兔干眼模型的建立及FK506治疗干眼的疗效及作用机制研究
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摘要
第一部分皮下注射氢溴酸东莨菪碱建立兔干眼模型的实验研究
目的:探讨兔皮下注射氢溴酸东莨菪碱,建立干眼模型的特点和眼表病理损害机制。
方法:健康新西兰大白兔18只,随机分为三组,每组6只。实验组皮下注射氢溴酸东莨菪碱,其中低剂量组(L,1.0mg/次)、高剂量组(H, 2.0mg/次),对照组注射等量生理盐水。注射时间点为每天8点11点、14点和18点,连续30天。用药后0、7,14,21,30d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色,印迹细胞检查。第30d空气栓塞法处死实验动物,取角膜、结膜及泪腺组织行HE染色,免疫组化检测FAS,BCL-2及NFκB)的表达水平。
结果:酚红棉线法测得高剂量(H)组造模前泪液分泌量为16.25±2.299mm,第21d时下降为6.75±1.982mm,对照组为16.50±2.619mm,差异具有统计学意义(p<0.05),同期低剂量组(L)为15.00±2.390mm,与对照组相比无明显差异。H组第3d出现BUT缩短,L组则第7d发生BUT的变化,此后BUT值均低于5s,对照组BUT大于20s。实验组均出现角膜荧光素染色阳性,H组出现时间早(3d),持续时间长。L组和H组PAS染色与对照组相比,杯状细胞形态紊乱、核淡染,部分核固缩,L组和H组之间无明显差异。HE染色显示H组泪腺周围见散在淋巴细胞和腺腔凋亡性改变,角膜上皮呈重度瘤样增生基质层变性伴水肿,结膜杯状细胞反应性增多,固有层见淋巴细胞浸润数量增加。L组角膜浅层上皮轻度增生,细胞排列稍紊乱,结膜和泪腺组织病理改变轻微。电镜观察高剂量组泪腺腺管管腔壁绒毛较对照组明显减少,角膜表层微绒毛较对照组减少,上皮细胞空泡变,细胞核固缩,结膜表面微绒毛减少,上皮细胞呈坏死凋亡改变,低剂量未见明显显微改变。凋亡抑制因子BCL-2在高剂量模型组泪腺、角膜和结膜组织中极少表达,对照组各组织均呈高度阳性表达,两组差异显著p<0.001;促凋亡因子Fas在模型组泪腺组织中呈中度表达,在角膜和结膜组织中呈中高度表达。炎症转录因子NFκB在模型组兔眼泪腺腺泡细胞胞浆和胞核轻度表达,腺腔管壁细胞胞核和胞浆中重度表达,角膜组织上皮细胞和基底细胞胞浆、胞核以及结膜上皮细胞中NFκB呈中度阳性表达,对照组各组织NFκB极少表达,两组差异有统计学意义(p<0.001)。
结论:兔皮下注射氢溴酸东莨菪碱,4次/天,2.0mg/次,可有效维持药物外周浓度,抑制泪腺的分泌,引起角结膜上皮细胞的损伤以及角结膜上皮和泪腺组织细胞凋亡因子和炎症因子阳性表达增加,成功建立以泪液缺乏为主,凋亡和炎症反应共同参与的干眼动物模型。
第二部分FK506对氢溴酸东莨菪碱至兔干眼模型的疗效
目的:观察FK506滴眼液干预兔干眼模型的疗效及其作用机制
方法:健康新西兰大白兔30只,,随即分为实验组18只和对照组12只。实验组采用上述方法每天8点11点、14点和18点皮下注射氢溴酸东莨菪碱注射液2.0mg/次,并从第四天开始左眼点0.05%FK506滴眼液,4次/天,右眼不做处理,对照组皮下注射生理盐水。实验第0,3,7,14,21,35d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色检查。分别在第7,14,和35d采取空气栓塞法分别随即处死模型组5只兔子,取双眼角膜、结膜和泪腺组织,PCR定量分析IL-1β、TNF-α和MMP-9等炎性因子在各组织的表达。第35d采用印记细胞学方法取双眼角结膜上皮细胞行免疫组化检测NFκB表达水平的差异。
结果:造模后3,7天实验兔双侧眼均角膜荧光素染色强阳性。第14d开始FK506治疗眼角膜荧光素染色评分低于未用药眼,至第35d,差异进一步增大具有统计学意义(P<0.01)。FK506治疗组眼的角结膜上皮NFκB表达显著低于对照眼,不同时间点治疗组结膜组织中IL-1β、TNF-α和MMP-9表达水平低于对照组,两者差异有统计学意义(p<0.05)。14天时双侧泪腺MMP-9水平造模后与对照组相比呈现增高,但用药干预眼与未治疗眼之间无明显差异。
结论:FK506通过抑制炎症信号分子NFκB的激活,降低角膜,结膜组织中IL-1β、TNF-α和MMP-9等炎症因子的水平,有效地抑制眼表免疫性炎症,进而减轻干眼对眼表组织的损害。
PartⅠAn experimental of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide
Objective To evaluate the characteristic of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide and explore the mechanism of ocular surface.
Methods The rabbits were randomly divided into three groups, 6 rabbits in each group. Subcutaneous injection of scopolamine hydrobromide was made in experimental group including 1.0mg in low dose group, 2.0mg in high dose group and normal saline in control group, at 8am, 11am, 14pm and 18pm each day for 30 days. Schirmer I test, tear membrane break-up time( BUT), corneal fluorescein staining and conjunctival impression cytology test with PAS staining to evaluate the phenotype of epithelia cells were performed before and 7、14、21、30 days after injection. On the thirtieth day, cornea, conjunctiva and lacrimal glands tissues biopsy were carried out for H-E staining and electron microscope observation, and expression of cytokine Fas BCL-2 and NFκB were detected by immuno- histochemistry.
Results Schirmer I test by phenol red thread test of H group was 16.25±2.299mm before injection and decreased to 6.75±1.982mm on the 21st day, with a statistically difference(p<0.05) compared to 16.50±2.619mm of the control group ,however, it was 15.00±2.390mm of L group with no statistically difference .BUT decreased at the 3rd day in high dose group and the 7th Day in low dose group after which it kept shorter than 5s, BUT kept greater than 20s in control group .Flurescein staining of corneal were found in two experimental groups. It was initially observed in H group at the 3rd day and last for a long time. Compared with the control group, the morphology of goblet cells were irregular and nucleus were slightly stained, some of which were pyknotic in two experimental groups. There was no significant difference between H and L group. H-E stainning showed that lymphocyte dispersedly distributed around lacrimal gland,apoptosis in glandular cavity, severe tumor-like hyperplasia in corneal epithelium, degeneration with edema in corneal stroma layer, conjunctival goblet cell increased reactively and increased the number of cell infiltration in lamina propria in H group, Slight hyperplasia, cell disarrangement in corneal and mild histopathology changes in conjunctiva and lacrimal gland. The decreased microvilli of lacrimal glandular and corneal, conjunctival epithelium cell with cell necrosis and nucleus pyknotic compared with the control group were observed by transmission electron microscope. BCL-2 was seldomly expressed in the tissues biopsys of experimental group and fas and NFκB was in a high level compare with he control group. Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in lacrimal secretion decrease and damage to epithelium of coneal and conjunctiva, which mainly caused by tear deficient and apoptosis and inflammation may also participate in.
PartⅡthe effect of FK506 in the treatment of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide
Objective To observe the therapeutic effect of FK506 eye drops to rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide .
Methods The rabbits were randomly divided into experimental group 18 and control group 12. Subcutaneous injection of scopolamine hydrobromide 2.0mg was made in experimental group at 8am, 11am, 14pm and 18pm each day .And since the day 4, the left eyes of the experimental group rabbits were treated with 0.5%FK506 eye drops four times each day,and,, the control group was injected with physiological saline.
Methods Schirmer I test, lacrimal membrane break-up time( BUT) and corneal fluorescein staining were measured in 3、7、14、21、35 days after injection. The level of IL-1β、TNF-αand MMP-9 mRNA was evaluated by real-time polymerase chain reaction in corneal, conjunctival and lacrimal gland. At day 35,we used impression cytology to obtain corneal, conjunctival epithelium cells and detected NFκB expression in which by immunohistochemistry.
Results The corneal fluorescein staining were observed in both eyes of model rabbits, there is no statistically difference at day 3,7 but the level of corneal fluorescein staining was much lower in the eyes treated with 0.5%FK506 than the other side since day 14, with a statistically difference(p<0.01). At day 35, NFκB expression in untreated side was significantly positive with high levels of IL-1β、TNF-αand MMP-9 mRNA in corneal and conjunctival,compare to the treated eye which NFκB was seldomly expressed with low level of cytokines , with a statistically difference(p<0.05).The expression of MMP-9 was increased in both side of rabbit eyes in day 14,with no statistically difference.
Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in rabbit dry eye model that with high level of IL-1β、TNF-αand MMP-9 mRNA and significantly positive expression of NFκB in corneal, conjunctival and lacrimal gland.FK506 can probably inhibit activation of inflammatory cytokines and improve the symptoms of dry eye.
目的:探讨兔皮下注射氢溴酸东莨菪碱,建立干眼模型的特点和眼表病理损害机制。
方法:健康新西兰大白兔18只,随机分为三组,每组6只。实验组皮下注射氢溴酸东莨菪碱,其中低剂量组(L,1.0mg/次)、高剂量组(H, 2.0mg/次),对照组注射等量生理盐水。注射时间点为每天8点11点、14点和18点,连续30天。用药后0、7,14,21,30d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色,印迹细胞检查。第30d空气栓塞法处死实验动物,取角膜、结膜及泪腺组织行HE染色,免疫组化检测FAS,BCL-2及NFκB)的表达水平。
结果:酚红棉线法测得高剂量(H)组造模前泪液分泌量为16.25±2.299mm,第21d时下降为6.75±1.982mm,对照组为16.50±2.619mm,差异具有统计学意义(p<0.05),同期低剂量组(L)为15.00±2.390mm,与对照组相比无明显差异。H组第3d出现BUT缩短,L组则第7d发生BUT的变化,此后BUT值均低于5s,对照组BUT大于20s。实验组均出现角膜荧光素染色阳性,H组出现时间早(3d),持续时间长。L组和H组PAS染色与对照组相比,杯状细胞形态紊乱、核淡染,部分核固缩,L组和H组之间无明显差异。HE染色显示H组泪腺周围见散在淋巴细胞和腺腔凋亡性改变,角膜上皮呈重度瘤样增生基质层变性伴水肿,结膜杯状细胞反应性增多,固有层见淋巴细胞浸润数量增加。L组角膜浅层上皮轻度增生,细胞排列稍紊乱,结膜和泪腺组织病理改变轻微。电镜观察高剂量组泪腺腺管管腔壁绒毛较对照组明显减少,角膜表层微绒毛较对照组减少,上皮细胞空泡变,细胞核固缩,结膜表面微绒毛减少,上皮细胞呈坏死凋亡改变,低剂量未见明显显微改变。凋亡抑制因子BCL-2在高剂量模型组泪腺、角膜和结膜组织中极少表达,对照组各组织均呈高度阳性表达,两组差异显著p<0.001;促凋亡因子Fas在模型组泪腺组织中呈中度表达,在角膜和结膜组织中呈中高度表达。炎症转录因子NFκB在模型组兔眼泪腺腺泡细胞胞浆和胞核轻度表达,腺腔管壁细胞胞核和胞浆中重度表达,角膜组织上皮细胞和基底细胞胞浆、胞核以及结膜上皮细胞中NFκB呈中度阳性表达,对照组各组织NFκB极少表达,两组差异有统计学意义(p<0.001)。
结论:兔皮下注射氢溴酸东莨菪碱,4次/天,2.0mg/次,可有效维持药物外周浓度,抑制泪腺的分泌,引起角结膜上皮细胞的损伤以及角结膜上皮和泪腺组织细胞凋亡因子和炎症因子阳性表达增加,成功建立以泪液缺乏为主,凋亡和炎症反应共同参与的干眼动物模型。
第二部分FK506对氢溴酸东莨菪碱至兔干眼模型的疗效
目的:观察FK506滴眼液干预兔干眼模型的疗效及其作用机制
方法:健康新西兰大白兔30只,,随即分为实验组18只和对照组12只。实验组采用上述方法每天8点11点、14点和18点皮下注射氢溴酸东莨菪碱注射液2.0mg/次,并从第四天开始左眼点0.05%FK506滴眼液,4次/天,右眼不做处理,对照组皮下注射生理盐水。实验第0,3,7,14,21,35d对实验组及对照组行泪液分泌试验、泪膜破裂时间(BUT)、角膜荧光素染色检查。分别在第7,14,和35d采取空气栓塞法分别随即处死模型组5只兔子,取双眼角膜、结膜和泪腺组织,PCR定量分析IL-1β、TNF-α和MMP-9等炎性因子在各组织的表达。第35d采用印记细胞学方法取双眼角结膜上皮细胞行免疫组化检测NFκB表达水平的差异。
结果:造模后3,7天实验兔双侧眼均角膜荧光素染色强阳性。第14d开始FK506治疗眼角膜荧光素染色评分低于未用药眼,至第35d,差异进一步增大具有统计学意义(P<0.01)。FK506治疗组眼的角结膜上皮NFκB表达显著低于对照眼,不同时间点治疗组结膜组织中IL-1β、TNF-α和MMP-9表达水平低于对照组,两者差异有统计学意义(p<0.05)。14天时双侧泪腺MMP-9水平造模后与对照组相比呈现增高,但用药干预眼与未治疗眼之间无明显差异。
结论:FK506通过抑制炎症信号分子NFκB的激活,降低角膜,结膜组织中IL-1β、TNF-α和MMP-9等炎症因子的水平,有效地抑制眼表免疫性炎症,进而减轻干眼对眼表组织的损害。
PartⅠAn experimental of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide
Objective To evaluate the characteristic of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide and explore the mechanism of ocular surface.
Methods The rabbits were randomly divided into three groups, 6 rabbits in each group. Subcutaneous injection of scopolamine hydrobromide was made in experimental group including 1.0mg in low dose group, 2.0mg in high dose group and normal saline in control group, at 8am, 11am, 14pm and 18pm each day for 30 days. Schirmer I test, tear membrane break-up time( BUT), corneal fluorescein staining and conjunctival impression cytology test with PAS staining to evaluate the phenotype of epithelia cells were performed before and 7、14、21、30 days after injection. On the thirtieth day, cornea, conjunctiva and lacrimal glands tissues biopsy were carried out for H-E staining and electron microscope observation, and expression of cytokine Fas BCL-2 and NFκB were detected by immuno- histochemistry.
Results Schirmer I test by phenol red thread test of H group was 16.25±2.299mm before injection and decreased to 6.75±1.982mm on the 21st day, with a statistically difference(p<0.05) compared to 16.50±2.619mm of the control group ,however, it was 15.00±2.390mm of L group with no statistically difference .BUT decreased at the 3rd day in high dose group and the 7th Day in low dose group after which it kept shorter than 5s, BUT kept greater than 20s in control group .Flurescein staining of corneal were found in two experimental groups. It was initially observed in H group at the 3rd day and last for a long time. Compared with the control group, the morphology of goblet cells were irregular and nucleus were slightly stained, some of which were pyknotic in two experimental groups. There was no significant difference between H and L group. H-E stainning showed that lymphocyte dispersedly distributed around lacrimal gland,apoptosis in glandular cavity, severe tumor-like hyperplasia in corneal epithelium, degeneration with edema in corneal stroma layer, conjunctival goblet cell increased reactively and increased the number of cell infiltration in lamina propria in H group, Slight hyperplasia, cell disarrangement in corneal and mild histopathology changes in conjunctiva and lacrimal gland. The decreased microvilli of lacrimal glandular and corneal, conjunctival epithelium cell with cell necrosis and nucleus pyknotic compared with the control group were observed by transmission electron microscope. BCL-2 was seldomly expressed in the tissues biopsys of experimental group and fas and NFκB was in a high level compare with he control group. Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in lacrimal secretion decrease and damage to epithelium of coneal and conjunctiva, which mainly caused by tear deficient and apoptosis and inflammation may also participate in.
PartⅡthe effect of FK506 in the treatment of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide
Objective To observe the therapeutic effect of FK506 eye drops to rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide .
Methods The rabbits were randomly divided into experimental group 18 and control group 12. Subcutaneous injection of scopolamine hydrobromide 2.0mg was made in experimental group at 8am, 11am, 14pm and 18pm each day .And since the day 4, the left eyes of the experimental group rabbits were treated with 0.5%FK506 eye drops four times each day,and,, the control group was injected with physiological saline.
Methods Schirmer I test, lacrimal membrane break-up time( BUT) and corneal fluorescein staining were measured in 3、7、14、21、35 days after injection. The level of IL-1β、TNF-αand MMP-9 mRNA was evaluated by real-time polymerase chain reaction in corneal, conjunctival and lacrimal gland. At day 35,we used impression cytology to obtain corneal, conjunctival epithelium cells and detected NFκB expression in which by immunohistochemistry.
Results The corneal fluorescein staining were observed in both eyes of model rabbits, there is no statistically difference at day 3,7 but the level of corneal fluorescein staining was much lower in the eyes treated with 0.5%FK506 than the other side since day 14, with a statistically difference(p<0.01). At day 35, NFκB expression in untreated side was significantly positive with high levels of IL-1β、TNF-αand MMP-9 mRNA in corneal and conjunctival,compare to the treated eye which NFκB was seldomly expressed with low level of cytokines , with a statistically difference(p<0.05).The expression of MMP-9 was increased in both side of rabbit eyes in day 14,with no statistically difference.
Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in rabbit dry eye model that with high level of IL-1β、TNF-αand MMP-9 mRNA and significantly positive expression of NFκB in corneal, conjunctival and lacrimal gland.FK506 can probably inhibit activation of inflammatory cytokines and improve the symptoms of dry eye.
引文
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[15] Anthony J.Bron, Frcophth, F Med Sci, et a1.Predicted phenotypes of dry eye: proposedconsequences of its natural history[J].The Ocular Surface,2009 ,7(2):78-92.
[16] Ghosh, S., May, M.J., Kopp, E.B., NFкB and Rel proteins: evolutionarily conserved mediators of immune responses[J]. Annu. Rev. Immunol. 1998, 16,225-260.
[17] Hiroshi Toshida, Doan H. Nguyen, Roger W. Beuerman, et a1.Evaluation of Novel Dry Eye Model: Preganglionic Parasympathetic Denervation in Rabbit[J]. Invest Ophthalmol Vis Sci. 2007,48:4468–4475.
[18] Driss Zoukhri. Effect of in?ammation on lacrimal gland function[J]. Experimental Eye Research, 2006,82 :885–898
[19] Solomon A, Dursun D, Liu Z, et al.Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease[J]. Invest Ophthalmol Vis Sci 2001; 42:2283-92.
[20] Oshida T , Iwata M, Sakimoto T ,et al . Tumor necrosis factor-alpha in tears of patients with Sjogren syndrome[J]. Nippon Ganka Gakkai Zasshi ,2004 ,108 :297-301.
[21] Li hui Luo, De-Quan Li, Amish Doshi, et al . Experimental Dry Eye Stimulates Production of In?ammatory Cytokines and MMP-9 and Activates MAPK Signaling Pathways on the Ocular Surface[J]. nvestigative Ophthalmology & Visual Science, December 2004, Vol. 45, No. 12
[22] Pflugfelder SC. Antiinflammatory therapy for dry eye[J]. Am J Ophthalmol , 2004 ,137 : 337-342.
[23] Kathleen Turner , Stephen C , PflugfelderSC ,et al .Interleukin-6 Levels in the Conjunctival epithelium of patients with dry eye disease treated with cyclosporine ophthalmic emulsion[J]. Cornea. 2000 ,19 :492-496.
[24] Nakamura Y, Sotozono C , Kinoshita S. Inflammatory cytokines in normal human tears[J]. Curr Eye Res ,1998 ,17 :673-667.
[25] Creuzot-Garcher C .Treating severe dry eye syndromes with autologous serum[J]. J Fr Ophtalmol, 2004,27(4):346–351,
[26] Sall K . Two multicenter, randomized studies of the efficacy and safety of cyclosporine ophthalmic emulsion in moderate to severe dry eye disease[J]. CsA Phase 3 Study Group. Ophthalmology,2000, 107(4):631–639,
[27] Stevenson D, Tauber J, Reis BL. Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate- to-severe dry eye disease: a dose-ranging, randomized trial[J]. The Cyclosporin A Phase 2 Study Group. Ophthalmology,2000, 107(5):967–974,
[28] Pflugfelder, S.C., Solomon, A., Dursun, D., et al. Dry eye and delayed tear clearance:‘a call to arms’[J]. Adv. Exp. Med. Biol,2002,506, 739–743.
[29] Schafer, M.K., Bette, M., Romeo, H., et al. Localization of kappa-opioid receptor mRNA in neuronal subpopulations of rat sensory ganglia and spinal cord[J].Neurosci. Lett,1994,167, 137–140.
[30] Fini ME, Parks WC, Rinehart WB, et al. Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury[J]. Am JPathol. 1996;149:1287–1302.
[1] Moss SE, Klein R, Klein BE.. Incidence of dry eye in an older population[J]. Arch Ophthalmol,2004, 122: 369-373.
[2] Darlene A.Dartt. Neural regulation of lacrimal gland secretory processes: Relevance in dry eye diseases[J]. Progress in Retinal and Eye Research, 2009, 28:155-177.
[3] Baudouin, MD. The Pathology of Dry Eye[J]. Survey of Ophthalmology, 2001, 45 (Suppl 2):211-220.
[4] Pflugfelder, S.C., Solomon, A., Dursun, D., Li, D.Q. Dry eye and delayed tear clearance:‘a call to arms’[J]. Adv. Exp. Med. Biol,2002,506:739–743.
[5] Schafer, M.K., Bette, M., Romeo, H., Schwaeble, W., Weihe, E. Localization of kappa-opioid receptor mRNA in neuronal subpopulations of rat sensory ganglia and spinal cord[J]. Neurosci. Lett,1994,167:37–140.
[6] Zoukhri, D., Hodges, R.R., Dartt, D.A.Lacrimal gland innervation is not altered with the onset and progression of disease in a murine model of Sj?gren’s syndrome[J]. Clin. Immunol. Immunopathol,1998,89:126–133.
[7] Zoukhri, D., Hodges, R.R., Rawe, I.M., Dartt, D.A., Ca2+Csignaling by cholinergic and alpha1-adrenergic agonists is up-regulated in lacrimal and submandibular glands in a murine model of Sjo¨gren’s syndrome[J]. Clin. Immunol. Immunopathol, 1998,89:134–140.
[8] Michael E. Stern, Jianping Gao, Karyn F. Siemasko,Roger W. Beuerman, Stephen C. Pflugfelder The role of the lacrimal functional unit in the pathophysiology of dry eye[J] . Experimental Eye Research,2004,78: 409–416.
[9] Sullivan, D.A.,. Tearful relationships? Sex, hormones and aqueousdeficient dry eye[J]. Ocul. Surf,2004,2:92–123.
[10] Leiba, H., Garty, N.B., Schmidt-Sole, J., Piterman, O., Azrad, A., Salomon, Y. The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter[J]. Eur. J. Pharmacol , 1990,181:71–82.
[11] Sullivan, D.A., Edwards, J.A. Androgen stimulation of lacrimal gland function in mouse models of Sj?gren’s syndrome[J]. Steroid Biochem. Mol. Biol , 1997, 60:237–245.
[12] Sullivan, D.A., Sullivan, B.D., Ullman, M.D., Rocha, E.M., Krenzer, K.L., Cermak, J.M., Toda, I., Doane, M.G., Evans, J.E., Wickham, L.A. Androgen influence on the meibomian gland[J]. Invest. Ophthalmol. Vis. Sci, 2000,41:3732–3742.
[13] Sullivan DA ,Krenzer KL ,Sullivan BD ,et al. Does androgen insuffi- ciency cause lacrimal gland inflammation and aqueous tear deficiency[J]. Invest Ophthalmol Vis Sci ,1999 ,40 :1261-1265.
[14] Azzarolo, A.M., Mircheff, A.K., Kaswan, R.L., Stanczyk, F.Z., Gentschein, E., Becker, L., Nassir, B., Warren, D.W. Androgen support of lacrimal gland function[J]. Endocrine , 1997,6:39–45.
[15] Wickham, L.A., Gao, J., Toda, I., Rocha, E.M., Ono, M., Sullivan, D.A. Identification of androgen, estrogen and progesterone receptor mRNAs in the eye. Acta[J]. Ophthalmol. Scand, 2000, 78:146–153.
[16] Humphreys Beher, M.G., Peck, A.B., Dang, H., Talal, N. The role of apoptosis in the initiation of the autoimmune response in Sj?gren’s syndrome[J]. Clin. Exp. Immunol,1999,116: 383–387.
[17] Tapinos, N.I., Polihronis, M., Tzioufas, A.G., Skopouli, F.N., Immunopathology of Sj?gren’s syndrome[J]. Ann. Med. Interne (Paris) ,1998,149:17–24.
[18] Manganelli, P., Fietta, P. Apoptosis and Sj?gren’s syndrome[J]. Semin. Arthritis Rheum, 2003,33:49–65.
[19] Mariette, X. [Pathophysiology of Sj?gren’s syndrome[J]. Ann. Med. nterne (Paris) , 2003,154:157–168.
[20] Zoukhri, D., Hodges, R.R., Byon, D., Kublin, C.L. Role of proinflammatory cytokines in theimpaired lacrimation associated with autoimmune xerophthalmia[J]. Invest. Ophthalmol. Vis. Sci, 2002,43:1429-1436.
[21] Anthony J.Bron, Frcophth, F Med Sci, et a1.Predicted phenotypes of dry eye: proposed consequences of its natural history[J].The Ocular Surface,2009 ,7(2):78-92.
[22] Corfield, A.P., Carrington, S.D., Hicks, S.J., Berry, M., Ellingham, R., Ocular mucins: purification, metabolism and functions[J]. Prog. Retin. Eye Res,1997,16:627–656.
[23] Franscois J.Hayashi A,Kusaka S.et a1.Experimental keratltis sicca.The corneal epihelium at the transmission and scanning electron microscope[J]. Ophthal Res,1976,8(4):414—418.
[24]谢汉平,张汉承.家兔干眼模型的角膜上皮超微结构及几种眼药的疗效[J].眼科研究,1992,10(1):10—12.
[25] 25 Gibard JP, Rossi SR,Gray KL, et a1 .A new rabbit model keratoconjunctivitis sicca[J]. Invest Ophthalmol Vis Sci ,1987,28( 2) :2 2 5—2 28.
[26] Hakim SG, Schroder C, Geerling G, et a1.Early and late immunohistochemical and ultrastructural changes associated with functional impairment of the lachrymal gland following external beam radiation[J].Int J Exp Pathol ,2006,87:65–71.
[27] Guo Z, Song D, Azzarolo AM, Schechter JE, et a1. Autologous lacrimal-lymphoid mixed-cell reactions induce dacryoadenitis in rabbits[J]. Exp Eye Res,2000,71:23–31.
[28] Choul Yong Park,Wenjuan Zhuang,Kaevalin Lekhanont,et al.Lacrimal gland inflammatory cytokine gene expression in the botulinum toxin B-induced murine dry eye model[J].Molecular Vision,2007,13:2222-32.
[29] Driss Zoukhri,Elizabeth Macari,Claire L. Kublin. A single injection of interleukin-1 induces reversible aqueous-tear deficiency, lacrimal gland in?ammation, and acinar and ductal cell proliferation[J]. Experimental Eye Research,2007, 84:894-904.
[30]肖启国.干眼模型的建立方法及评价[J].眼科研究,2004,22(4):438-440.
[31]肖启国,刘祖国,张梅等.局部滴用阿托品建立兔干眼模型的评价[J].眼科研究,2005,23 (4):340-343.
[32] Sabrina Viau ,arie-Annick Maire,Bruno Pasquis,et al. Time course of ocular surface and lacrimal gland changes in a new scopolamine-induced dry eye model[J]. Graefes Arch Clin Exp Ophthalmol ,2008, 246:857–867.
[33] Hiroshi Toshida,Doan H. Nguyen,Roger W. Beuerman,et al. Evaluation of Novel Dry Eye Model: Preganglionic Parasympathetic Denervation in Rabbit[J]. Invest Ophthalmol Vis Sci,2007,48:4468–4475.
[34] Gilbard JP, Rossi SR.Tear film and ocular surface changes in a rabbit model of neurotrophic keratitis.Ophthalmology,1990,97( 3):308-312.
[35] Xiu Jun Song,De-Quan Li,William Farley,et al. Neurturin-Deficient Mice Develop Dry Eye and Keratoconjunctivitis Sicca[J]. Investigative Ophthalmology & Visual Science, 2003, 44 (10) : 4223-4229.
[36] Gilbard JP, Rossi SR, Heyda KG: Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit[J]. Ophthalmology, 1989,96:1180–1186.
[37] Stefan Schrader,Austin K. Mircheff, Gerd Geerling..Animal Models of Dry Eye[J]. Dev Ophthalmol,2008, 41:298–312.
[38] Stefano Barabino ,Maurizio Rolando , Lu Chen,et al. Exposure to a dry environment induces strain-specific responses in mice[J]. Experimental Eye Research ,2007, 84:973– 977.
[39] Nakamura S, Shibuya M, Nakashima H, et al.D-β-Hydroxybutyrate protects against corneal epithelial disorders in a rat dry eye model with jogging board[J]. Invest Ophthalmol Vis Sci ,2005,46:2379–2387.
[40] Cuiju Xiong, Dong Chen, Jingbo Liu,et al. A Rabbit Dry Eye Model Induced by Topical Medication of a Preservative Benzalkonium Chloride[J]. Investigative Ophthalmology & Visual Science, 2008, 49 (5): 1850-1856.
[41] Nakamura S, Okada S, Umeda Y, Saito F: Development of a rabbit model of tear film instability and evaluation of viscosity of artificial tear preparations[J]. Cornea 2004;23:390–397.
[42] Shahriari HA,Tokhmehchi F, Reza M. Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model[J].Cornea,2008,Dec;27(10):1148-50.
[43]马轶群,杨青,于海涛.维生素A缺乏兔干眼病模型角膜上皮细胞凋亡及相关蛋白表达的关系[J].眼科新进展, 2008,28 (8) : 5862-589.
[2] Christophe Baudouin, MD The Pathology of Dry Eye [J]. Survey of Ophthalmology,2001, 45(3):S211-220.
[3] Sullivan, D.A., Sullivan, B.D., Ullman, M.D., Rocha, E.M., Krenzer, K.L., Cermak, J.M., Toda, I., Doane, M.G., Evans, J.E., Wickham, L.A. Androgen influence on the meibomian gland[J]. Invest. Ophthalmol. Vis. Sci, 2000,41:3732–3742.
[4] Michael E. Stern, Jianping Gao, Karyn F. Siemasko,Roger W. Beuerman, Stephen C. Pflugfelder The role of the lacrimal functional unit in the pathophysiology of dry eye[J] . Experimental Eye Research,2004,78: 409–416.
[5] Stefan Schrader ,Austin K. Mircheff, Gerd Geerling. Animal Models of Dry Eye[J]. Dev Ophthalmol. 2008, vol 41, pp 298–312.
[6] 6 . Susi B,Luana P,Patrizia C,et a1.Development of a simple dry eye model in the albino rabbit and evaluation of some tear substitutes[J].Ophthalmic Res,1999, 31(2):229-235.
[7]肖启国,刘祖国,张梅等.局部滴用阿托品建立兔干眼模型的评价[J].眼科研究,2005 , 23 (4) :340 -343.
[8] Sabrina Viau ,Marie-Annick Maire ,Bruno Pasquis .Time course of ocular surface and lacrimal gland changes in a new scopolamine-induced dry eye model[J]. Graefes Arch Clin Exp Ophthalmol, 2008, 246:857–867.
[9] 9 Dilek Dursun, Min Wang, Dagoberto Monroy.A Mouse Model of Keratoconjunctivitis Sicca[J]. Invest Ophthalmol Vis Sci, 2002, 43(3):632-638.
[10] Humphreys Beher, M.G., Peck, A.B., Dang, H., Talal, N. The role of apoptosis in the initiation of the autoimmune response in Sj?gren’s syndrome[J]. Clin. Exp. Immunol,1999,116: 383–387.
[11] Tapinos, N.I., Polihronis, M., Tzioufas, A.G., Skopouli, F.N., Immunopathology of Sj?gren’s syndrome[J]. Ann. Med. Interne (Paris) ,1998,149:17–24.
[12] Manganelli, P., Fietta, P. Apoptosis and Sj?gren’s syndrome[J]. Semin. Arthritis Rheum, 2003,33:49–65.
[13] Mariette, X. [Pathophysiology of Sj?gren’s syndrome[J]. Ann. Med. nterne (Paris) , 2003,154:157–168.
[14] Baudouin, MD. The Pathology of Dry Eye[J]. Survey of Ophthalmology, 2001, 45 (Suppl 2):211-220.
[15] Anthony J.Bron, Frcophth, F Med Sci, et a1.Predicted phenotypes of dry eye: proposedconsequences of its natural history[J].The Ocular Surface,2009 ,7(2):78-92.
[16] Ghosh, S., May, M.J., Kopp, E.B., NFкB and Rel proteins: evolutionarily conserved mediators of immune responses[J]. Annu. Rev. Immunol. 1998, 16,225-260.
[17] Hiroshi Toshida, Doan H. Nguyen, Roger W. Beuerman, et a1.Evaluation of Novel Dry Eye Model: Preganglionic Parasympathetic Denervation in Rabbit[J]. Invest Ophthalmol Vis Sci. 2007,48:4468–4475.
[18] Driss Zoukhri. Effect of in?ammation on lacrimal gland function[J]. Experimental Eye Research, 2006,82 :885–898
[19] Solomon A, Dursun D, Liu Z, et al.Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease[J]. Invest Ophthalmol Vis Sci 2001; 42:2283-92.
[20] Oshida T , Iwata M, Sakimoto T ,et al . Tumor necrosis factor-alpha in tears of patients with Sjogren syndrome[J]. Nippon Ganka Gakkai Zasshi ,2004 ,108 :297-301.
[21] Li hui Luo, De-Quan Li, Amish Doshi, et al . Experimental Dry Eye Stimulates Production of In?ammatory Cytokines and MMP-9 and Activates MAPK Signaling Pathways on the Ocular Surface[J]. nvestigative Ophthalmology & Visual Science, December 2004, Vol. 45, No. 12
[22] Pflugfelder SC. Antiinflammatory therapy for dry eye[J]. Am J Ophthalmol , 2004 ,137 : 337-342.
[23] Kathleen Turner , Stephen C , PflugfelderSC ,et al .Interleukin-6 Levels in the Conjunctival epithelium of patients with dry eye disease treated with cyclosporine ophthalmic emulsion[J]. Cornea. 2000 ,19 :492-496.
[24] Nakamura Y, Sotozono C , Kinoshita S. Inflammatory cytokines in normal human tears[J]. Curr Eye Res ,1998 ,17 :673-667.
[25] Creuzot-Garcher C .Treating severe dry eye syndromes with autologous serum[J]. J Fr Ophtalmol, 2004,27(4):346–351,
[26] Sall K . Two multicenter, randomized studies of the efficacy and safety of cyclosporine ophthalmic emulsion in moderate to severe dry eye disease[J]. CsA Phase 3 Study Group. Ophthalmology,2000, 107(4):631–639,
[27] Stevenson D, Tauber J, Reis BL. Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate- to-severe dry eye disease: a dose-ranging, randomized trial[J]. The Cyclosporin A Phase 2 Study Group. Ophthalmology,2000, 107(5):967–974,
[28] Pflugfelder, S.C., Solomon, A., Dursun, D., et al. Dry eye and delayed tear clearance:‘a call to arms’[J]. Adv. Exp. Med. Biol,2002,506, 739–743.
[29] Schafer, M.K., Bette, M., Romeo, H., et al. Localization of kappa-opioid receptor mRNA in neuronal subpopulations of rat sensory ganglia and spinal cord[J].Neurosci. Lett,1994,167, 137–140.
[30] Fini ME, Parks WC, Rinehart WB, et al. Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury[J]. Am JPathol. 1996;149:1287–1302.
[1] Moss SE, Klein R, Klein BE.. Incidence of dry eye in an older population[J]. Arch Ophthalmol,2004, 122: 369-373.
[2] Darlene A.Dartt. Neural regulation of lacrimal gland secretory processes: Relevance in dry eye diseases[J]. Progress in Retinal and Eye Research, 2009, 28:155-177.
[3] Baudouin, MD. The Pathology of Dry Eye[J]. Survey of Ophthalmology, 2001, 45 (Suppl 2):211-220.
[4] Pflugfelder, S.C., Solomon, A., Dursun, D., Li, D.Q. Dry eye and delayed tear clearance:‘a call to arms’[J]. Adv. Exp. Med. Biol,2002,506:739–743.
[5] Schafer, M.K., Bette, M., Romeo, H., Schwaeble, W., Weihe, E. Localization of kappa-opioid receptor mRNA in neuronal subpopulations of rat sensory ganglia and spinal cord[J]. Neurosci. Lett,1994,167:37–140.
[6] Zoukhri, D., Hodges, R.R., Dartt, D.A.Lacrimal gland innervation is not altered with the onset and progression of disease in a murine model of Sj?gren’s syndrome[J]. Clin. Immunol. Immunopathol,1998,89:126–133.
[7] Zoukhri, D., Hodges, R.R., Rawe, I.M., Dartt, D.A., Ca2+Csignaling by cholinergic and alpha1-adrenergic agonists is up-regulated in lacrimal and submandibular glands in a murine model of Sjo¨gren’s syndrome[J]. Clin. Immunol. Immunopathol, 1998,89:134–140.
[8] Michael E. Stern, Jianping Gao, Karyn F. Siemasko,Roger W. Beuerman, Stephen C. Pflugfelder The role of the lacrimal functional unit in the pathophysiology of dry eye[J] . Experimental Eye Research,2004,78: 409–416.
[9] Sullivan, D.A.,. Tearful relationships? Sex, hormones and aqueousdeficient dry eye[J]. Ocul. Surf,2004,2:92–123.
[10] Leiba, H., Garty, N.B., Schmidt-Sole, J., Piterman, O., Azrad, A., Salomon, Y. The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter[J]. Eur. J. Pharmacol , 1990,181:71–82.
[11] Sullivan, D.A., Edwards, J.A. Androgen stimulation of lacrimal gland function in mouse models of Sj?gren’s syndrome[J]. Steroid Biochem. Mol. Biol , 1997, 60:237–245.
[12] Sullivan, D.A., Sullivan, B.D., Ullman, M.D., Rocha, E.M., Krenzer, K.L., Cermak, J.M., Toda, I., Doane, M.G., Evans, J.E., Wickham, L.A. Androgen influence on the meibomian gland[J]. Invest. Ophthalmol. Vis. Sci, 2000,41:3732–3742.
[13] Sullivan DA ,Krenzer KL ,Sullivan BD ,et al. Does androgen insuffi- ciency cause lacrimal gland inflammation and aqueous tear deficiency[J]. Invest Ophthalmol Vis Sci ,1999 ,40 :1261-1265.
[14] Azzarolo, A.M., Mircheff, A.K., Kaswan, R.L., Stanczyk, F.Z., Gentschein, E., Becker, L., Nassir, B., Warren, D.W. Androgen support of lacrimal gland function[J]. Endocrine , 1997,6:39–45.
[15] Wickham, L.A., Gao, J., Toda, I., Rocha, E.M., Ono, M., Sullivan, D.A. Identification of androgen, estrogen and progesterone receptor mRNAs in the eye. Acta[J]. Ophthalmol. Scand, 2000, 78:146–153.
[16] Humphreys Beher, M.G., Peck, A.B., Dang, H., Talal, N. The role of apoptosis in the initiation of the autoimmune response in Sj?gren’s syndrome[J]. Clin. Exp. Immunol,1999,116: 383–387.
[17] Tapinos, N.I., Polihronis, M., Tzioufas, A.G., Skopouli, F.N., Immunopathology of Sj?gren’s syndrome[J]. Ann. Med. Interne (Paris) ,1998,149:17–24.
[18] Manganelli, P., Fietta, P. Apoptosis and Sj?gren’s syndrome[J]. Semin. Arthritis Rheum, 2003,33:49–65.
[19] Mariette, X. [Pathophysiology of Sj?gren’s syndrome[J]. Ann. Med. nterne (Paris) , 2003,154:157–168.
[20] Zoukhri, D., Hodges, R.R., Byon, D., Kublin, C.L. Role of proinflammatory cytokines in theimpaired lacrimation associated with autoimmune xerophthalmia[J]. Invest. Ophthalmol. Vis. Sci, 2002,43:1429-1436.
[21] Anthony J.Bron, Frcophth, F Med Sci, et a1.Predicted phenotypes of dry eye: proposed consequences of its natural history[J].The Ocular Surface,2009 ,7(2):78-92.
[22] Corfield, A.P., Carrington, S.D., Hicks, S.J., Berry, M., Ellingham, R., Ocular mucins: purification, metabolism and functions[J]. Prog. Retin. Eye Res,1997,16:627–656.
[23] Franscois J.Hayashi A,Kusaka S.et a1.Experimental keratltis sicca.The corneal epihelium at the transmission and scanning electron microscope[J]. Ophthal Res,1976,8(4):414—418.
[24]谢汉平,张汉承.家兔干眼模型的角膜上皮超微结构及几种眼药的疗效[J].眼科研究,1992,10(1):10—12.
[25] 25 Gibard JP, Rossi SR,Gray KL, et a1 .A new rabbit model keratoconjunctivitis sicca[J]. Invest Ophthalmol Vis Sci ,1987,28( 2) :2 2 5—2 28.
[26] Hakim SG, Schroder C, Geerling G, et a1.Early and late immunohistochemical and ultrastructural changes associated with functional impairment of the lachrymal gland following external beam radiation[J].Int J Exp Pathol ,2006,87:65–71.
[27] Guo Z, Song D, Azzarolo AM, Schechter JE, et a1. Autologous lacrimal-lymphoid mixed-cell reactions induce dacryoadenitis in rabbits[J]. Exp Eye Res,2000,71:23–31.
[28] Choul Yong Park,Wenjuan Zhuang,Kaevalin Lekhanont,et al.Lacrimal gland inflammatory cytokine gene expression in the botulinum toxin B-induced murine dry eye model[J].Molecular Vision,2007,13:2222-32.
[29] Driss Zoukhri,Elizabeth Macari,Claire L. Kublin. A single injection of interleukin-1 induces reversible aqueous-tear deficiency, lacrimal gland in?ammation, and acinar and ductal cell proliferation[J]. Experimental Eye Research,2007, 84:894-904.
[30]肖启国.干眼模型的建立方法及评价[J].眼科研究,2004,22(4):438-440.
[31]肖启国,刘祖国,张梅等.局部滴用阿托品建立兔干眼模型的评价[J].眼科研究,2005,23 (4):340-343.
[32] Sabrina Viau ,arie-Annick Maire,Bruno Pasquis,et al. Time course of ocular surface and lacrimal gland changes in a new scopolamine-induced dry eye model[J]. Graefes Arch Clin Exp Ophthalmol ,2008, 246:857–867.
[33] Hiroshi Toshida,Doan H. Nguyen,Roger W. Beuerman,et al. Evaluation of Novel Dry Eye Model: Preganglionic Parasympathetic Denervation in Rabbit[J]. Invest Ophthalmol Vis Sci,2007,48:4468–4475.
[34] Gilbard JP, Rossi SR.Tear film and ocular surface changes in a rabbit model of neurotrophic keratitis.Ophthalmology,1990,97( 3):308-312.
[35] Xiu Jun Song,De-Quan Li,William Farley,et al. Neurturin-Deficient Mice Develop Dry Eye and Keratoconjunctivitis Sicca[J]. Investigative Ophthalmology & Visual Science, 2003, 44 (10) : 4223-4229.
[36] Gilbard JP, Rossi SR, Heyda KG: Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit[J]. Ophthalmology, 1989,96:1180–1186.
[37] Stefan Schrader,Austin K. Mircheff, Gerd Geerling..Animal Models of Dry Eye[J]. Dev Ophthalmol,2008, 41:298–312.
[38] Stefano Barabino ,Maurizio Rolando , Lu Chen,et al. Exposure to a dry environment induces strain-specific responses in mice[J]. Experimental Eye Research ,2007, 84:973– 977.
[39] Nakamura S, Shibuya M, Nakashima H, et al.D-β-Hydroxybutyrate protects against corneal epithelial disorders in a rat dry eye model with jogging board[J]. Invest Ophthalmol Vis Sci ,2005,46:2379–2387.
[40] Cuiju Xiong, Dong Chen, Jingbo Liu,et al. A Rabbit Dry Eye Model Induced by Topical Medication of a Preservative Benzalkonium Chloride[J]. Investigative Ophthalmology & Visual Science, 2008, 49 (5): 1850-1856.
[41] Nakamura S, Okada S, Umeda Y, Saito F: Development of a rabbit model of tear film instability and evaluation of viscosity of artificial tear preparations[J]. Cornea 2004;23:390–397.
[42] Shahriari HA,Tokhmehchi F, Reza M. Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model[J].Cornea,2008,Dec;27(10):1148-50.
[43]马轶群,杨青,于海涛.维生素A缺乏兔干眼病模型角膜上皮细胞凋亡及相关蛋白表达的关系[J].眼科新进展, 2008,28 (8) : 5862-589.