**3.1. Experimental BD in small animals**

A systemic inflammatory response is present in most patients at the diagnosis of BD [44]. The intense cellular and molecular activation that quickly follows the acute onset of BD involves acute transcriptional upregulation of inflammatory cytokines, both on a systemic and intraorgan level [16]. In liver, aside from microcirculatory changes, early effects of I/R injury and BD are mediated via Kupffer cells. When I/R injury occurs, Kupffer cells and neutrophils are activated, together with an increased expression of adhesion molecules and infiltration of monocytes and lymphocytes [9, 45]. Cell death by necrosis and apoptosis is initiated. In addition to mitochondrial damage, synthesis of oxygen-free radicals is boosted and accompanied by a decreasing activity of antioxidant enzymes. All of this results in immediate organ damage, contributing to hepatic failure after transplantation. In BD models many of these events have been also documented, exacerbating the hepatic damage that already occurs by I/R. In this sense, expression of adhesion molecules in endothelial and epithelial cells, initiating the release of proinflammatory cytokines and the infiltration of immune competent cells is induced by BD [16, 46–50]. VCAM-1 expression was found after 6 hours of BD in both hypotensive and normotensive donors. The pattern of VCAM-1 expression was similar to that described by others during periods of inflammation or rejection of the liver. Also, the ICAM-1 expression observed in all brain-dead rats was similar to expression patterns during episodes of inflammation [42, 51–55]. Leukocyte recruitment to the underlying parenchyma was facilitated, as expected, with upregulated VCAM-1 and ICAM-1 expression, with a significant increase in infiltrating leukocytes in the liver tissue of brain-dead rats. Also, naive as well as activated macrophages (i.e., ED1- and ED2-positive cells) were significantly increased in brain-dead donors versus controls [42]. The detrimental changes observed during experimental BD were confirmed in more limited clinical studies. Inflammatory changes were found in cadaveric donor livers that showed increased proportion of CD3+ lymphocytes, CD68+ monocytes, and macrophages relative to livers from living donors [33, 56, 57]. Clinical events after BD included more frequent acute rejection episodes and increased lymphocytic infiltrates [33, 56]. In human liver transplants, donor BD triggered upregulation of inflammatory cytokines IL-6, IL-10, TNF-α, TGFβ, IFN-γ, and MIP-1α. Those findings correlated with more pronounced cellular infiltrates, a higher incidence of primary graft dysfunction, and frequent acute rejection episodes [16, 58]. Findings in small animal models of BD parallel clinical observations [16], for instance upregulation of IL-10 and iNOS mRNA in liver has been demonstrated after 6 h of BD [39]. On the contrary, another study has not found iNOS induction in brain-dead animals. This contradictory result may be explained by the fact that the phase of

138 Organ Donation and Transplantation - Current Status and Future Challenges

BD in this later study was maintained for only 2–3 h [39].

**liver graft quality**

**3. Experimental models used to study the effect of brain death on** 

Future research in experimental models of LT using BD donors is required to understand the pathophysiology of BD and elucidate the consequences of BD on graft function and survival [6]. The knowledge of the underlying mechanisms on the detrimental effects of BD is a critical question to promote better preservation of the organ and to improve outcome after LT [1]. Irreversible pontine ischemia is the essential hallmark of experimental BD. As hemodynamic instabilities related to cardiovascular collapse may bias experimental results, invasive monitoring is important for accurate determination of volume and maintenance of physiological blood pressure. The initial spike in blood pressure during brain stem herniation, apnea, and transient spontaneous reflexes with subsequent absence of spinal reflexes are characteristic criteria of the central catastrophe in experimental models of BD [16, 24, 28, 29, 31, 59]. To produce BD in small laboratory animals, a Fogarty balloon catheter (2–4 French) is inserted through a parietal bore hole into the subdural space and inflated. Reported inflation volumes for induction of the condition range from 200 to 500 μL in rats and 80–103 μL in mice. The inflated balloon catheter is left in place during the entire period of observation to avoid intracerebral hemorrhage and hemodynamic collapse. Computerized axial tomography or magnetic resonance imaging in brain-dead animals can document that the catheter inflation causes the hindbrain to herniate through the foramen magnum [16, 21, 28, 29, 60, 61]. Two types have been described: the so-called 'sudden onset' BD model and the 'gradual onset' model. In the gradual onset model described by [26], induction of BD was started by gradually increasing the intracranial pressure by inflating the balloon with 16 μL saline per minute. During balloon inflation, a hypotensive period occurred followed by a short peak and a subsequent drop in blood pressure. When the blood pressure returned to its basal level during an increasing peak, inflation of the balloon was stopped and anesthesia was withdrawn. The state of BD was confirmed 30 min after the onset of BD, by the absence of corneal reflexes and a positive apnea test, and such condition was maintained during 1 h or for 4 h [26]. In contrast to the sudden onset model, the gradual onset model simulates cerebral hemorrhage by slowly increasing the intracranial pressure, resulting in less hemodynamic instability and maintenance of normotension during BD for several hours. So far, only a few studies in abdominal organs have described BD induction using a gradual expansion of an intracranial balloon [24, 26].

Authors using a sudden onset model of BD [60] have demonstrated upregulations of proinflammatory markers in different organs, including the liver. BD was produced by rapid balloon inflation of a Fogarty arterial embolectomy catheter introduced into the subdural space through an occipital burr hole; this maneuver suddenly increases intracranial pressure and causes herniation of the brain stem within 20 min in all animals. The rats were tracheostomized and mechanically respirated for periods up to 6 h. [60]. Authors conclude that the experimental system described provides a potentially clinically relevant model in which to study the systemic effects of BD in detail and suggests means to prevent changes in peripheral organs [60]. Thus, it has been postulated that sudden onset BD reflects the situation of the hemodynamically instable donors [23]. In a sudden onset' BD model described by [23], through a frontolateral trepanation a balloon catheter was introduced in the subdural space with the tip pointing caudally. Inflating the balloon for 1 min increased the intracranial pressure thereby inducing rapid progressive brain injury leading to BD, which was based on the sharp rise and subsequent drop of blood pressure and heart rate. The state of BD was confirmed 30 min after induction by the absence of corneal reflexes and an apnea test and the effect of BD was studied at 1 h and 6 h after BD induction [23]. Hemodynamic changes in this model, mimicking acute isolated cerebral trauma, included first a sudden increase and subsequently a gradual drop of both heart rate and mean arterial pressure [23]. This BD model appears to be a reliable tool to mimic the type of fast occurring brain injury due to isolated cerebral trauma in man. In this BD model that reflects the situation of the hemodynamically instable donor, expression of immediate early genes was found in tissues of liver and kidney coinciding with progressive dysfunction of these organs and suggest a progressive detrimental effect of BD on donor organ quality, especially in livers from hemodynamically instable donors [23].

**Table 1** summarizes the experimental models used in small animals to study the effect of BD

Experimental Brain Death Models in Liver Transplantation

http://dx.doi.org/10.5772/intechopen.75438

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In the baboon, BD is produced by creating intracranial hypertension. Under full inhalation anesthesia, a Foley catheter was introduced into the subdural space through a frontal burr hole in the skull and filled with 20–30 mL of saline, then BD occurred within 20 min [62, 63]. BD has been also induced in pigs by ligation of both brachiocephalic arteries, from which arise the carotid and vertebral arteries. Both experimental models of BD led to a series of major pathophysiological changes that may be collectively referred to as the autonomic storm. Though there was a brief initial period of excessive parasympathetic activity, evidenced by a marked bradycardia, most of the effects of this autonomic activity were brought about by the

Porcine BD models have been widely used in transplant surgery studies. A research group [64] performed a study with a lengthening of the induction phase to 60 min with gradual epidural balloon inflation up to 15 mL. Furthermore, they suggested that prolonged BD process might give the necessary preconditions to trigger a systemic inflammatory response that might contribute to organ dysfunction. Compared to the clinical situation, other authors [27] considered that 60 min was still a relatively short time. They therefore wanted to prolong the BD induction phase up to 200 min by stepwise increase of intracranial volume, followed by a 30-min observation period. Analysis of the monitoring results showed a classic intracranial pressure-volume relationship. Furthermore, intracranial compliance decreased gradually when intracranial pressure increased, which reflects a decreasing ability to compensate for added intracranial volume [27]. The described so-called Cushing response with arterial blood pressure increase, bradycardia, and respiratory irregularities was also demonstrated [27]. Authors believe that their model has the advantage of applying gradual progressive changes in intracranial pressure, cerebral perfusion pressure, and brain tissue oxygenation, leading to cellular injury in parenchymatous organs. Therefore, the model provides the possibility

**Article Type of BD Time of sustained BD Support during BD Cold ischemia**

Ref. [23] Sudden 1 or 6 h Ventilation Not realized

Ref. [31] Gradual and sudden 6 h Ventilation Not realized

Ref. [60] Sudden 6 h Ventilation Not realized

**Table 1.** Experimental models in small animals used to study the effect of BD on liver graft quality.

Inotropic support

Inotropic support

Not realized

Not realized

Ref. [1] Sudden 6 h Ventilation 6 h

Ref. [26] Gradual 1 or 4 h Ventilation

Ref. [42] Sudden 1 or 6 h Ventilation

on liver graft quality.

**3.2. Experimental BD in large animals**

sympathetic nervous system [63].

**Small animals**

In these experimental models described above, only the effect of induction of BD on liver tissue was evaluated. Unfortunately, the other conditions (ischemia and reperfusion) that are present in the clinical practice of transplantation were not included. It is important to evaluate such conditions, since they negatively affect liver grafts already damaged by BD when these grafts, especially steatotic ones, are obtained from cadaveric donors.

Given the prevalence of hepatic steatosis in the population, this represents a large potential pool of donors. However, the clinical problem is still unresolved as steatotic livers are more susceptible to I/R injury and, when used, have poorer outcome than non-steatotic livers [6]. To date, only one experimental study has evaluated the effect of BD on optimal and steatotic liver grafts, which were subsequently subjected to cold ischemia and transplantation. In such research [1], authors have tested the influence of hepatic steatosis and BD separately and in combination in an experimental model of LT [1]. A balloon catheter was introduced through the drill hole in the extradural space with the tip pointing caudally. The intracranial pressure was increased by inflating the balloon for 1 min. The increase in intracranial pressure induced rapid brain injury, leading to immediate BD, simulating a condition comparable to acute isolated cerebral trauma in humans. The state of BD was confirmed by the absence of corneal reflexes and an apnea test and liver grafts were extracted from donors at 6 h after the induction of BD [1]. Steatotic and non-steatotic liver grafts were preserved during 6 h and then were transplanted and submitted to reperfusion for 4 h. Authors report for the first time that the injurious effects of BD in LT are exacerbated in the presence of hepatic steatosis and occur before liver grafts are retrieved from donors [1]. In addition, the mechanisms responsible for the detrimental effects of BD were different depending of the type of the liver, which would interfere with protective pharmacological or surgical strategies applied in liver grafts, avoiding its potential benefits [6]. In such a study, BD-induced dysfunction in the cholinergic anti-inflammatory pathway and prevented the benefits induced by ischemic preconditioning, a surgical strategy that shows benefits when it is applied in non-BD clinical situations. In fact, the study indicated that the combination of acetylcholine and ischemic preconditioning could be considered as a feasible and easy-to-perform intervention to reduce the adverse effects of BD and improve the quality of liver grafts. So authors propose that the time frame between the declaration of BD and organ retrieval provides an important window for cytoprotective intervention, which may counteract the detrimental effects of BD [1].

**Table 1** summarizes the experimental models used in small animals to study the effect of BD on liver graft quality.
