**2. Effects of brain death on liver graft undergoing transplantation**

of brain and brain stem function, usually caused by major hemorrhage, hypoxia or metabolic dysregulation [5, 6]. Hemodynamic events, hormonal changes and inflammation and immune activation occur consequently to BD [6–9]. It has been described that BD markedly reduces the tolerance of liver grafts to preservation and reperfusion injury and reduces graft survival [2–4, 10]. The detrimental consequences of BD have been clinically described; however the underlying mechanisms and their relevance in LT remain poorly understood. Indeed, few studies have evaluated the effect of BD on LT, and most of the experimental studies focused in establishing surgical or pharmacological strategies to reduce liver graft damage associated

Shortage of donor organs remains a major obstacle to the widespread application of LT in patients with end-stage liver disease [11–14]. To overcome this problem, transplant centers developed strategies to expand the organ donor pool [15]. This shortage could be alleviated by routine use of sub-optimal donor livers including elderly donors, steatotic donors, split livers and donor with viral infections or with malignancy. These sub-optimal livers exhibit a greater risk of organ dysfunction and primary non-function compared with optimal livers, when used as grafts in transplantation [1, 4, 9]. Multiple methods are currently being investigated to minimize the effects of ischemia-reperfusion (I/R) injury to allow the use of sub-optimal liver grafts, including anti-inflammatory approaches to attenuate cytokines, blockade of adhesion molecules, antiapoptotic strategies, among others. However, these studies are performed in non-BD surgical conditions. Only a recent study describes, for the first time, a potential treatment in steatotic liver grafts undergoing LT from cadaveric

Since consequences of BD are linked to an inferior graft function and a more potent immune response, studies in animal models could be critical in uncovering its basic mechanisms and to provide a rationale for the development of novel targets that may prevent the deteriorating consequences of BD in clinical practice [16]. Virtually, all experimental organ transplantation studies generally utilize young, healthy living animals as donors; in clinical practice; however, a relatively low percentage of organs are acquired from living sources. Among other variables, the difference between living and brain-dead donors includes the effect of profound physiological and structural derangements [17]. Although a wide variety of animal BD models have been used to study the effect of BD on donor organ quality (without transplantation) [18–26], the lack of standardization of BD models makes difficult to compare results from different research groups. Undoubtedly, there is a need for a more controlled and standardized BD model allowing studies of organs in a situation closely resembling the reality in

In the first part of this chapter, we highlight the actual knowledge and new insights into the pathophysiology in BD focusing on liver graft undergoing transplantation. Following, the different experimental models reported in literature used to study the effect of BD on quality of optimal and sub-optimal liver grafts are presented, focusing on the strengths and limitations. Since recognize the underlying mechanisms of detrimental effects of BD is a critical question to design therapeutic strategies to protect liver grafts undergoing transplantation, we discuss if the existing BD animal models mimic the clinical findings in transplants with BD donors,

and highlight which BD model could be more appropriated.

with transplantation have been performed in the absence of BD [6].

136 Organ Donation and Transplantation - Current Status and Future Challenges

donors [1].

the intensive care unit [27].

Liver I/R injury is a local proinflammatory response mediated by the immune system. The central nervous system plays a fundamental role in the regulation of molecular markers triggering inflammation and tissue damage, and BD results in a breakdown of these mechanisms, hence initiating the cascade of I/R injury liver transplants and exacerbating the hepatic damage caused by I/R [9]. However, although the detrimental consequences of BD have been clinically described, the underlying mechanisms and their relevance in LT remain poorly understood [6]. Following, systemic events that happen after BD and the specific effects of BD on hepatic graft undergoing transplantation are presented.

BD is followed immediately by an acute and transient rise in blood pressure (Cushing reflex), that is immediately followed by a transient bradycardia communicated through parasympathetic activation [16, 24, 28–31]. Later after BD, deteriorating hemodynamics and a compromised perfusion of abdominal organs is becoming evident. Accordingly, a shift from aerobic to anaerobic metabolism and acidosis is registered, clinically reflected by elevated serum levels of lactate and free fatty acids and promoted by decreasing insulin secretion and hyperglycemia [21, 32, 33]. Alterations of specific mitochondrial functions may lead to an impaired production of ATP and a reduced uptake of substrates for mitochondrial metabolism and eventually limit resistance and survival of cells and organs to damaging insults [34]. It is well known that ATP degradation during hepatic ischemia leads to an acceleration of glycolysis [35]. Although glycolysis is essential for cell survival, it may also be detrimental because of the accumulation of glycolytic products such as lactate [35]. In the liver, the increase in cAMP levels due to ischemia triggers the activation of glycolysis. This causes the accumulation of hexose 6-phosphates, which proceed down the glycolytic pathway to form lactate [35–38]. Some authors [39] have shown that normotensive BD had no influence on the general viability of the liver, as measured by ATP content. In this research work, no differences were found in ATP content in livers from BD-induced rats and control rats, implicating that the phase of BD up to 6 h does not reduce general liver viability. However, it has also been described that the ATP content after 3 h of reoxygenation after graft harvesting was significantly increased in liver biopsies from brain-dead rats, may be because of a stress response, caused by increased catecholamine levels induced by BD and exaggerated reoxygenation times of liver tissues, exacerbating the I/R injury [39].

Hemodynamic instability, hormonal alterations, blood coagulation factor consumption, lung tissue changes, hypothermia, and electrolyte disturbance generated by BD invariably influence hepatic functions. Increased AST and ALT concentrations indicated liver dysfunction in brain-dead pigs and hepatocyte edema, hepatic sinusoid compression, and other microscopic observations in such animals demonstrated damage to liver cells [40, 41]. Also, BD causes time-dependent general dysfunction in rats, as indicated by elevated LDH and creatinine, AST and α-GST levels [42]. The disruption of liver function seems to be due to circulatory collapse and hemodynamic instability [41]. Morphological changes in the liver following BD are even less well defined. Recent experimental findings show that hepatocytes in livers from brain-dead donors show an altered cell membrane permeability and integrity [17, 43]. It has also been reported increased caspase-3 activity indicating that apoptosis occurs in liver tissue of brain-dead donors [3].

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

A wide variety of BD models have been described in the literature, and in most of them an increase in intracranial pressure is generated by an expanding intracranial balloon, finally leading to BD [26]. This section will discuss about experimental animal models used to evalu-

Experimental Brain Death Models in Liver Transplantation

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

139

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

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

ate the effects of BD on optimal and steatotic liver grafts.

**3.1. Experimental BD in small animals**

balloon [24, 26].
