碱烧伤诱导兔部分性角膜缘干细胞失代偿模型
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摘要
目的:探讨诱导兔部分性角膜缘干细胞失代偿(limbal stem cell deficiency,LSCD)动物模型的一种新方法。方法:分别采用C57小鼠和新西兰白兔制作完全性和部分性角膜缘干细胞失代偿动物模型。小鼠(n=30)采用1mol/L氢氧化钾溶液浸泡的滤纸片(直径3mm)置于左眼中央角膜表面30s,随后用生理盐水冲洗。白兔(n=19)切除瞬膜(第三眼睑)后,采用1mol/L氢氧化钾溶液浸泡的滤纸片(直径5mm)置于左眼颞上方角膜表面30s,随后用生理盐水冲洗。烧伤眼术后采用0.5%盐酸左氧氟沙星滴眼液4次/d。烧伤前、烧伤后第1、2、4wk,2mo采用裂隙灯显微镜观察、摄像,记录角膜溃疡、穿孔等并发症。术后2mo采用印迹细胞学检测角膜杯状细胞分布,根据裂隙灯显微镜检查所见和角膜印迹细胞学检查判断LSCD严重程度。术后2mo处死动物,角结膜切片观察角膜新生血管、杯状细胞分布。意外死亡动物不计入总数,计算并比较完全性LSCD和部分性LSCD的模型诱导成功率。结果:30只小鼠中6只意外死亡,2只于烧伤后出现角膜穿孔,其余22只发生完全性LSCD,诱导成功率92%,烧伤后2mo小鼠角膜可见新生血管广泛分布于角膜浅层及深基质层,病理切片可见角膜新生血管。19只白兔,7只意外死亡,其余12只发生不同程度LSCD(部分性LSCD,平均累及1.17±0.39个象限),未发生角膜穿孔情况,诱导成功率100%(P=0.543)。正常角膜区域无杯状细胞,LSCD区域角膜上皮印迹细胞学PAS染色可见杯状细胞,平均密度58.60±12.58个细胞/HP。结论:中央角膜碱烧伤可以诱导产生完全性LSCD,部分动物会因为角膜溃疡穿孔而导致模型诱导失败,LSCD往往比较严重,且合并深层角膜新生血管。颞上方角膜碱烧伤可以诱导产生部分性LSCD,合并较少的角膜病变,角膜新生血管位于浅层。
?AIM: To explore a new method to induce the animal model of rabbit partial limbal stem cell deficiency(LSCD).?METHODS: LSCD was induced through corneal alkali burn, C57 mice and New Zealand rabbits were used to establish the animal models. Corneal alkali burn manipulation was accomplished in experimental animals under general anesthesia combined with surface anesthesia in the operated eye. Specifically, mice(n=30) were used to induce complete LSCD model. In brief, the filter paper(diameter of 3 mm) that immersed in 1 mol/L potassium hydroxide solution was placed on the central corneal surface of the left eye for 30 s, followed by washing with saline. In addition, rabbits(n=19) were utilized to establish the partial LSCD model. Briefly, the nictitating membrane(third eyelid) was resected, and the filter paper(diameter of 5 mm) that immersed in 1 mol/L potassium hydroxide solution was placed on the superior temporal peripheral corneal surface of the left for 30 s, followed by washing with saline. After surgery, the model eyes were treated with 0.5% Levofloxacin Hydrochloride Eye Drops four times a day. In addition, the slit-lamp microscope was adopted for observation and photo-taking before burn, as well as at 1, 2, 4 wk and 2 mo after burn; meanwhile, complications such as corneal ulcer and perforation were recorded. 2 mo after surgery, the corneal goblet cell distribution was detected with impression cytology, and the severity of LSCD was classified according to slit-lamp microscopic findings and corneal impression cytology. The animals were sacrificed 2 mo after surgery, cornea and conjunctiva sections were made to observe angiogenesis and goblet cell distribution in cornea. Animals died accidentally were not counted into the total number, and the successful induction rates of complete LSCD and partial LSCD models were compared.?RESULTS: Six out of the 30 mice died accidentally, while 2 developed corneal perforation after burn, and the remaining 22 had developed complete LSCD only, yielding the successful induction rate of 92%. 2 mo after burn, extensive angiogenesis distribution in the superficial and deep corneal stromal layers could be observed, and pathological sections revealed corneal angiogenesis. Seven out of the 19 rabbits died accidentally, while the remaining 12 had various degrees of LSCD only(partial LSCD, average involving 1.17±0.39 quadrants). Additionally, no corneal perforation was observed, and the successful induction rate was 100%. The result of Fisher's exact test P value is 0.543, without statistical difference. No goblet cells were observed in the normal corneal region, while goblet cells were observed in the LSCD region, with the average density of 58.60±12.58 cell/HP.?CONCLUSION: Central corneal alkali burn can induce complete LSCD; however, some animals will experience failure in model induction due to corneal ulcer and perforation, LSCD is generally serious and may be combined with angiogenesis in deep cornea. Alkali burn in superior temporal cornea can induce partial LSCD, which may be combined with relatively minor corneal lesion, and the corneal angiogenesis is located in the superficial layer.
?AIM: To explore a new method to induce the animal model of rabbit partial limbal stem cell deficiency(LSCD).?METHODS: LSCD was induced through corneal alkali burn, C57 mice and New Zealand rabbits were used to establish the animal models. Corneal alkali burn manipulation was accomplished in experimental animals under general anesthesia combined with surface anesthesia in the operated eye. Specifically, mice(n=30) were used to induce complete LSCD model. In brief, the filter paper(diameter of 3 mm) that immersed in 1 mol/L potassium hydroxide solution was placed on the central corneal surface of the left eye for 30 s, followed by washing with saline. In addition, rabbits(n=19) were utilized to establish the partial LSCD model. Briefly, the nictitating membrane(third eyelid) was resected, and the filter paper(diameter of 5 mm) that immersed in 1 mol/L potassium hydroxide solution was placed on the superior temporal peripheral corneal surface of the left for 30 s, followed by washing with saline. After surgery, the model eyes were treated with 0.5% Levofloxacin Hydrochloride Eye Drops four times a day. In addition, the slit-lamp microscope was adopted for observation and photo-taking before burn, as well as at 1, 2, 4 wk and 2 mo after burn; meanwhile, complications such as corneal ulcer and perforation were recorded. 2 mo after surgery, the corneal goblet cell distribution was detected with impression cytology, and the severity of LSCD was classified according to slit-lamp microscopic findings and corneal impression cytology. The animals were sacrificed 2 mo after surgery, cornea and conjunctiva sections were made to observe angiogenesis and goblet cell distribution in cornea. Animals died accidentally were not counted into the total number, and the successful induction rates of complete LSCD and partial LSCD models were compared.?RESULTS: Six out of the 30 mice died accidentally, while 2 developed corneal perforation after burn, and the remaining 22 had developed complete LSCD only, yielding the successful induction rate of 92%. 2 mo after burn, extensive angiogenesis distribution in the superficial and deep corneal stromal layers could be observed, and pathological sections revealed corneal angiogenesis. Seven out of the 19 rabbits died accidentally, while the remaining 12 had various degrees of LSCD only(partial LSCD, average involving 1.17±0.39 quadrants). Additionally, no corneal perforation was observed, and the successful induction rate was 100%. The result of Fisher's exact test P value is 0.543, without statistical difference. No goblet cells were observed in the normal corneal region, while goblet cells were observed in the LSCD region, with the average density of 58.60±12.58 cell/HP.?CONCLUSION: Central corneal alkali burn can induce complete LSCD; however, some animals will experience failure in model induction due to corneal ulcer and perforation, LSCD is generally serious and may be combined with angiogenesis in deep cornea. Alkali burn in superior temporal cornea can induce partial LSCD, which may be combined with relatively minor corneal lesion, and the corneal angiogenesis is located in the superficial layer.
引文
1 Movahedan A,Afsharkhamseh N,Sagha HM,et al.Loss of Notch1 disrupts the barrier repair in the corneal epithelium.PloS One 2013;8(7):e69113
2 Tseng SCG.Concept and application of limbal stem cells.Eye (Lond) 1989;3 (Pt 2):141-157
3 Kasetti RB,Gaddipati S,Tian S,et al.Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice.Sci Rep 2016;6:35202
4 Ma Y,Xu Y,Xiao Z,et al.Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.Stem Cells 2006;24(2):315-321
5亓晓琳.小鼠眼部碱烧伤后骨髓间充质干细胞归巢到角膜组织的检测及影响因素分析.青岛大学2013
6 Shortt AJ,Secker GA,Notara MD,et al.Transplantation of ex vivo cultured limbal epithelial stem cells:a review of techniques and clinical results.Surv Ophthalmol 2007;52(5):483-502
7 Le Q,Xu J,Deng SX.The diagnosis of limbal stem cell deficiency.Ocul Surf 2018;16(1):58-69
8 Avila M,Espaňa M,Moreno C,et al.Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model.Cornea 2001;20(4):414-420
9 Sitalakshmi G,Sudha B,Madhavan HN,et al.Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits an animal model.Tissue Eng Part A 2009;15(2):407-415
10 Yeh SI,Ho TC,Chen SL,et al.Pigment Epithelial-Derived Factor Peptide Regenerated Limbus Serves as Regeneration Source for Limbal Regeneration in Rabbit Limbal Deficiency.Invest Ophthalmol Vis Sci 2016;57(6):2629-2636
11 Ti SE,Anderson D,Touhami A,et al.Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.Invest Ophthalmol Vis Sci 2002;43(8):2584-2592
12 Gomes JA,Geraldes Monteiro B,Melo GB,et al.Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells.Invest Ophthalmol Vis Sci 2010;51(3):1408-1414
13 Monteiro BG,Serafim RC,Melo GB,et al.Human immature dental pulp stem cells share key characteristic features with limbal stem cells.Cell Prolif 2009;42(5):587-594
14 Ono K,Yokoo S,Mimura T,et al.Autologous transplantation of conjunctival epithelial cells cultured on amniotic membrane in a rabbit model.Mol Vis 2007;13:1138-1143
15 Talbot M,Carrier P,Giasson CJ,et al.Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction.Mol Vis 2006;12:65-75
16 Li G,Zhang Y,Cai S,et al.Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model.Sci Rep 2018;8(1):6566
17 Yeh SI,Ho TC,Chen SL,et al.Pigment Epithelial-Derived Factor Peptide Facilitates the Regeneration of a Functional Limbus in Rabbit Partial Limbal Deficiency.Invest Ophthalmol Vis Sci 2015;56(4):2126-2134
18 Kadar T,Horwitz V,Sahar R,et al.Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits.Curr Eye Res 2011;36(12):1098-1107
19王向军.角膜缘干细胞自体移植的实验研究.第三军医大学 2007
20徐彬.组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究.中国海洋大学 2012
21 Zhang H,Lin S,Zhang M,et al.Comparison of Two Rabbit Models with Deficiency of Corneal Epithelium and Limbal Stem Cells Established by Different Methods.Tissue Eng Part C Methods 2017;23(11):710-717
22 Selver OB,Durak I,Gürdal M,et al.Corneal recovery in a rabbit limbal stem cell deficiency model by autologous grafts of tertiary outgrowths from cultivated limbal biopsy explants.Mol Vis 2016;22:138-149
23 Holan V,Trosan P,Cejka C,et al.A Comparative Study of the Therapeutic Potential of Mesenchymal Stem Cells and Limbal Epithelial Stem Cells for Ocular Surface Reconstruction.Stem Cells Transl Med 2015;4(9):1052-1063
24 Chae JJ,Ambrose WM,Espinoza FA,et al.Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency.Acta Ophthalmol 2014;93(1):e57-66
25 Zhou Q,Liu XY,Ruan YX,et al.Construction of corneal epithelium with human amniotic epithelial cells and repair of limbal deficiency in rabbit models.Hum Cell 2015;28(1):22-36
26 Li FJ,Nili E,Lau C,et al.Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.Exp Eye Res 2016;147:1-11
27郭滨,任丛,温莹.眼部碱烧伤导致兔角膜缘干细胞缺乏动物模型的建立与评价.中国实用眼科杂志 2015;33(12):1411-1414
28 Afsharkhamseh N,Movahedan A,Gidfar S,et al.Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.Exp Eye Res 2016;145:88-92
29 Afsharkhamseh N,Ghahari E,Eslani M,et al.A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse.J Vis Exp 2016;(117)
30 Dua HS,Azuara-Blanco A.Limbal stem cells of the corneal epithelium.Surv Ophthalmol 2000;44:415-425
2 Tseng SCG.Concept and application of limbal stem cells.Eye (Lond) 1989;3 (Pt 2):141-157
3 Kasetti RB,Gaddipati S,Tian S,et al.Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice.Sci Rep 2016;6:35202
4 Ma Y,Xu Y,Xiao Z,et al.Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.Stem Cells 2006;24(2):315-321
5亓晓琳.小鼠眼部碱烧伤后骨髓间充质干细胞归巢到角膜组织的检测及影响因素分析.青岛大学2013
6 Shortt AJ,Secker GA,Notara MD,et al.Transplantation of ex vivo cultured limbal epithelial stem cells:a review of techniques and clinical results.Surv Ophthalmol 2007;52(5):483-502
7 Le Q,Xu J,Deng SX.The diagnosis of limbal stem cell deficiency.Ocul Surf 2018;16(1):58-69
8 Avila M,Espaňa M,Moreno C,et al.Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model.Cornea 2001;20(4):414-420
9 Sitalakshmi G,Sudha B,Madhavan HN,et al.Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits an animal model.Tissue Eng Part A 2009;15(2):407-415
10 Yeh SI,Ho TC,Chen SL,et al.Pigment Epithelial-Derived Factor Peptide Regenerated Limbus Serves as Regeneration Source for Limbal Regeneration in Rabbit Limbal Deficiency.Invest Ophthalmol Vis Sci 2016;57(6):2629-2636
11 Ti SE,Anderson D,Touhami A,et al.Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.Invest Ophthalmol Vis Sci 2002;43(8):2584-2592
12 Gomes JA,Geraldes Monteiro B,Melo GB,et al.Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells.Invest Ophthalmol Vis Sci 2010;51(3):1408-1414
13 Monteiro BG,Serafim RC,Melo GB,et al.Human immature dental pulp stem cells share key characteristic features with limbal stem cells.Cell Prolif 2009;42(5):587-594
14 Ono K,Yokoo S,Mimura T,et al.Autologous transplantation of conjunctival epithelial cells cultured on amniotic membrane in a rabbit model.Mol Vis 2007;13:1138-1143
15 Talbot M,Carrier P,Giasson CJ,et al.Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction.Mol Vis 2006;12:65-75
16 Li G,Zhang Y,Cai S,et al.Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model.Sci Rep 2018;8(1):6566
17 Yeh SI,Ho TC,Chen SL,et al.Pigment Epithelial-Derived Factor Peptide Facilitates the Regeneration of a Functional Limbus in Rabbit Partial Limbal Deficiency.Invest Ophthalmol Vis Sci 2015;56(4):2126-2134
18 Kadar T,Horwitz V,Sahar R,et al.Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits.Curr Eye Res 2011;36(12):1098-1107
19王向军.角膜缘干细胞自体移植的实验研究.第三军医大学 2007
20徐彬.组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究.中国海洋大学 2012
21 Zhang H,Lin S,Zhang M,et al.Comparison of Two Rabbit Models with Deficiency of Corneal Epithelium and Limbal Stem Cells Established by Different Methods.Tissue Eng Part C Methods 2017;23(11):710-717
22 Selver OB,Durak I,Gürdal M,et al.Corneal recovery in a rabbit limbal stem cell deficiency model by autologous grafts of tertiary outgrowths from cultivated limbal biopsy explants.Mol Vis 2016;22:138-149
23 Holan V,Trosan P,Cejka C,et al.A Comparative Study of the Therapeutic Potential of Mesenchymal Stem Cells and Limbal Epithelial Stem Cells for Ocular Surface Reconstruction.Stem Cells Transl Med 2015;4(9):1052-1063
24 Chae JJ,Ambrose WM,Espinoza FA,et al.Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency.Acta Ophthalmol 2014;93(1):e57-66
25 Zhou Q,Liu XY,Ruan YX,et al.Construction of corneal epithelium with human amniotic epithelial cells and repair of limbal deficiency in rabbit models.Hum Cell 2015;28(1):22-36
26 Li FJ,Nili E,Lau C,et al.Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.Exp Eye Res 2016;147:1-11
27郭滨,任丛,温莹.眼部碱烧伤导致兔角膜缘干细胞缺乏动物模型的建立与评价.中国实用眼科杂志 2015;33(12):1411-1414
28 Afsharkhamseh N,Movahedan A,Gidfar S,et al.Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.Exp Eye Res 2016;145:88-92
29 Afsharkhamseh N,Ghahari E,Eslani M,et al.A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse.J Vis Exp 2016;(117)
30 Dua HS,Azuara-Blanco A.Limbal stem cells of the corneal epithelium.Surv Ophthalmol 2000;44:415-425