视神经挤压对小鼠视网膜神经节细胞存活和再生的影响

Evan G. Cameron; Xin  Xia; Joana  Galvao; Masoumeh  Ashouri; Michael S. Kapiloff; Jeffrey L. Goldberg

doi:10.21769/BioProtoc.3559

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration

视神经挤压对小鼠视网膜神经节细胞存活和再生的影响

EC Evan G. Cameron ^* email

XX Xin Xia ^* email

JG Joana Galvao

MA Masoumeh Ashouri

MK Michael S. Kapiloff

JG Jeffrey L. Goldberg

(*contributed equally to this work) 发布: 2020年03月20日第10卷第6期 DOI: 10.21769/BioProtoc.3559 浏览次数: 8699

评审: Oneil G. BhalalaXiaoyu LiuAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Jul 2019

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

妊娠晚期短暂性产前缺氧的简化范式研究发育性低氧损伤的功能和结构结果

Elyse C. Gadra and Ana G. Cristancho

2022年10月05日 1562 阅读

条件性舔抑制：评估独立于小鼠运动活动的情境、线索和情境线索复合恐惧反应

Youcef Bouchekioua [...] Yu Ohmura

2022年12月05日 1013 阅读

利用金纳米团簇和组织清除方法进行原位微炎症检测

Fayrouz Naim [...] Masaaki Murakami

2023年04月05日 1560 阅读

Abstract

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.

Keywords: Glaucoma (青光眼)

Retinal ganglion cells (RGCs) (视网膜神经节细胞)

Degeneration (退化)

Neuroprotection (神经保护)

Regeneration (再生)

AAV2 (AAV2)

Background

Glaucoma is the leading cause of irreversible blindness worldwide characterized by the progressive degeneration and loss of retinal ganglion cells (RGCs), the central projecting neurons that form the optic nerve connecting the retina to the brain (Quigley, 2011; Tham et al., 2014). Glaucomatous RGC cell death is thought to be induced, in part, by an increase in intraocular pressure (IOP) and concomitant compression of RGC axons as they exit the retina through the optic nerve head (Quigley, 2011; Chang and Goldberg, 2012). Several models of glaucoma in rodents have been developed to study the cellular and molecular mechanisms that underlie RGC degeneration including optic nerve crush (ONC), intracameral injection of microbeads, and intravitreal injection of silicon oil (Sappington et al., 2010; Tang et al., 2011; Templeton and Geisert, 2012; Ito et al., 2016; Zhang et al., 2019). While both the microbead and silicon injection models recapitulate the increase in IOP and induce progressive RGC cell death associated with glaucoma, they are not conducive to study axon regeneration due to variability of the insult and incomplete degeneration of RGC axons. Alternatively, ONC has served as a useful preclinical model to study both neuronal survival and regeneration as it induces significant RGC death with little variability and severs all axons allowing confidence that any fibers found past the site of injury are regenerating, rather than spared. Adeno-associated virus serotype 2 (AAV2) specifically and efficiently transduces RGCs following intravitreal injection, making it the principal means to deliver genes (recombinant DNA, shRNA, etc.) into RGCs (Martin et al., 2002; Nickells et al., 2017). Indeed, we’ve reported the use of AAV2 to deliver therapeutic peptides and shRNA to promote RGC survival and axon regeneration following ONC injury (Moore et al., 2009; Apara et al., 2017; Galvao et al., 2018; Boczek et al., 2019). Here, we describe a comprehensive protocol detailing the use of AAV2 in combination with ONC as a means to study the mechanisms that promote neuroprotection and regeneration, and to identify and characterize candidate molecules with therapeutic potential for the treatment of glaucoma and other optic neuropathies.

Materials and Reagents

Falcon 3 ml transfer pipette (Corning, catalog number: 357524 )
6-inch Cotton Tipped Applicators (VWR, catalog number: 89031-270 )
VWR^® Microcentrifuge Tubes (VWR, catalog number: 87003-294 )
VWR^® Superfrost^® Plus Micro Slide (VWR, catalog number: 48311-703 )
Polycin^® Ophthalmic Ointment, USP (Perrigo, catalog number: NDC 0574-4021-35 )
Absorbent Bench Underpads (VWR, catalog number: 82020-845 )
Kimwipes (Fisher Scientific, catalog number: 06-666 )
Falcon^® 48-well clear flat bottom plate (Corning, catalog number: 353078 )
35 x 10 mm tissue culture dish (Corning, catalog number: 353001 )
SecureSeal^TM imaging spacer (Grace Bio-Labs, catalog number: 654006 )
22 x 22 mm (No. 1.5) micro coverslip (VWR, catalog number: 48366-227 )
Adult (> P21) male and female C57BL/6J mice (JAX, catalog number: 000664 )
Fluriso^TM Isofluorane, USP (VetOne, catalog number: NDC 13985-528-60 )
Ketamine (or similar as approved by your local regulations)
Xylazine (or similar as approved by your local regulations)
Buprenorphine HCl (or similar as approved by your local regulations)
Refresh Tears^® Lubricant Eye Drops (Allergan, catalog number: NDC 0023-0798-01 , or similar)
Proparacaine Hydrochloride Ophthalmic Solution, USP (Sandoz, catalog number: NDC 61314-016-01 )
Cholera Toxin Subunit B (CTB), Alexa Fluor^TM 555 Conjugate (Thermo Fisher, catalog number: C34776 )
10x Phosphate Buffered Saline (Thermo Fisher, catalog number: AM9624 )
Paraformaldehyde 16% Solution (Fisher Scientific, catalog number: 50-980-488 )
FocusClear^TM (CelExplorer, catalog number: FC-101 )
MountClear^TM (CelExplorer, catalog number: MC-301 )
Triton^TM X-100 (Sigma, catalog number: X100 )
Non-specific Goat serum (Thermo Fisher, catalog number: 16-210-064 )
Anti-RBPMS, Guinea Pig (Phospho Solutions, catalog number: 1832-RBPMS )
Anti-Guinea Pig-Alexa Fluor 647 (Thermo Fisher, catalog number: A21450 )
ProLong^® Gold Antifade Mountant (Thermo Fisher, catalog number: P36934 )
Flat mount blocking buffer (see Recipes)

Equipment

Surgical Microscope (WPI, catalog number: PSMB5N )
Laboratory Stereo Microscope (VWR, catalog number: 10836-004 )
Hot Bead Sterilizer (FST, catalog number: 1800050 )
Mouse Heating Pad (Stryker, catalog number: TP600/700 )
Tabletop Isoflurane Anesthesia System with Induction Box (Harvard Apparatus, catalog number: 72-6468 )
Dumont #5 Straight Forceps (FST, catalog number: 11251-10 )
Dumont #7 Curved Forceps (FST, catalog number: 11271-30 )
Dumont #N5 Forceps Cross Action Inox Thin Tips Size .05 x .01 Biologie Tips (Roboz Surgical, catalog number: RS-5020 )
Hamilton Syringe (Hamilton Company, 5 μl, 700 series, catalog number: 7634-01 )
Hamilton 33 gauge needle (Hamilton Company, catalog number: 7803-05 , 33 gauge, point 4, 0.375 inches)
Vannas Spring Scissors-2.5 mm Cutting Edge (FST, catalog number: 15000-08 )
Surgical Scissors (FST, catalog number: 91402-14 )
Zeiss 880 LSM Confocal Microscope (Zeiss)

Software

Fiji ImageJ (https://imagej.net/Fiji)
Adobe Photoshop (https://www.adobe.com/)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Cameron, E. G., Xia, X., Galvao, J., Ashouri, M., Kapiloff, M. S. and Goldberg, J. L. (2020). Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration. Bio-protocol 10(6): e3559. DOI: 10.21769/BioProtoc.3559.
Boczek, T., Cameron, E. G., Yu, W., Xia, X., Shah, S. H., Castillo Chabeco, B., Galvao, J., Nahmou, M., Li, J., Thakur, H., Goldberg, J. L. and Kapiloff, M. S. (2019). Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment. J Neurosci 39(28): 5466-5480.