Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Abstract

Source: Kim, M., et al. Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury. J. Vis. Exp. (2017).

The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.    

1. Mouse Model of Neonatal HI Brain Injury

1. Anesthetize the pups with isoflurane.
   1. Place the pups (less than 5) into an anesthetizing box and close the lid.
   2. Turn on the anesthetizing system for approximately 15 min; adjust the gas and isoflurane using a tabletop anesthesia machine. Adjust the oxygen flowmeter to 1.5 L/min. Adjust the isoflurane vaporizer to 3-5% for the induction of anesthesia.
   3. After 15 min, adjust the isoflurane vaporizer to 1-2% for the maintenance of anesthesia.
2. Lay a fully anesthetized pup under a dissection microscope (abdomen facing the researcher) and secure it with tape.
3. Make a ~0.7-mm incision in the neck using sterilized scissors.
4. Carefully remove the adipose tissue using sterilized forceps and expose the unilateral right carotid artery.
5. Ligate the unilateral right carotid artery with a 5-0 absorbable suture.
6. Suture the incision in the neck with 5-0 suture.
7. Place each pup in a 37 °C warm hypoxic chamber for 1 h for recovery. Do not close the chamber lid.
8. 1 h after the surgery, when the pups are fully awake, close the hypoxia chamber lid and decrease the gas levels to establish hypoxic conditions (8% O₂ and 92% N₂).
9. After 90 min of hypoxia, return the pups to their cages.
10. One week after the HI brain injury, repeat step 1.
    1. After the anesthesia, make an incision in the scalp with sterilized scissors and forceps to identify the brain lesion in the posterolateral area of the right hemisphere.
       Note: This treatment induces hypoxia in pups. The presence and extent of brain injury in all mice is visually assessed with the naked eye through the semi-transparent skull. As determined by the size or volume of the discoloration (i.e., the brain lesion), pups are classified into groups. If there is no visible cortical injury, the mouse is classified into the "no cortical injury" group. If there is a visible cortical injury (i.e., a lesion in the right hemisphere), the mouse is classified into the "cortical injury" group. Since classification of the mice into groups is done one week after the operation, the groupings can be modified when the morphologies of the brain samples are clearly defined at the time of sacrifice.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hypoxic chamber	Jeung Do Bio & Plant Co	Experimental Builder
Harvard Apparatus Fluovac anesthetizing system	Harvard Apparatus
Anesthetizing box			acryl box
I-Fran Liquid (Isofluorane)	Hana Pharm. Co., Ltd.		General Anesthetics ( isoflurane 100ml)
CD-1 mice	Orient Co., Ltd.
Blue Nylon Mono Non-Absorbbable suture 5-0 50cm	Ailee Co., Ltd.	NB 521