.05)。B、C组间EGF和TNF-α的表达均无差异(P>0.05); 与C组比较,B组中IL-6的表达量更高(P<0.05)。

结论：摘除泪腺和泪腺注射肉毒杆菌毒素A均可以构建稳定的水液缺乏型大鼠干眼模型,建议根据实验设计及实验目的去选择合适的动物模型。     

Efficacy of botulinum toxin type a after topical anesthesia

PURPOSE: To determine whether the use of topical anesthesia has an impact on botulinum toxin type A (BTX-A) efficacy. METHODS: Forty patients (20 receiving BTX-A for facial cosmetic rhytid reduction and 20 for benign essential blepharospasm) were evaluated in a double-blind, randomized, triple-crossover study at 2.5- to 4.5-month intervals. The discomfort and efficacy of BTX-A injections after betacaine application to half the face (random assignment) were compared against the discomfort and efficacy of a placebo ointment on the other half of the face. This was followed by cryoanalgesia to the entire face. RESULTS: Patients ranged from 27 to 81 years of age (mean, 53 years), and 34 were female. Of the 120 total injection comparisons, a better BTX-A effect on one side of the face was reliably identified by 80% and 77% of blepharospasm and cosmetic patients, respectively, with the placebo-treated side providing better BTX-A effect approximately 90% of the time (p < 0.001). Patients reported a more painful side during injection in just 18 of the 120 trials, and only 1 of 40 patients believed the administration of analgesia was worth the trouble. CONCLUSIONS: Pretreatment with topical betacaine followed by skin cooling seems to have a deleterious impact on BTX-A effect without a significantly beneficial patient-perceived reduction in injection discomfort.     

Effect of topical 0.05% cyclosporine A on the tear protein lacritin in a rat model of dry eye

AIM: To observe the effect of topical 0.05% cyclosporine A (CsA) on the ocular surface and tear protein lacritin in a botulinum B-induced dry eye rat model. METHODS: A total of 36 female SD rats were randomly divided into 3 groups, botulinum B was injected into the right lacrimal gland of all rats. Group A and group B were treated with 0.05% CsA and 0.1% sodium hyaluronate, respectively, 3 times daily. The control group was not treated. Basal tear flow, corneal epithelial defects, and lacritin levels were measured. RESULTS: Tear secretion in all rats was reduced on day 3 and was even lower on day 7 postoperation (P<0.05). Tear secretion in group A increased by day 14 and was at the preoperative level on day 42. Tear secretion in group B and control rats was lower on days 14 and 42 compared with preoperative level (P<0.05). Corneal fluorescein staining in group A was higher on day 3, peaked on day 7, and then decreased gradually from day 7 until day 14, returning to normal by day 42 post-procedure. However, in group B, corneal fluorescein staining had improved, but was not fully recovered by day 42. Corneal fluorescein staining was more intense than before the operation and then in the control group at all time points. Tear protein lacritin levels reached the lowest levels on day 7 in all groups. In group A, tear protein lacritin levels began to increase on day 14 and were normal on day 42. In group B, tear protein lacritin levels began to increase on day 14, but had not completely recovered on day 42. In the control group, tear protein lacritin levels remained low post-procedure. CONCLUSION: CsA 0.05% prompts tear protein lacritin expression in a rat model of dry eye and improves the signs and symptoms of dry eye disease.     

Corneal epithelial proliferation and thickness in a mouse model of dry eye

Although several studies have previously focused on the conjunctival epithelial response to surface dryness, little is known about the effect of a dry environment on corneal epithelium, which is the most clinically significant tissue affected in dry eye. The aim of this study was to quantitatively evaluate the effect of desiccating stress on the number of proliferating corneal epithelial cells and corneal epithelial thickness in mice placed in a controlled-environment chamber (CEC) that induces dry eye. Corneal epithelial cell proliferation and thickness were studied in 8- to 12-week-old female BALB/c mice placed in the CEC (temperature: 22.3 ± 0.7 °C; relative humidity: 22.5 ± 4.5%; airflow: 15 L/min) for 7 days and compared to a control group of mice with no dry eye. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle. To detect the spatial distribution of proliferative cells, Ki-67⁺ cells were counted in three areas of the epithelium: center, periphery, and limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin-eosin. Results from each experimental group were compared using the Mann-Whitney test. The number of Ki-67⁺ cells observed in the corneal epithelium of mice exposed to the CEC was significantly higher in each area (center: 32.1 ± 1.1; periphery: 94.2 ± 5.3; limbus: 4.0 ± 1.5) than in the control group (center: 13.2 ± 1.0, p = 0.02; periphery: 42.9 ± 2.3, p = 0.02; limbus: 0.0, p = 0.01). In mice subjected to desiccating stress, a significant number of Ki-67⁺ positive cells were detected in the basal and suprabasal cell layers (central area 46%; periphery 30.8%: limbus 0%), whereas in the control group the cells were exclusively distributed through the basal cell layer. Ki-67⁺ cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was found to be significantly thicker in dry eye mice (54.94 ± 6.09 μm) as compared to the controls (43.9 ± 6.23 μm, p < 0.0001) by a mean of 25%. These results demonstrate that desiccating stress increases corneal epithelial turnover and thickness, similar to what is observed in other chronic inflammatory states of other epithelialized surfaces. The CEC can facilitate the study of the regulation of epithelial cell function and turnover at the molecular and cellular levels under desiccating stress conditions.     

维生素A治疗苯扎氯铵诱导的小鼠干眼的实验研究

目的通过随机对照实验探讨维生素A对苯扎氯铵诱导的小鼠干眼的治疗作用。方法实验研究。选用BALB＆小鼠50只,使用0．25％的苯扎氯铵溶液局部点眼以诱导干眼。在第21天时根据泪膜破裂时间（BUT）、角膜荧光素钠染色、炎症指数等参数选择干眼表现程度相近的小鼠30只（60眼）并随机分为A、B、C三组,每组10只（20眼）。A、B组分别使用5000IU／（g·mD维生素A微乳剂和空白微乳剂滴眼,C组为空白对照组。在治疗的第0、1、3、5、7天检测BUT和角膜上皮荧光素钠染色、眼表炎症程度及基础泪液分泌量。7d治疗结束后取出小鼠眼球,进行HE及过碘酸．希夫（PAS）染色,角蛋白10（K10）免疫荧光标记,以及WesternBlot检测角膜组织中K10表达水平。对数据采用单因素方差分析。结果在药物治疗的第0、1、3、5天各项临床指标差异无统计学意义。治疗第7天时,A、B、C组BUT值分别为（4．20±0．89）s、（3．32±1．01）s和（3．28±0．74）s;其中A组的BUT值较B组和C组长,差异有统计学意义（F=6．825,P=-0．002）。A、B、C组角膜荧光素钠染色评分分别为3．74±1．31、5．47±1．81和5．83±1．54,其中维生素A微乳组较空白微乳组和空白对照组角膜荧光素钠染色分级轻．差异具有统计学意义（F=8．853,P〈0．01）。各组间的角膜炎症指数评分、基础泪液分泌量差异均无统计学意义。组织病理检查示维生素A组与空白微乳组和空白对照组比较,其角膜上皮形态较规整;PAS染色示维生素A组杯状细胞数量更接近正常小鼠。维生素A组的角膜上皮层几乎均不表达K10．而空白微乳组及空白对照组呈阳性表达。Westem Blot检测示维生素A组角膜中的K10水平低于空白微乳组和空白对照组。结论维生素A可以延长苯扎氯铵诱导的干眼小鼠的BUT,降低角膜上皮染色程度,抑制角膜上皮鳞状化生,并能增加结膜杯状细胞数量,有望用于干眼治疗。     

The controlled-environment chamber: a new mouse model of dry eye

PURPOSE: To develop a controlled-environment chamber (CEC) for mice and verify the effects of a low-humidity setting on ocular surface signs in normal mice. METHODS: Eight- to 12-week-old BALB/c mice were used in a controlled-environment chamber (CEC) where relative humidity (RH), temperature (T), and airflow (AF) are regulated and monitored. Mice were placed into the CEC and exposed to specific environmentally controlled conditions (RH = 18.5% +/- 5.1%, AF = 15 L/min, T = 21-23 degrees C) for 3, 7, 14, and 28 days. Control mice were kept in a normal environment (RH = 50%-80%, no AF, T = 21-23 degrees C) for the same duration. Aqueous tear production by means of the cotton thread test, corneal fluorescein staining (score, 0-15), and goblet cell density in the superior and inferior conjunctiva were measured by a masked observer. RESULTS: No statistically significant differences between the groups were found at baseline. Decreased tear secretion and increased corneal fluorescein staining were significantly present on day 3, 7, 14, and 28 in animals kept in the CEC. Goblet cell density was significantly decreased in the superior conjunctiva on day 7, and on day 3, 7, and 14 in the inferior conjunctiva in the CEC-kept mice compared with control animals. CONCLUSIONS: This study indicates that exposure of normal mice to a low-humidity environment in a CEC can lead to significant alterations in tear secretion, goblet cell density, and acquisition of dry eye-related ocular surface signs.     

A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride

PURPOSE: To establish a rabbit dry eye model with topical medication of the ocular preparation preservative benzalkonium chloride (BAC). METHODS: Sixteen white rabbits were used. One eye of each rabbit was chosen randomly for topical administration of 0.1% BAC twice daily for 14 days. The other untreated eyes served as controls. Schirmer test, fluorescein, and rose bengal staining were performed before and after BAC treatment on days 3, 5, 7, and 14. Conjunctiva impression cytology specimens were collected on days 0, 7, and 14. The rabbits were killed after day 14. Immunofluorescence staining was performed to detect mucin-5 subtype AC (MUC5AC) on conjunctival cryosections. Cornea and conjunctiva structures were evaluated by light and electron microscopy. RESULTS: Compared with untreated controls, BAC-treated eyes showed significant decreases in Schirmer scores (P = 0.01) and increases in fluorescein scores (P < 0.001) on days 5, 7, and 14. A significant increase in rose bengal scores was noticed as early as day 3 (P = 0.001). Decreases in goblet cell density occurred on days 7 and 14 (P = 0.001). Decreased MUC5AC and histopathologic and ultrastructural disorders of the cornea and conjunctiva were also observed in the BAC group. CONCLUSIONS: These findings demonstrated that an ophthalmic preservative, benzalkonium chloride, induced a dry eye syndrome in rabbits with damage to the cornea and conjunctiva, decreased aqueous tear basal secretion, goblet cell loss, and MUC5AC deficiency. This rabbit model was consistent with human dry eye syndrome in both aqueous tear and mucin deficiency and may be appropriate for studying dry eye syndrome.