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.
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.
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 stem5,6. 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.
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
- 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).
- 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.
- Make a cervical midline incision with dissecting scissors.
- 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.
- Place three 8-0 silk ligatures beneath the right common carotid artery.
- Place a clamp on the proximal ligature and bring tension in the artery so that the flow is suspended.
- Close the most distal ligature.
- 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.
- 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.
- Dissect the pre-tracheal musculature bluntly using forceps.
- Place two 8-0 silk ligatures beneath the trachea.
- Tracheotomize using micro scissors as proximally as possible to avoid unilateral ventilation. Use a horizontal cutting line between two tracheal cartilages.
- Insert the ventilation tube and fixate with both prepared ligatures.
NOTE: The proximal end of the trachea does not need to be ligated.
- Close the skin with a running suture (e.g., 6-0 monofilament, non-absorbable).
- Ventilate the mouse with a frequency of 150/min and a tidal volume of 200 µL.
3. Brain death induction
- Arrange the mouse to the prone position.
- Remove the skin from the skull using surgical scissors and forceps to hold the skin.
- Drill a 1 mm caliber borehole paramedially above the left parietal cortex. Stop drilling before breaching the inner compact bone and the dura mater.
- Penetrate the final tissue bridge of the skull using blunt forceps, removing sharp edges.
- 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.
- 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.
- 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.
- Stop the inflation of the balloon catheter.
- Put a heating blanket over the mouse to avoid hypothermia.
4. During BD period
- Monitor and document blood pressure regularly. Exclude mice with a prolonged hypotensive phase (mean arterial pressure [MAP] < 50 mmHg for >30 min).
- 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.
- 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
- Perform steps 1.1−3.3.
NOTE: Do not open the inner compact bone. Do not insert or inflate a balloon catheter.
- 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.
- 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.
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 peak followed by a prolonged hypotensive phase. To avoid detrimental physiological effects of hypotension, mice with a prolonged insufficient blood pressure (MAP < 50 mmHg for more than 30 min) were excluded.
Another well-established observation is that BD induction leads to the activation of the immune system. After 4 h of BD duration organ-specific upregulation of immune markers at the mRNA level (Figure 2) as well as immune cell migration was observed7.
Figure 1: Invasive measurement of mean arterial pressure (MAP) following BD induction or sham procedure. At the time point of BD induction, blood pressure was significantly higher in BD animals (white squares) than in sham animals (black squares) which is explained via the well described cytokine storm; thereafter, normalization of blood pressure levels became evident followed by a period of declining pressure levels starting after 90 min of BD induction (n = 19 animals/group ± SD). Statistically significant differences were tested with two-way ANOVA and the Bonferroni post-test; **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
Figure 2: BD induction activates the immune system and causes direct organ damage prior to transplantation. We show representative results of the natural cytotoxicity triggering receptor 1 (NCR1 also NK-p46), which increases in kidneys as a consequence of BD at the mRNA level. No changes were observed in livers or hearts. Data are presented as mean ± SE of n = 7−8 animals/group. Statistically significant differences between BD and sham were tested using the Mann-Whitney U-test; **p = 0.0037. Please click here to view a larger version of this figure.
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 have already led to a variety of new insights in BD-related research7,8,9.
Whereas in the 20th century the first models for BD research were developed in baboons, dogs and pigs11,12, it was not until 1996 when the first rat model was described13. It took a further 14 years until a murine model of BD was first published by the Vienna group in 20106. Although each individual step is not challenging for a trained microsurgeon, the model described here remains fragile and may take some weeks to establish.
Critical steps of this protocol are as follows. Use sterile disposable items (especially the arterial catheter and connecting tubes) to avoid unwanted immune stimulation. Avoid any blood loss as hypotensive phase may become uncontrollable. Use small cannulas for blood pressure measurement or reduce the lumen to avoid blood reflux. Fix the ventilation tube as well as the arterial cannula to avoid dislocation when turning the mouse or during seizure activity. The borehole must not be sharp-edged and should not be too large. The balloon catheter should be in contact the borehole. Otherwise brain tissue may protrude and hinder the intended elevation in intra-cranial pressure. During the BD-period, administer intravenous fluid steadily. If mice become hypotensive too early in the process (MAP < 50 mmHg after 2−3 h), additional fluid resuscitation is unlikely to provide sustained maintenance of blood pressure.
Most problems occurred during BD induction via the balloon catheter itself or became obvious over the course of the BD period. The common endpoint of all inaccuracies will be hypotension despite fluid administration. Depending on the blood pressure stability, BD-duration can be prolonged or shortened. In the literature, varying periods ranging from 3−6 h have been implemented6,7,8. The success of BD induction can also vary between different mouse strains. Although anatomical variants between mice strains should be limited, we were not able to establish the same model in BALB/c mice. Although the effort was limited to only few mice and no other strains were tested, we recommend the use of C57BL/6, which has been used in most previous research8,9. The authors have no satisfactory explanation why the model did not work in BALB/c strain. Only one publication has so far demonstrated a BD model using large (35 g) female OF-1 mice6.
In summary, the mouse model described here represents a valuable tool for studying a multitude of pathophysiological changes caused by BD. Though challenging, the model can be optimized within a reasonable timeframe and performed in a standardized manner with a high success rate.
The authors have nothing to disclose.
|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|
|Homeothermic Blanket Systems with Flexible Probe||Harvard Apparatus Inc.||55-7020|
|Pump 11 Elite Infusion Only Single||Harvard Apparatus Inc.||70-4500|
|Ventilator for mice (MiniVent Model 845)||Harvard Apparatus Inc.||73-0043|
- Hart, A., et al. OPTN/SRTR 2017 Annual Data Report: Kidney. American Journal of Transplantation. 19, (Suppl 2), 19 (2019).
- The Quality Standards Subcommittee of the American Academy of Neurology. Practice parameters for determining brain death in adults (summary statement). Neurology. 45, (5), 1012-1014 (1995).
- Terasaki, P. I., Cecka, J. M., Gjertson, D. W., Takemoto, S. High survival rates of kidney transplants from spousal and living unrelated donors. New England Journal Medicine. 333, (6), 333-336 (1995).
- Pratschke, J., Neuhaus, P., Tullius, S. G. What can be learned from brain-death models? Transplant International. 18, (1), 15-21 (2005).
- Wilhelm, M. J., et al. Activation of the heart by donor brain death accelerates acute rejection after transplantation. Circulation. 102, (19), 2426-2433 (2000).
- Pomper, G., et al. Introducing a mouse model of brain death. Journal of Neuroscience Methods. 192, (1), 70-74 (2010).
- Ritschl, P. V., et al. Donor brain death leads to differential immune activation in solid organs but does not accelerate ischaemia-reperfusion injury. Journal of Pathology. 239, (1), 84-96 (2016).
- Atkinson, C., et al. Donor brain death exacerbates complement-dependent ischemia/reperfusion injury in transplanted hearts. Circulation. 127, (12), 1290-1299 (2013).
- Oberhuber, R., et al. Treatment with tetrahydrobiopterin overcomes brain death-associated injury in a murine model of pancreas transplantation. American Journal of Transplantation. 15, (11), 2865-2876 (2015).
- Floerchinger, B., et al. Inflammatory immune responses in a reproducible mouse brain death model. Transplant Immunology. 27, (1), 25-29 (2012).
- Steen, P. A., Milde, J. H., Michenfelder, J. D. No barbiturate protection in a dog model of complete cerebral ischemia. Annals of Neurology. 5, (4), 343-349 (1979).
- Cooper, D. K., Novitzky, D., Wicomb, W. N. The pathophysiological effects of brain death on potential donor organs, with particular reference to the heart. Annals of the Royal College of Surgeons of England. 71, (4), 261-266 (1989).
- Herijgers, P., Leunens, V., Tjandra-Maga, T. B., Mubagwa, K., Flameng, W. Changes in organ perfusion after brain death in the rat and its relation to circulating catecholamines. Transplantation. 62, (3), 330-335 (1996).