Intravitreal Injection of Adeno-Associated Virus to Assess Retinal Barrier Disruption in Mice

要約

Source: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015)

This video demonstrates the intravitreal injection of an adeno-associated viral vector into a mouse model with a compromised inner limiting membrane. The resulting fluorescent protein expression reveals the extent of retinal barrier disruption based on viral penetration into deeper retinal layers.

プロトコル

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intravitreal Injection of Adeno-Associated Virus (AAV)

1. Anesthetize C57BL6J mice with ketamine (50 mg/kg) and xylazine (10 mg/kg) and verify the loss of righting reflex, the withdrawal reflex, and tail pinch response, indicating a proper anesthetization. Dilate pupils by corneal application of neosynephrine 5% and mydriaticum 0.5% eye drops. Use vet ointment on the eyes to prevent dryness while under anesthesia.
2. Pass an ultrafine 30 G disposable needle through the equator and next to the limbus, into the vitreous cavity. Inject 1 µL stock containing 1 to 4 x 10¹¹ viral particles of ShH10-GFP or AAV5-GFP with direct observation of the needle in the center of the vitreous cavity (Figure 1). Use one eye as a control for Inner Limiting Membrane experiments. Inject both eyes for BRB (Blood-Retinal Barrier) experiments. Apply an anti-inflammatory and antibacterial topical treatment to the injected eyes.
3. Dilate pupils with neosynephrine and mydriaticum eye drops for imaging. Perform fundus examinations with an eye fundus camera to follow GFP (Green Fluorescent Protein) expression 1 week, 2 weeks, 1 month, and 2 months after intravitreal injection (Figure 1). Sacrifice mice by CO₂ inhalation 1 to 2 months post-injection.

結果

Figure 1: Schematic representation of the general protocol. After AAV production, particles are injected into the vitreous or into the penile vein of anesthetized adult mice. Fundus images with the Micron III camera are obtained to monitor GFP expression. GFP expression is analyzed in retinal cryosections and flat-mounted retinas using immunohistochemistry and confocal microscopy. The AAV passage through the BRB is assessed by PCR analysis on blood samples coming from intravitreally or intrapenally injected mice, in order to detect the presence of the GFP sequence, an indicator of the AAV particles presence into the bloodstream.

材料

この記事で使用された材料の一覧

名前	会社	カタログ番号	コメント
C57BL6J mice strain	JANVIER LABS		mice
Ketamine 500	Virbac France		anesthetic
Xylazine Rompun 2%	Bayer Healthcare		anesthetic
Neosynephrine 5% Faure	Europhta		dilatant
Mydriaticum 0.5%	Thea		dilatant
Sterdex	Novartis		anti-inflammatory
Micron III	Phoenix Research Labs		Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.
Insulin Syringes	Terumo	SS30M3109
Syringe 10 µl Hamilton	Dutscher	74487	Seringue 1701
Needle RN G33, 25 mm, PST 2	Fisher Scientific	11530332	Intravitreal Injection
UltraMicroPump UMP3	World Precision Instruments	UMP3	Versatile injector uses microsyringes to deliver picoliter volumes
UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller	UMP3-1	Digital controller