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Animal Models of Corneal Injury
角膜损伤动物模型

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Abstract

The cornea is an excellent model system to use for the analysis of wound repair because of its accessibility, lack of vascularization, and simple anatomy. Corneal injuries may involve only the superficial epithelial layer or may penetrate deeper to involve both the epithelial and stromal layers. Here we describe two well-established in vivo corneal wound models: a mechanical wound model that allows for the study of re-epithelialization and a chemical wound model that may be used to study stromal activation in response to injury (Stepp et al., 2014; Carlson et al., 2003).

Keywords: Cornea(角膜), Mouse(鼠标), Wound(伤口), Epithelium(上皮), Stroma(基质)

Materials and Reagents

Note: All reagents may be maintained at room temperature.

  1. FVB mouse (5-10 weeks old)
  2. Isoflurane (Abbott Laboratories, catalog number: 5260-04-05 )
  3. Proparacaine hydrochloride ophthalmic solution (0.5%) (Bausch & Lomb, NDC: 24208-730- 06 )
  4. Fluorescein sodium and benoxinate hydrochloride ophthalmic solution (0.25%/0.4%)  (Akorn, NDC: 17478-640-10 )
  5. Weck-cel cellulose eye spears (Medtronic, catalog number: 0008680 )
  6. NaOH solution (see Recipes)

Chemical injury

  1. Sterile water
  2. 1x sterile phosphate buffered saline buffer (1x PBS)
  3. Sodium hydroxide (NaOH) 1.0 N (normal) solution (Sigma-Aldrich, catalog number: S2770 )
  4. Fluorescein solution (see Recipes)

Equipment

  1. Heating pad for mouse
  2. Algerbrush II with 0.5 mm Burr (Katena, catalog number: K 2-4900 )
  3. Trephine with handle (1.5 mm) (Beaver-visitec, catalog number: 9748 )
  4. Alcohol swabs - isopropyl alcohol 70% (BD, catalog number: 326895 )
  5. Filter paper (Thermo Fisher Scientific, catalog number: 09-795AA )
  6. Ear punch (Roboz, catalog number: 65-9902 )
  7. Forceps (Dumont #5)
  8. Pipette with tips (1 ml)
  9. Timer
  10. Stereo microscope (for scratch/epithelial injury, need filter set to visualize GFP; for both injuries, camera attachment is optional) (Leica, catalog number: MZ16F )
  11. Anesthesia machine with nose cone attachment appropriate for mice (Summit Anesthesia Solutions, catalog number: AS-01-0007 )

Procedure

Ethical statement: All procedures discussed here are in accordance with and were approved by the University of California, San Francisco Institutional Animal Care and Use Committee.

For scratch/epithelial injury

  1. Set up the surgical area.
    1. Place the isoflurane chamber near the microscope and place the anesthesia platform with the nose cone under the microscope objective. Place a mouse heating pad by the nose cone.
    2. Place the Proparacaine, Algerbrush, weck-cels, and trephine on the lab bench near the surgical area. Have 20 μl of 1:40 diluted Fluorescein solution drawn up in a pipette (see Recipes).
    3. Clean off the Algerbrush burr by rubbing with an alcohol swab.
  2. Anesthetize the mouse in the isoflurane chamber. A typical approach for anesthesia involves placing the animal in an induction chamber connected to an oxygen source and isoflurane vaporizer, and adjusting oxygen flow to 0.9 liters/min and the isoflurane vaporizer to 1-2%. As soon as the mouse becomes unresponsive and has shallow breathing, it may be transferred to the anesthesia platform with the nose cone and placed on a heating pad. Position the mouse head so that the eye to be injured is facing up towards the microscope objective.
  3. Squeeze the bottle of Proparacaine and place 1 drop of Proparacaine on the cornea. Wait 30 sec.
  4. Use a weck-cel to dry off the cornea by gently sweeping across cornea once and dabbing both corners of the eye.
  5. Apply periocular pressure with one hand to proptose the mouse eye. (Optional step: Take a brightfield picture of the cornea prior to injury.)
  6. Use the other hand to mark the cornea with the trephine as central as possible with gentle pressure. Hold the handle of trephine with your thumb and second fingers and place the entire circular edge on the cornea. Gently turn the trephine with mild pressure approximately 3 clock hours to mark the cornea. Avoid making multiple marks.
  7. Turn the Algerbrush on and make an epithelial defect in the center of the cornea by applying gentle pressure in a circular manner and observing a break in the surface epithelial cell layer. Carefully extend the epithelial defect close to the trephine mark.
  8. Turn the Algerbrush off and use the dull blades of the Algerbrush burr to gently remove the remaining corneal epithelium out to the trephine mark. Apply Fluorescein solution to the cornea and confirm size of epithelial defect using the microscope GFP filter.
  9. Optional step: Apply periocular pressure to proptose the mouse eye. Take a fluorescence picture of the wounded cornea after applying Fluorescein solution and using the GFP filter (Figure 1).


    Figure 1. Corneal scratch/epithelial injury. Photograph of an epithelial defect of the central cornea after scratch injury stained with Fluorescein solution. The border of the scratch wound is in shown in yellow. [adapted from Figure 2 of Chan et al. (2013)]

  10. Place 1 drop of Proparacaine on the cornea.
  11. Remove mouse from the nose cone and allow mouse to awaken in a recovery cage. Monitor the mouse for pain and eye infections.

For chemical injury

  1. Set up the surgical area.
    1. Place the isoflurane chamber near the microscope and place the anesthesia platform with the nose cone under the microscope objective.
    2. Place the Proparacaine, filter paper, forceps, weck-cels, and NaOH on the lab bench near the surgical area.
    3. Have the pipette with 500 μl PBS drawn up readily available. (Optional: Have 20 μl Fluorescein solution drawn up in a pipette.)
    4. Prepare 2 mm filter paper discs using the ear punch.


      Figure 2. Filter paper disc. An ear punch can be used to create uniform 2 mm filter paper discs.

    5. Set the timer for 10 sec and 30 sec.
  2. Anesthetize the mouse in the isoflurane chamber. A typical approach for anesthesia involves placing the animal in an induction chamber connected to an oxygen source and isoflurane vaporizer, and adjusting oxygen flow to 0.9 L/min and the isoflurane vaporizer to 1-2%. As soon as the mouse becomes unresponsive and has shallow breathing, it may be transferred to the anesthesia platform with the nose cone and placed on a heating pad. Position the mouse head so that the eye to be injured is facing up towards the microscope objective.
  3. Place 1 drop of Proparacaine on the cornea. Wait 30 sec.
  4. Use a weck-cel to dry off the cornea by gently sweeping across cornea once and dabbing both corners of the eye.
  5. Use the forceps to submerge the filter paper into the NaOH solution for exactly 10 seconds.
  6. Apply periocular pressure with one hand to proptose the mouse eye. (Optional step: Take a brightfield picture of the cornea prior to injury.)
  7. Use forceps in the other hand to apply the NaOH-soaked filter paper to the center cornea for exactly 30 sec. Perform this step using the stereomicroscope to precisely place the filter paper as central as possible.
  8. Remove the filter disc from the cornea using forceps.
  9. Immediately flush the eye with 500 μl PBS to wash away residual NaOH (apply PBS, dry with weck-cel, apply PBS, dry with weck-cel, repeat until all 500 μl PBS has been used).
  10.  Optional step: Apply periocular pressure to proptose the mouse eye. Take a fluorescence picture of the wounded cornea after applying Fluorescein solution and using the GFP filter.


    Figure 3. Corneal chemical injury. Photograph of an epithelial defect of the central cornea after chemical injury stained with Fluorescein solution [adapted from Figure 1 of Chan et al. (2013)]

  11. Place 1 drop of Proparacaine on the cornea.
  12. Remove mouse from the nose cone and allow mouse to awaken in a recovery cage. Monitor mouse for pain and eye infections.

Notes

  1. When using the scratch injury model to compare epithelial repair rates between mice, it is recommended that littermate and/or age-matched mice are used.

Recipes

  1. NaOH solution
    Mix the following ingredients in a 1.5 ml Eppendorf tube the same day of the procedure: 50 μl 1 N NaOH and 450 μl sterile H2O to gain a total volume of 500 μl.
  2. Fluorescein solution (optional for chemical injury)
    Prepare the 1:40 dilution in a 1.5 ml Eppendorf tube the same day of the procedure: 2 μl Fluorescein sodium/benoxinate hydrochloride ophthalmic solution and 78 μl sterile PBS to gain a total volume of 80 μl.

Acknowledgements

This work was supported by grants from the National Institutes of Health (K08 EY018858 and R01 EY002162 to M.F.C. and R01 CA057621and P01 AI053194 to Z.W.).

References

  1. Carlson, E. C., Wang, I. J., Liu, C. Y., Brannan, P., Kao, C. W. and Kao, W. W. (2003). Altered KSPG expression by keratocytes following corneal injury. Mol Vis 9: 615-623.
  2. Chan, M. F., Li, J., Bertrand, A., Casbon, A. J., Lin, J. H., Maltseva, I. and Werb, Z. (2013). Protective effects of matrix metalloproteinase-12 following corneal injury. J Cell Sci 126(Pt 17): 3948-3960.
  3. Stepp, M. A., Zieske, J. D., Trinkaus-Randall, V., Kyne, B. M., Pal-Ghosh, S., Tadvalkar, G. and Pajoohesh-Ganji, A. (2014). Wounding the cornea to learn how it heals. Exp Eye Res 121: 178-193.

简介

角膜是一个优秀的模型系统,用于分析伤口修复,因为其可及性,缺乏血管化和简单的解剖结构。 角膜损伤可以仅涉及表面上皮层,或者可以更深地穿透以涉及上皮层和基质层。 在这里我们描述了两个建立良好的体内角膜伤口模型:允许研究重新上皮化的机械伤口模型和可用于研究基质激活以响应损伤的化学伤口模型 (Stepp等人,2014; Carlson等人,2003)。

关键字:角膜, 鼠标, 伤口, 上皮, 基质

材料和试剂

注意:所有试剂均可在室温下保存。

  1. FVB小鼠(5-10周龄)
  2. 异氟烷(Abbott Laboratories,目录号:5260-04-05)
  3. 盐酸丙卡因滴眼液(0.5%)(Bausch& Lomb,NDC:24208-730-06)
  4. 荧光素钠和盐酸肟酸盐眼溶液(0.25%/0.4%) (Akorn,NDC:17478-640-10)
  5. Weck-cel纤维素眼刺(Medtronic,目录号:0008680)
  6. NaOH溶液(参见配方)

化学伤害

  1. 无菌水
  2. 1x无菌磷酸盐缓冲盐水缓冲液(1x PBS)
  3. 氢氧化钠(NaOH)1.0N(正常)溶液(Sigma-Aldrich,目录号:S2770)
  4. 荧光素溶液(参见配方)

设备

  1. 鼠标加热垫
  2. 具有0.5mm毛刺的Algerbrush II(Katena,目录号:K 2-4900)
  3. 戴手柄(1.5毫米)(Beaver-visitec,目录号:9748)的
  4. 酒精拭子 - 异丙醇70%(BD,目录号:326895)
  5. 过滤纸(Thermo Fisher Scientific,目录号:09-795AA)
  6. 耳孔(Roboz,目录号:65-9902)
  7. 镊子(Dumont#5)
  8. 移液器吸头(1毫升)
  9. 计时器
  10. 立体显微镜(对于刮伤/上皮损伤,需要过滤器设置以可视化GFP;对于这两种损伤,照相机附件是可选的)(Leica,目录号:MZ16F)
  11. 具有适用于小鼠的鼻锥附件的麻醉机(Summit Anesthesia Solutions,目录号:AS-01-0007)

程序

伦理声明:此处讨论的所有程序均符合加利福尼亚大学旧金山大学动物护理和使用委员会的批准。

对于刮伤/上皮损伤

  1. 设置手术区域。
    1. 将异氟烷室放在显微镜附近,放置 麻醉平台与鼻锥下显微镜物镜。 通过鼻锥放置鼠标加热垫。
    2. 放置 Proparacaine,Algerbrush,weck-cels和环钻在附近的实验室长椅上 手术区。有20μl的1:40稀释荧光素溶液绘制  (见食谱)。
    3. 通过用酒精棉签擦拭清洁Algerbrush毛刺。
  2. 麻醉鼠标在异氟烷室。麻醉的典型方法包括将动物置于与氧源和异氟烷蒸发器连接的诱导室中,并将氧气流量调节至0.9升/分钟,将异氟烷蒸发器调节至1-2%。一旦小鼠变得无反应并且具有浅呼吸,它可以用鼻锥转移到麻醉平台并放置在加热垫上。定位鼠标头,使受伤的眼睛朝向显微镜物镜。
  3. 挤压一瓶Proparacaine,并在角膜上放1滴Proparacaine。等待30秒。
  4. 使用weck-cel干燥角膜,通过轻轻地扫过角膜一次,并涂抹眼睛的两个角落。
  5. 用一只手施加眼周压力以支撑小鼠眼睛。 (可选步骤:在受伤前拍摄角膜的明场照片。)
  6. 使用另一只手以尽可能轻轻的压力将环钻标记为中心的角膜。用拇指和第二根手指握住环钻的手柄,将整个圆形边缘放在角膜上。用温和的压力轻轻转动环钻约3小时,以标记角膜。避免产生多个标记。
  7. 打开Algerbrush,通过以圆形方式施加轻柔的压力并观察表面上皮细胞层的断裂,在角膜中心产生上皮缺损。小心地延长上皮缺损接近环钻标记。
  8. 关闭Algerbrush,使用Algerbrush毛刺的钝叶片轻轻地将剩余的角膜上皮移出到环钻标记。应用荧光素溶液到角膜,并使用显微镜GFP滤波器确认上皮缺损的大小。
  9. 可选步骤:应用眼周压力支撑鼠标眼睛。在应用荧光素溶液和使用GFP过滤器后,获取受伤角膜的荧光图片(图1)。


    图1.角膜刮伤/上皮损伤。用荧光素溶液染色的刮擦损伤后中央角膜的上皮缺损的照片。划痕伤口的边界以黄色显示。 [根据Chan等人(2013)的图2修改]

  10. 将1滴丙美卡因置于角膜上。
  11. 从鼻锥上删除鼠标,让鼠标在恢复笼中醒来。监测鼠标的疼痛和眼部感染。

化学伤害

  1. 设置手术区域。
    1. 将异氟烷室放在显微镜附近,放置 麻醉平台与鼻锥下显微镜物镜。
    2. 在手术区附近的实验室工作台上放置Proparacaine,滤纸,镊子,Weck-cels和NaOH。
    3. 有500微升PBS吸管随时可用。 (可选:在移液管中制备20μl荧光素溶液。)
    4. 使用耳塞准备2 mm滤纸盘。


      图2.过滤纸盘。 一个耳塞可用于制作均匀的2毫米滤纸盘。

    5. 将定时器设置为10秒和30秒。
  2. 麻醉鼠标在异氟烷室。 麻醉的典型方法包括将动物置于与氧源和异氟烷蒸发器连接的诱导室中,并将氧气流量调节至0.9L/min,将异氟烷蒸发器调节至1-2%。 一旦小鼠变得无反应并且具有浅呼吸,它可以用鼻锥转移到麻醉平台并放置在加热垫上。 定位鼠标头,使受伤的眼睛朝向显微镜物镜。
  3. 将1滴丙美卡因置于角膜上。 等待30秒。
  4. 使用weck-cel干燥角膜,通过轻轻地扫过角膜一次,并涂抹眼睛的两个角落。
  5. 使用镊子将滤纸浸入NaOH溶液中10秒钟。
  6. 用一只手施加眼周压力以支撑小鼠眼睛。 (可选步骤:在受伤前拍摄角膜的明场照片。)
  7. 另一方面,使用镊子将NaOH浸泡的滤纸应用于中心角膜正好30秒。 使用立体显微镜执行此步骤,以将滤纸精确地放置在尽可能的中心位置。
  8. 使用镊子从角膜中取出滤光片。
  9. 立即用500μlPBS冲洗眼睛以洗去残余的NaOH(应用PBS,用weck-cel干燥,施加PBS,用weck-cel干燥,重复直到所有500μlPBS已经使用)。
  10.  可选步骤:应用眼周压力支撑鼠标眼睛。 采取荧光图片的伤口角膜后应用荧光素溶液和使用GFP过滤器。


    图3.角膜化学损伤。用荧光素溶液染色的化学损伤后中央角膜的上皮缺损的照片[从Chan等人(2013)的图1改编) ]

  11. 将1滴丙美卡因置于角膜上。
  12. 从鼻锥上删除鼠标,让鼠标在恢复笼中醒来。 监测鼠标的疼痛和眼部感染。

笔记

  1. 当使用划痕损伤模型来比较小鼠之间的上皮修复率时,建议使用同窝仔和/或年龄匹配的小鼠。

食谱

  1. NaOH溶液
    在操作的同一天在1.5ml Eppendorf管中混合以下成分:50μl1N NaOH和450μl无菌H 2 O以获得500μl的总体积。
  2. 荧光素溶液(化学伤害可选)
    准备1:40稀释在1.5毫升Eppendorf管同一天的程序:2微升荧光素钠/肟酸盐酸盐眼科溶液和78微升无菌PBS获得总体积80微升。

致谢

这项工作得到国立卫生研究院(K08 EY018858和R01 EY002162到M.F.C.和R01 CA057621和P01 AI053194到Z.W.)的赠款支持。

参考文献

  1. Carlson,E.C.,Wang,I.J.,Liu,C.Y.,Brannan,P.,Kao,C.W.and Kao,W.W。(2003)。 角膜损伤后角膜细胞改变KSPG表达。 Mol Vis 9:615-623。
  2. Chan,M.F.,Li,J.,Bertrand,A.,Casbon,A.J.,Lin,J.H.,Maltseva,I.and Werb,Z。(2013)。 角膜损伤后基质金属蛋白酶-12的保护作用。细胞科学 126(Pt 17):3948-3960。
  3. Stepp,M.A.,Zieske,J.D.,Trinkaus-Randall,V.,Kyne,B.M.,Pal-Ghosh,S.,Tadvalkar,G.and Pajoohesh-Ganji,A。(2014)。 伤害角膜以了解其如何治疗。 Exp Eye Res < em> 121:178-193。

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引用:Chan, M. F. and Werb, Z. (2015). Animal Models of Corneal Injury. Bio-protocol 5(13): e1516. DOI: 10.21769/BioProtoc.1516.
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