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
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	<li>Hart, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OPTN/SRTR+2017+Annual+Data+Report:+Kidney.">OPTN/SRTR 2017 Annual Data Report: Kidney.</a> American Journal of Transplantation. 19 (Suppl 2), 19 (2019).</li><li>The Quality Standards Subcommittee of the American Academy of Neurology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practice+parameters+for+determining+brain+death+in+adults+(summary+statement).">Practice parameters for determining brain death in adults (summary statement).</a> Neurology. 45 (5), 1012-1014 (1995).</li><li>Terasaki, P. I., Cecka, J. M., Gjertson, D. W., Takemoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+survival+rates+of+kidney+transplants+from+spousal+and+living+unrelated+donors.">High survival rates of kidney transplants from spousal and living unrelated donors.</a> New England Journal Medicine. 333 (6), 333-336 (1995).</li><li>Pratschke, J., Neuhaus, P., Tullius, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+be+learned+from+brain-death+models?.">What can be learned from brain-death models?.</a> Transplant International. 18 (1), 15-21 (2005).</li><li>Wilhelm, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+heart+by+donor+brain+death+accelerates+acute+rejection+after+transplantation.">Activation of the heart by donor brain death accelerates acute rejection after transplantation.</a> Circulation. 102 (19), 2426-2433 (2000).</li><li>Pomper, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+a+mouse+model+of+brain+death.">Introducing a mouse model of brain death.</a> Journal of Neuroscience Methods. 192 (1), 70-74 (2010).</li><li>Ritschl, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+brain+death+leads+to+differential+immune+activation+in+solid+organs+but+does+not+accelerate+ischaemia-reperfusion+injury.">Donor brain death leads to differential immune activation in solid organs but does not accelerate ischaemia-reperfusion injury.</a> Journal of Pathology. 239 (1), 84-96 (2016).</li><li>Atkinson, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+brain+death+exacerbates+complement-dependent+ischemia/reperfusion+injury+in+transplanted+hearts.">Donor brain death exacerbates complement-dependent ischemia/reperfusion injury in transplanted hearts.</a> Circulation. 127 (12), 1290-1299 (2013).</li><li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+tetrahydrobiopterin+overcomes+brain+death-associated+injury+in+a+murine+model+of+pancreas+transplantation.">Treatment with tetrahydrobiopterin overcomes brain death-associated injury in a murine model of pancreas transplantation.</a> American Journal of Transplantation. 15 (11), 2865-2876 (2015).</li><li>Floerchinger, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+immune+responses+in+a+reproducible+mouse+brain+death+model.">Inflammatory immune responses in a reproducible mouse brain death model.</a> Transplant Immunology. 27 (1), 25-29 (2012).</li><li>Steen, P. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arterial catheter (BD Neoflon 26G)</td>
			<td>BD</td>
			<td>391349</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Pressure Transducers (APT300)</td>
			<td>Harvard Apparatus Inc.</td>
			<td>73-3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Fogarty Arterial Embolectomy Catheter N&#176; 3</td>
			<td>Edwards Lifesciences Corporation</td>
			<td>120403F</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic Blanket Systems with Flexible Probe</td>
			<td>Harvard Apparatus Inc.</td>
			<td>55-7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketansol</td>
			<td>Graeub</td>
			<td>6680110</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>FST</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>12060-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene 5-0</td>
			<td>Ethicon</td>
			<td>8698H</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump 11 Elite Infusion Only Single</td>
			<td>Harvard Apparatus Inc.</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>FST</td>
			<td>14075-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>Transpore Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Underpads</td>
			<td>Molinea.A</td>
			<td>274301</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator for mice (MiniVent Model 845)</td>
			<td>Harvard Apparatus Inc.</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylasol</td>
			<td>Graeub</td>
			<td>7630109</td>
			<td></td>
		</tr>
	</tbody>
</table>

brain death induction in mice using intra-arterial blood pressure monitoring and ventilation via tracheostomy

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.

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...

Watch this Scientific Journal Video about Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy at JoVE.com

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

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 ...

Although donation after donor brain death is the main source for solid-organ transplantation, the irreversible loss of brain function induces pathophysiological changes that lead to a systemic inflammatory response. This murine model of brain-death induction allows the use of a large variety of analytical tools and knockout models to study the pathways of brain-death regulation. For arterial catheterization after confirming a lack of response to pedal reflex and removing the upper abdominal hair, disinfect the exposed skin with 70%ethanol and use dissecting scissors to make a midline incision in the skin.Using forceps, blunt dissect the submandibular glands and neck muscle tissue and separate the tissues to expose the common carotid artery. Place three 8-0 silk ligatures beneath the right common carotid artery and place a clamp on the proximal ligature. Bring tension in the artery so that the flow is suspended and close the most distal ligature.Insert a 26-gauge arterial catheter through a small preformed skin hole on the cranial aspect of the incision. Squeeze the catheter with the forceps if the lumen is too large to reduce the backflow and secure the catheter with all three sutures, then use a single 5-0 monofilament nonabsorbable to fix the catheter to the skin near the preformed skin hole. To perform a tracheostomy, use forceps to blunt dissect the pretracheal musculature and place two 8-0 silk ligatures beneath the trachea.Insert the ventilation tube between two tracheal cartilages to avoid unilateral ventilation and secure the tube with both prepared ligatures, then close the skin with a 6-0 monofilament nonabsorbable running suture and ventilate the mouse with a frequency of 150 per minute and a tidal volume of 200 microliters. To induce brain death in the experimental animal, arrange the mouse to the prone position and, holding the skin with forceps, use surgical scissors to remove the skin from the skull. Drill a one-millimeter caliper borehole perimedially above the left parietal cortex and use blunt forceps to penetrate the final tissue bridge of the skull.After removing any sharp edges, insert a balloon catheter prefilled with saline with all of the air evacuated. When the catheter is entirely within the cranial cavity, use a syringe pump to begin inflating the catheter at approximately 0.1 millimeters per minute over a period of 10 to 15 minutes. When brain death occurs, stop the inflation of the balloon catheter and place a heating blanket over the mouse to avoid hypothermia.After brain death has been confirmed, monitor and document the blood pressure regularly, infusing 100 microliters of saline every 30 minutes to stabilize the blood pressure of the animal. After four hours of brain death, exclude any animals without beating hearts and harvest the mouse organs and tissues of interest according to standard protocols. After brain-death induction, the blood pressure exhibits an initial hypertensive peak followed by a prolonged hypotensive phase.Another well-established observation is that brain-death induction leads to activation of the immune system. Indeed, after four hours of brain death in this model, an organ-specific upregulation of immune markers is observed at the mRNA level. After a predefined period of brain death, the organs may be harvested for their direct analysis or for subsequent organ transplantation.This model will enable in-depth studies into the influence of brain-death induced injury and has already revealed new insights into complement activation and leukocyte migration.

brain-death-induction-mice-using-intra-arterial-blood-pressure

Research

JoVE Journal

Neuroscience

5.4K vistas.  Charité - Universitätsmedizin Berlin. Aunque la donación después de la muerte cerebral del donante es la principal fuente de trasplante de órganos sólidos, la pérdida irreversible de la función cerebral induce cambios fisiopatológicos que conducen a una respuesta inflamatoria sistémica. Este modelo murino de inducción de la muerte cerebral permite el uso de una gran variedad de herramientas analíticas y modelos de knockout para estudiar las vías de regulación de la muerte cerebral. Para el cateterismo arterial despué...