Jul 12, 2022
Unilateral intranigral AAV alpha synuclein mouse model
- Pranay Srivastava1,
- Waijiao Cai2,
- Xiqun Chen1
- 1Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase MD 20815 USA;
- 2Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA, Department of Neurology Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200032 China
Protocol Citation: Pranay Srivastava, Waijiao Cai, Xiqun Chen 2022. Unilateral intranigral AAV alpha synuclein mouse model. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzp5xv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58411
Keywords: ASAPCRN
Abstract
This protocol details methods for the unilateral intranigral AAV alpha-synuclein injection
Materials
Virus and reagents:
1. AAV alpha-synuclein, dilute the stock to 7.8 × 1012 gc/ml in sterile PBS. Viruses can be kept at 4C for 2 weeks and -80C for avery long time. Avoid freeze-thaw from -80C.
2 Avertin-HCl (2% 2,2,2-tribromoethanol and 1% amyl alcohol, at 4C, shielded from light), 0.15 ml/10 g body weight.
MOUSE SN injection
From ƛAP +0.09 cmML +0.12 cm DV -0.44 cm
| Mouse# | AP +0.09 | ML +0.12 | DV -0.44 | ∆breg/ƛ | Note | |
Clean all equipment and surgical tools with 70% ethanol.
Turn on the heating pad and cover it with a paper towel.
Clean and prepare injector needle.
Rinse 3x with 70% ethanol.
Rinse 3x with saline.
Animals: adult mice.
Coordinators: from lambda AP +0.09 cm, ML+0.12 cm, DV -0.44 cm
Injection rate: 12 µl/hour, total 2 µl in 10 min.
Anesthesia: IP animals with Avertin solution
15 ml/10 g body weight. After approximately 3-5 minutes animal should be completely anesthetized. Verify this by observing no response to tail pinch.
Fill the glass micropipette needle with virus solution (20 µl) by drawing the syringe plunger slowly. You can see a line between mineral oil and the solution. Adjust the rate to 12 µl/hour, volume to 2 µl. Pre-run the micro pump until you see a drop of liquid in the needle tip. Resume the volume to 2 µl.
Make a midline scalp incision and identify both the bregma and lambda coronal sutures. Place animal on platform in prone position and place head in ear bars (ear bars should be aligned with the area just anterior to external ear). Place snout in incisor bar and tighten loosely. Equilibrate bregma and lambda by measuring height of each using the stereotaxic arm to lower cannula until it contacts the exposed skull surface. A difference of less than 0.02 cm is acceptable. Adjust by raising or lowering snout height using incisor bar (be careful not to tighten snout holder since it will force wrong head angle).
From lambda dial in the following coordinates: AP: +0.09 cm, ML: +0.12 cm, lower needle tip to exposed dura and dial in: DV: -0.44 cm.
Infuse AAV alpha synuclein, at a rate of 12 µl/hour for 10 min (total 2 µL). Wait 2 minute and slowly withdraw needle 1 minutes after the end of the infusion
Suture scalp and place animal in incubator for recovery from anesthesia.