Intracardial perfusion for electrophysiology in rat

Disclaimer

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).

Before start

Gather tools and set up the area and double check before beginning any surgical procedure.

Steps

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

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

decapitate rat

10.

carefully remove skull

11.

gently free brain from skull, cut cranial nerves when necessary

12.

let brain gently release from skull into ice cold high sucrose solution

Cleanup

13.

clean surgical tools carefully and let air dry

14.

sharps disposed of in sharps biohazard red box

15.

let blood dissolve in bleach in flask in hood to decontaminate

16.

dispose of other biohazard material in a double bagged biohazard red bag .add sticker label. dispose of in biohazard waste bins.

17.

clean surfaces in hood with ethanol and paper towels

18.

bring rat body in body bag with labeled tag to BSB 099 cold room

19.

bring cage and accessories with it to BSB dirty cage area for washingµL