May 10, 2024

Public workspaceAAV Craniotomy

DOI
dx.doi.org/10.17504/protocols.io.5jyl829q7l2w/v1
  • 1Weill Cornell Medical College
Eileen Ruth Torres
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl829q7l2w/v1
Protocol Citation: Santiago Unda, Roberta Marongiu, Mihaela Stavarache, Michael G. Kaplitt 2024. AAV Craniotomy. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl829q7l2w/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: May 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 99452
Keywords: ASAPCRN, surgery, mouse, AAV
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
This protocol is for injection of AAV into a mouse brain using a stereotactic frame and Hamilton syringe.
Guidelines
Note that sterile technique must be followed during this procedure to avoid health complications.
Anesthesia
Anesthesia
Weigh mice.
Administer intraperitoneal mixture of ketamine (ButlerAnimal Health Supply) and xylazine (Lloyd Laboratories) at concentrations of 110 and 10 mg/kg of body weight, respectively.
Confirm induction of anesthesia.
Injection
Injection
Place mouse into stereotactic frame (David Kopf Instruments) and place eye lubricant.
Remove fur from skull area using either electric shaver or depilatory cream.
Sterilize skull area and then cut open with scalpel.
Adjust brain so that it is level. Locate bregma and zero X and Y axes on stereotaxic frame.
Move to injection coordinates and drill small hole into the skull.
Zero the Z axis on the stereotaxic frame.
Prepare AAV solution by diluting AAV in PBS with 500mM Mg2/Cl to a final dilution of total of 2x1011 genomic particles/mL.
Using a 10 μL stereotactic syringe with a 33 G needle attached to a micro-infusion pump (World Precision Instruments), withdraw the AAV solution.
With the loaded syringe, navigate the needle to the desired X, Y, Z coordinates.
Infuse the brain with AAV at a flow rate of 0.1 - 0.4 μL/min.
To prevent reflux, after each infusion, leave the injection needle in place for 5 min, withdraw a short distance (0.3 - 0.5 mm), and then leave in the new position for an additional 2 min before complete removal of the needle.
Close skin with non-absorbable sutures.
AAV Preparation
AAV Preparation
Inject a total of 2x1011 genomic particles/mL of vector is injected into the appropriate coordinates.
Post-procedure
Post-procedure
Give meloxicam 2mg/kg subcutaneously.
Perform health checks every day for the first 72 hrs post-injection.
Remove sutures 15 days post-injection.