Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立
Summary
慢性眼高压是在大鼠和小鼠中应用 circumlimbal 缝合, 导致视网膜神经节细胞与青光眼的功能和结构恶化。
Abstract
circumlimbal 缝合是一种以慢性抬高眼压 (眼压) 诱发啮齿动物实验性青光眼的技术, 是一种众所周知的青光眼危险因素。该协议说明了该技术在长埃文斯大鼠和 C57BL/6 小鼠的分步指导。在全身麻醉下, 在结膜、赤道周围和眼睛的后缘上应用 “钱包绳” 缝合。他的眼睛是一个未经处理的控制。在我们的研究期间, 这是一个8周的老鼠和12周的老鼠, 眼压保持升高, 在有意识的动物的反弹测量定期测量没有局部麻醉。在这两种类型中, 缝合眼睛显示视网膜电图的特点与优先内视网膜功能障碍一致。光学相干断层扫描显示视网膜神经纤维层有选择性细化。组织学的大鼠视网膜在横断面发现减少细胞密度在神经节细胞层, 但没有变化的其他细胞层。用神经节细胞特异标记 (RBPMS) 检测平板小鼠视网膜的染色, 证实神经节细胞丢失。circumlimbal 缝合术是一种简单、微创、经济有效的诱发眼内高压的方法, 可导致大鼠和小鼠神经节细胞损伤。
Introduction
动物模型为实验研究青光眼发病机制的细胞过程提供了一个重要的平台, 并评估潜在的治疗干预措施。几种诱导模型的建立, 以产生持续眼压 (眼压) 升高, 最重要的危险因素, 青光眼。用于提高眼压的方法包括: 高渗盐水注射液在巩膜静脉1, 激光光凝的小梁网2或角膜缘静脉3, 和眼内注射液等物质鬼血红细胞4, 微球5,6和粘弹性剂7。每种方法都有其优点和局限性。
一个好的青光眼模型应该模仿疾病的过程, 以最小的并发症, 如创伤, 炎症和媒体混浊。这些并发症经常与用于诱发眼压升高的程序有关, 并会混淆结局的解释。例如, 即使在未引入异物的情况下, 前房穿刺也已被证明是造成外伤和炎症的原因, 而不是典型的青光眼改变8、9的代表性。除了避免炎症的重要性外, 保持光学清晰度有助于体内成像和电生理监测疾病进展。虽然目前尚不清楚这些并发症会对疾病调查有多大影响, 但在模型诱导过程中, 最好避免穿透眼球。circumlimbal 缝合方法避免了地球的穿透, 促进了体内视网膜结构和功能的纵向评估。更重要的是, 这个模型不同于以往的能力, 以返回眼压的基线值, 在必要时去除缝合。眼压正常化可能有助于研究可逆和不可逆转的神经节细胞损伤10,11,12,13,14的细胞和分子相关性。
本文着重介绍了模型归纳技术。该模型诱导的大鼠和小鼠视网膜损伤的特征可以在其他15、16、17、18、19中更详细地找到。
Protocol
所有实验程序都是根据澳大利亚国家卫生和医学研究理事会制定的《澳大利亚保护和使用动物行为守则》进行的。伦理学的批准是从霍华德 Florey 研究所动物伦理委员会 (批准号 13-044-um 和 13-068-um 为老鼠和老鼠, 分别)。 1. 有意识大鼠的眼压测量 设置实验室回弹血压计到大鼠的设置。襁褓的老鼠在一块柔软的布里使动物平静下来。暴露头部和颈部。轻轻地握住躯干, 一?…
Representative Results
以下结果在老鼠18和老鼠16以前被报告了并且这里总结。circumlimbal 缝合在大鼠和小鼠中产生了类似的眼压升高模式 (图 2)。缝合术后立即发现一只小鼠眼压峰值, 高达 58.1 2.7 mmHg, 38.7, 2.2 mmHg。在大鼠中, 随着时间的推移, 眼压量逐渐减少 44, 为 6 mmHg 和 32, 2 毫米汞, 3 和24小时, 分别为15。在这最初的眼…
Discussion
circumlimbal 缝合是慢性眼高压症的新模型。除了有代表性的研究结果来源于16、18, 这一动物模型在最近的一些研究中被利用了15、23、24、25 ,26。以上这些报告的比较表明, 该方法产生可重复的结果, 包括眼压升高的幅度, 以及在模型?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由澳大利亚国家卫生和医学研究理事会 (1046203)、澳大利亚研究理事会未来的奖学金 (FT130100338) 资助。
Materials
| normal saline | Baxter International Inc | AHB1323 | Maintain corneal hydration during surgery |
| Chlorhexadine 0.5% | Orion Laboratories | 27411, 80085 | Disinfection of surgical instrument |
| Isoflurane 99.9% | Abbott Australasia Pty Ltd | CAS 26675-46-7 | Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for suture procedure |
| ocular lubricant | Alcon Laboratories | 1618611 | Proprietory Name: Genteal, ocular lubricant to keep the other eye moist |
| Needle holder (microsurgery) | World Precision Instruments | 555419NT | To hold needle during ocular surgery |
| Proxymetacaine 0.5% | Alcon Laboratories | CAS 5875-06-9 | Topical ocular analgesia |
| Scissors (microsurgery) | World Precision Instruments | 501232 | To cut excessive suture stump during ligation |
| Surgical drape | Vital Medical Supplies | GM29-612EE | Ensure sterile enviornment during surgery |
| Suture needle for rats (microsurgery) | Ninbo medical needles | 151109 | 8-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm |
| Suture needle for mice (microsurgery) | Ninbo medical needles | 160905 | 10-0 nylon suture attached with round needle, cutting edge 3/8, dual-needle, suture length 30cm |
| Tweezers (microsurgery) | World Precision Instruments | 500342 | Manipulate tissues during ocular surgery |
| rebound tonometer | TONOLAB, iCare, Helsinki, Finland | TV02 | for intraocular pressure monitoring |
Referências
- Morrison, J. C., et al. A rat model of chronic pressure-induced optic nerve damage. Experimental Eye Research. 64 (1), 85-96 (1997).
- Feng, L., Chen, H., Suyeoka, G., Liu, X. A laser-induced mouse model of chronic ocular hypertension to characterize visual defects. Journal of Visualized Experiments. (78), (2013).
- Chiu, K., Chang, R., So, K. F. Laser-induced chronic ocular hypertension model on SD rats. Journal of Visualized Experiments. (10), 549 (2007).
- Quigley, H. A., Addicks, E. M. Chronic experimental glaucoma in primates. I. Production of elevated intraocular pressure by anterior chamber injection of autologous ghost red blood cells. Investigative Ophthalmology & Visual Science. 19 (2), 126-136 (1980).
- Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. Journal of Visualized Experiments. (96), (2015).
- Weber, A. J., Zelenak, D. Experimental glaucoma in the primate induced by latex microspheres. Journal of Neuroscience Methods. 111 (1), 39-48 (2001).
- Moreno, M. C., et al. A new experimental model of glaucoma in rats through intracameral injections of hyaluronic acid. Experimental Eye Research. 81 (1), 71-80 (2005).
- Hoyng, P. F., Verbey, N., Thorig, L., van Haeringen, N. J. Topical prostaglandins inhibit trauma-induced inflammation in the rabbit eye. Investigative Ophthalmology & Visual Science. 27 (8), 1217-1225 (1986).
- Kezic, J. M., Chrysostomou, V., Trounce, I. A., McMenamin, P. G., Crowston, J. G. Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse. Investigative Ophthalmology & Visual Science. 54 (4), 3028-3036 (2013).
- Waisbourd, M., et al. Reversible structural and functional changes after intraocular pressure reduction in patients with glaucoma. Graefe’s Archive for Clinical and Experimental Ophthalmology. 254 (6), 1159-1166 (2016).
- Foulsham, W. S., Fu, L., Tatham, A. J. Visual improvement following glaucoma surgery: a case report. BMC Ophthalmology. 14, 162 (2014).
- Anderson, A. J., Stainer, M. J. A control experiment for studies that show improved visual sensitivity with intraocular pressure lowering in glaucoma. Ophthalmology. 121 (10), 2028-2032 (2014).
- Ventura, L. M., Feuer, W. J., Porciatti, V. Progressive loss of retinal ganglion cell function is hindered with IOP-lowering treatment in early glaucoma. Investigative Ophthalmology & Visual Science. 53 (2), 659-663 (2012).
- Zhao, D., et al. ARVO abstract number 3696 – B0043. annual meeting of Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA. , (2018).
- Liu, H. H., et al. Chronic ocular hypertension induced by circumlimbal suture in rats. Investigative Ophthalmology & Visual Science. 56 (5), 2811-2820 (2015).
- Zhao, D., et al. Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels. Frontiers in Neuroscience. 11, 41 (2017).
- Nguyen, C. T., et al. Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats. Journal of Visualized Experiments. (113), (2016).
- Van Koeverden, A. K., He, Z., Nguyen, C. T., Vingrys, A. J., Bui, B. V. Systemic hypertension is not protective against chronic IOP elevation in a rodent model. Scientific Reports. 8 (1), 7107 (2018).
- Rodriguez, A. R., de Sevilla Muller, L. P., Brecha, N. C. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina. Journal of Comparative Neurology. 522 (6), 1411-1443 (2014).
- Aihara, M., Lindsey, J. D., Weinreb, R. N. Twenty-four-hour pattern of mouse intraocular pressure. Exp Eye Research. 77 (6), 681-686 (2003).
- Jia, L., Cepurna, W. O., Johnson, E. C., Morrison, J. C. Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats. Investigative Ophthalmology & Visual Science. 41 (6), 1380-1385 (2000).
- Nadal-Nicolas, F. M., Jimenez-Lopez, M., Sobrado-Calvo, P., Nieto-Lopez, L., Canovas-Martinez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Investigative Ophthalmology & Visual Science. 50 (8), 3860-3868 (2009).
- Liu, H. H., Flanagan, J. G. A Mouse Model of Chronic Ocular Hypertension Induced by Circumlimbal Suture. Investigative Ophthalmology & Visual Science. 58 (1), 353-361 (2017).
- Liu, H. H., He, Z., Nguyen, C. T., Vingrys, A. J., Bui, B. V. Reversal of functional loss in a rat model of chronic intraocular pressure elevation. Ophthalmic & Physiological Optics. 37 (1), 71-81 (2017).
- Liu, H. H., Zhang, L., Shi, M., Chen, L., Flanagan, J. G. Comparison of laser and circumlimbal suture induced elevation of intraocular pressure in albino CD-1 mice. PLoS One. 12 (11), 0189094 (2017).
- Shen, H. H., et al. Intraocular Pressure Induced Retinal Changes Identified Using Synchrotron Infrared Microscopy. PLoS One. 11 (10), 0164035 (2016).
Citar este artigo
He, Z., Zhao, D., van Koeverden, A. K., Nguyen, C. T., Lim, J. K. H., Wong, V. H. Y., Vingrys, A. J., Bui, B. V. A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice. J. Vis. Exp. (140), e58287, doi:10.3791/58287 (2018).