May 23, 2023
Rodent intracerebroventricular AAV injections
- 1California Institute of Technology
Protocol Citation: Miguel Chuapoco 2023. Rodent intracerebroventricular AAV injections. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorm4dv4o/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 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 81664
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
NIH Pioneer
Grant ID: DP1NS111369
NIH BRAIN Armamentarium
Grant ID: UF1MH128336
Abstract
This abstract outlines the procedure for AAV injections into the lateral ventricle of C57BL/6J mice (intracerebroventricular injections)
Protocol materials
DBA/2J miceThe Jackson LaboratoryCatalog #000671
Step 1
C57BL/6JThe Jackson LaboratoryCatalog #000664
Step 1
BALB/cJThe Jackson LaboratoryCatalog #000651
Step 1
Rodent surgery
Rodent surgery
Raise and house mice in compliance with your local Institutional Animal Care and Use Committee (IACUC) and ensure that all protocols and procedures have been approved by the appropriate ethical and regulatory committees.
Obtain mice from The Jackson Laboratory (male, 6-8 weeks).
C57BL/6JThe Jackson LaboratoryCatalog #000664 BALB/cJThe Jackson LaboratoryCatalog #000651 DBA/2J miceThe Jackson LaboratoryCatalog #000671
Anesthetize mice using isoflurane (5% for induction, 1-3% for maintenance) with 95% O2/5% CO2 (1 L/min).
Head fix mice in a stereotaxic frame, shave head using clippers, and sterilize the area with chlorohexidine.
Administer 0.05 mL of 2.5 mg/mL bupivacaine subcutaneously above the skull.
Using a surgical blade, make an midline incision to reveal the skull. Clean the skull of blood and connective tissue and level the head in the stereotaxic frame.
Intraoperatively administer 1 mg/kg of buprenorphine SR and 5 mg/kg of ketoprofen via subcutaneous injection.
Drill 2 bilateral burr holes above the lateral ventricles using the following coordinates:
- Anterior-posterior: 0.6 mm posterior to bregma
- Medial-lateral: 1.15 mm from the midline
Using a 10 μL NanoFil syringe with a 33 guage beveled needle, aspirate viral vectors (5 x 1010 or 1.5 x 1011 vector genomes in 3-5 μL) using a microsyringe pump.
Equipment
SUB-MICROLITER INJECTION SYSTEM
NAME
NanoFil Syringe
TYPE
World Precision Instruments
BRAND
NANOFIL
SKU
LINK
Equipment
33 guage beveled NanoFil needle
NAME
NANOFIL NEEDLES
TYPE
World Precision Instruments
BRAND
NF33BV-2
SKU
LINK
Equipment
UMP3 ULTRAMICROPUMP
NAME
Microsyringe pump and controller
TYPE
World Precision Instruments
BRAND
UMP3-3
SKU
LINK
Slowly lower the needle through the burr hole into the lateral ventricle (1.6 mm from the pial surface) and allow the needle to sit in place for approximately 5 minute. Inject viral vector at a rate of 300 nL/min.
Allow needle to stay in place for 5 minutes after injection has been complete. Slowly remove needle, and suture.
For 5-days post-surgery, mice were treated with 30 mg/kg of ibuprofen and 60 mg/kg of trimethoprim/sulfamethoxazole via water.