Oct 21, 2022
Version 2
Intracardial perfusion for electrophysiology in rat V.2
- 1UCSD, Scripps
Protocol Citation: Alicia Avelar 2022. Intracardial perfusion for electrophysiology in rat. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb7ey3vpk/v2Version created by Alicia Avelar
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: August 20, 2021
Last Modified: October 21, 2022
Protocol Integer ID: 52561
Keywords: intracranial, perfusion, electrophysiology, rat,
Abstract
This protocol is to use for clearing blood from the brain and helping to maintain viable cells for electrophysiology experiments. No PFA or any tools that have had contact with PFA should be used. This intracranial perfusion can help maintain viability of neurons in older animals and those who have undergone treatments that damage brain cells (for example: chronic methamphetamine and Parkinson's disease like treatments).
Guidelines
The rat should be unable to feel pain, but still breathing so the neurons are as healthy and in a physiological condition close to when the animal was alive when you attempt to do electrophysiology experiments later using the brain slices.
Materials
anesthesia machine and isoflurane solution
forceps
sharp surgical scissors, I prefer a very small blade but use what works for you
tweezers
needles/dissection pins to secure limbs before surgery
ronguers
bone cutters
weigh spatula with long thin end to help gently remove brain from skull
4, 60 mL syringes for the artificial cerebral spinal fluid and high sucrose solution
4, 18 gauge needles
ice bucket with ice to keep solutions cold
container (beaker for example) to hold high sucrose solution and brain once removed from skull
razor in scraper handle or rat size guillotine for decapitation
styrofoam lid (concave) or dissection pan for placing rat during surgery
hood- location for anesthesia and surgery
biohazard disposal supplies (sharps box, rat body bag, and red biohazard bags and labels)
ethanol
paper towels
PPE (gloves, lab coat, even safety glasses might be a good idea)
scale if you want to weigh the animal
Safety warnings
Sharp surgical tools and needles, be careful and get fully trained in the surgery before attempting alone
Before start
Gather tools and set up the area and double check before beginning any surgical procedure.
Anesthesia
Anesthesia
anesthetize rat with isoflurane anesthesia machine.
when rat is lying down immobile, doesn’t right it’s position when moved, and is breathing slowly put isoflurane nose cone on rat and test for pain with forceps toe pinch of both feet
Surgery and intracranial perfusion
Surgery and intracranial perfusion
open rat thoracic cavity under sternum
hold sternum back with forceps
cut diaphragm
cut sides of rib cage
small cut to open right atria
inject into heart ventricle ~ 120 mL ice cold artificial cerebral spinal fluid, then ~ 120 mL ice cold high sucrose solution
Brain extraction
Brain extraction
decapitate rat
carefully remove skull
gently free brain from skull, cut cranial nerves when necessary
let brain gently release from skull into ice cold high sucrose solution
Cleanup
Cleanup
clean surgical tools carefully and let air dry
sharps disposed of in sharps biohazard red box
let blood dissolve in bleach in flask in hood to decontaminate
dispose of other biohazard material in a double bagged biohazard red bag .add sticker label. dispose of in biohazard waste bins.
clean surfaces in hood with ethanol and paper towels
bring rat body in body bag with labeled tag to BSB 099 cold room
bring cage and accessories with it to BSB dirty cage area for washing0 µL