Inducing Brain Death in a Mouse Model Using a Balloon Catheter

Protocolo

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

1. Arterial catheterization

1. Anesthetize the mouse with an intraperitoneal injection of ketamine and xylazine and an analgesic according to local practice (e.g., buprenorphine). Pinch the hind limbs with forceps to confirm the correct depth of anesthesia.
   NOTE: For this study, male C57BL/6N mice at the age of 8−12 weeks were used (weight 20−25 g). The authors have tested male BALB/c of the same age unsuccessfully but have not tried other strains.
2. Remove the hair in the regions of interest (head, neck) using an electric razor. Ensure that no loose hair remains to prevent wound contamination. Subsequently, disinfect the surgical field with 70% ethanol/chlorhexidine/ betadine and place the mouse supine with the head facing the surgeon.
3. Make a cervical midline incision with dissecting scissors.
4. Dissect the submandibular glands and neck muscle tissue and separate them in order to expose the common carotid artery. Use mostly blunt dissection via forceps.
5. Place three 8-0 silk ligatures beneath the right common carotid artery.
6. Place a clamp on the proximal ligature and bring tension into the artery so that the flow is suspended.
7. Close the most distal ligature.
8. Insert the arterial catheter through a small, preformed skin hole on the cranial aspect of the incision. Squeeze and deform the lumen of the catheter if it appears too large to reduce blood backflow. Fixate with all three ligatures.
9. Fixate the catheter to the skin to avoid dislocation. Do this by using a suture (e.g., 5-0 monofilament, non-absorbable) that connects the catheter to the skin in the area of the preformed skin hole.

2. Tracheostomy

1. Dissect the pre-tracheal musculature bluntly using forceps.
2. Place two 8-0 silk ligatures beneath the trachea.
3. Tracheotomize using micro scissors as proximally as possible to avoid unilateral ventilation. Use a horizontal cutting line between two tracheal cartilages.
4. Insert the ventilation tube and fixate with both prepared ligatures.
   NOTE: The proximal end of the trachea does not need to be ligated.
5. Close the skin with a running suture (e.g., 6-0 monofilament, non-absorbable).
6. Ventilate the mouse with a frequency of 150/min and a tidal volume of 200 µL.

3. Brain death (BD) induction

1. Arrange the mouse to the prone position.
2. Remove the skin from the skull using surgical scissors and forceps to hold the skin.
3. Drill a 1 mm caliber borehole paramedially above the left parietal cortex. Stop drilling before breaching the inner compact bone and the dura mater.
4. Penetrate the final tissue bridge of the skull using blunt forceps, removing sharp edges.
5. Insert the balloon catheter, so that it is entirely within the cranial cavity. Ensure that the balloon is prefilled with saline and all air is evacuated.
6. Begin inflation at ~0.1 mL/min over a period of 10−15 min (total volume of 0.8−1.2 mL) with the help of a syringe pump.
   NOTE: The mouse will exhibit myoclonus, mydriasis, further seizure activity, and agonal gasps.
7. Pronounce the mouse brain dead once the tail of the mouse has gone stiff and erected.
   NOTE: BD is confirmed by a characteristic initial blood pressure peak (Cushing reflex), the absence of brain stem reflexes, and spontaneous breathing. Regular apnea testing should be avoided during experiments, as mice may become circulatory unstable due to a lack of oxygen.
8. Stop the inflation of the balloon catheter.
9. Put a heating blanket over the mouse to avoid hypothermia.

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Materiales

Name	Company	Catalog Number	Comments
Arterial catheter (BD Neoflon 26G)	BD	391349
Blood Pressure Transducers (APT300)	Harvard Apparatus Inc.	73-3862
Fogarty Arterial Embolectomy Catheter N° 3	Edwards Lifesciences Corporation	120403F
Forceps	FST	11271-30
Homeothermic Blanket Systems with Flexible Probe	Harvard Apparatus Inc.	55-7020
Ketansol	Graeub	6680110
Micro scissor	FST	15018-10
Needle holder	FST	12060-02
Prolene 5-0	Ethicon	8698H
Pump 11 Elite Infusion Only Single	Harvard Apparatus Inc.	70-4500
Scissor	FST	14075-11
Stereotactic microscope	Olympus	SZX7
Transpore Tape	3M	1527-1
Underpads	Molinea.A	274301
Ventilator for mice (MiniVent Model 845)	Harvard Apparatus Inc.	73-0043
Xylasol	Graeub	7630109