Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

Podsumowanie

We present a murine model of brain death induction in order to evaluate the influence of its pathophysiological effects on organs as well as on consecutive grafts in the context of solid organ transplantation.

Streszczenie

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain death (BD) still represents the major source for solid transplants. Unfortunately, the irreversible loss of brain function is known to induce multiple pathophysiological changes, including hemodynamic as well as hormonal modifications, finally leading to a systemic inflammatory response. Models that allow a systematic investigation of these effects in vivo are scarce. We present a murine model of BD induction, which could aid investigations into the devastating effects of BD on allograft quality. After implementing intra-arterial blood pressure measurement via the common carotid artery and reliable ventilation via a tracheostomy, BD is induced by steadily increasing intracranial pressure using a balloon catheter. Four hours after BD induction, organs may be harvested for analysis or for further transplantation procedures. Our strategy enables the comprehensive analysis of donor BD in a murine model, therefore allowing an in-depth understanding of BD-related effects in solid organ transplantation and potentially paving the way to optimized organ preconditioning.

Wprowadzenie

Transplantation is currently the only curative treatment for end-stage organ failure. Until now, brain death (BD) patients have been the main source for organ donations, although living donation and donation after circulatory death are valuable alternatives¹. BD is defined by an irreversible coma (with a known cause), the absence of brain stem reflexes and apnea². Unfortunately, BD organs demonstrate inferior results in long-term graft survival independent of human leukocyte antigen (HLA)-mismatch and cold ischemic time³. Meanwhile, intensive research on this antigen-independent risk factor has been performed resulting in three main aspects of pathophysiological changes mediated as a consequence of BD: hemodynamic, hormonal, and inflammatory⁴.

To date, experimental BD models in rodents have been mostly performed using rats. In order to gain greater insight into the immunological consequences on solid organs following BD, we aimed to establish a murine model of BD, as currently only mouse models allow for comprehensive investigations into genetic or immunological factors. In this context, the mouse system provides a larger variety of analytical tools.

The principle of BD induction as described here is based on an increase in intracranial pressure induced by the inflation of a balloon catheter inserted under the skull. Increased intracranial pressure mimics the physiological mechanism of BD by blocking the perfusion of the cerebrum, cerebellum, and brain stem^5,6. To guarantee sufficient perfusion of peripheral organs, blood pressure measurement is obligatory during the procedure. The catheter used for this purpose at the same time serves for saline administration in order to stabilize the blood pressure by fluid substitution. As BD is accompanied by cessation of spontaneous breathing, sufficient ventilation must be ensured. An electric blanket maintains physiological core body temperature.

In summary, this model will enable in-depth studies into the influence of BD-induced injury, on leukocyte migration⁷, compliment activation⁸, ischemic reperfusion injury⁹, and other factors.

Access restricted. Please log in or start a trial to view this content.

Protokół

Animal experiments were performed in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). All experiments were approved by the Austrian Ministry of Education, Science and Culture (BMWF-66.011/0071-II/3b/2012).

1. Arterial catheterization

1. Anesthetize the mouse with an intraperitoneal injection of ketamine and xylazine¹⁰ 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 (see Discussion).
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 in 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) which 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 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 instable 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.

4. During BD period

1. Monitor and document blood pressure regularly. Exclude mice with a prolonged hypotensive phase (mean arterial pressure [MAP] < 50 mmHg for >30 min).
2. Infuse 0.1 mL of saline every 30 min to stabilize the blood pressure of the mouse.
   NOTE: In total 0.8 mL of saline was administered to each mouse in this study.
3. After 4 h of BD duration, harvest mouse organs/tissues. Exclude the mouse from the experiment if the heart of the mouse is not beating at the end of the experiment.

5. Sham procedure

1. Perform steps 1.1−3.3.
   NOTE: Do not open the inner compact bone. Do not insert or inflate a balloon catheter.
2. Stay close to the mouse and continuously observe the animal. Repeatedly pinch the hind limbs with forceps to confirm the correct depth of anesthesia. Apply additional anesthesia subcutaneously (approximately 1/2 of the starting dose) if the mouse shows signs of awakening, which, to our experience, happens approximately after 2−3 h post-anesthesia.  
3. Apply the same amount of intravenous saline as in the BD mice.
   NOTE: The sham mouse will regain spontaneous breathing after cessation of ventilation. The BD mouse will not. Apnea testing should be done while establishing the model, but during experiments it should be avoided due to unnecessary physiological stress.

Access restricted. Please log in or start a trial to view this content.

Wyniki

The murine BD model was successfully performed more than 100 times with a success rate of over 90%. Additionally, post interventional organ transplantation of heart and kidney has been safely performed⁷.

BD induces a variety of pathophysiological changes that may be further investigated using this model. As shown in Figure 1, the blood pressure shows an initial hypertensive p...

Access restricted. Please log in or start a trial to view this content.

Dyskusje

BD, a risk factor for allograft quality in multi-organ donors, entails a plethora of pathophysiological changes, which can only be sufficiently assessed using in vivo models. Hemodynamic changes, cytokine storm, hormonal changes and their ultimate impact on organ graft quality and survival cannot be analyzed in vitro⁴. The majority of basic transplantation as well as immunological research is dependent on sophisticated diagnostic tools, which are widely available only in mice models. Mice models h...

Access restricted. Please log in or start a trial to view this content.

Materiały

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