**3.2. Experimental BD in large animals**

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].

grafts, especially steatotic ones, are obtained from cadaveric donors.

140 Organ Donation and Transplantation - Current Status and Future Challenges

intervention, which may counteract the detrimental effects of BD [1].

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

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 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 sympathetic nervous system [63].

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


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

of studying the effects of BD processes on organ quality and function as well as outcomes after transplantation. Furthermore, the model also may be used for explorative studies of brain injury mechanisms and for evaluating new neuromonitoring devices [27]. In another experimental model [65], BD has been induced by surgically placing an epidural balloon and gradually increasing the inflation to increase intracranial pressure to 15, 25, 35 and 45 mm Hg, maintaining each pressure level for 30 min, in piglets [65]. However, opposite results in comparison with the experimental model using a BD induction phase of 200 min [27] were observed, this means a decrease of the arterial blood pressure and tachycardia. The reason for this difference is unclear, but it is well-known from the clinical situation that a Cushing response is not always observed in patients with BD developing [27].

More recently, [66] have established a clinically relevant, reproducible, large animal model of BD in pigs, based on a controlled cerebral hemorrhage. To induce intracranial hemorrhage, a needle was inserted through a burr hole over the left hemisphere, and stereotaxically placed in the internal capsule at the level of the lateral ventricle. Blood was withdrawn from the central arterial catheter in a 10 ml unheparinized syringe and then was injected in the brain with a rate of 40 ml/h. The infusion of blood continued for 60 min to maintain an intracranial pressure sufficient to ensure BD. Pigs in the control group underwent surgical preparations similar to the BD group, including burr holes and tissue glue but without injection of blood. Computerized tomographic angiography was performed 120–180 min after BD [66]. Irreversible damage to the brain stem was validated by a negative atropine test, disappearance of corneal and pupillary light reflexes, and a negative cerebral perfusion pressure sustained for more than 60 min. The disappearance of intracranial pulse pressure waves supports the diagnosis, as this is a clinical parameter associated with BD [66].

affects the function of liver grafts that are procured from a cadaveric donor and subsequently transplanted [68]. Therefore, to achieve a successful design of therapeutic strategies to protect liver grafts against harmful effects of BD, it is important to take into account only experimental

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

Ref. [64] Gradual 6 h Ventilation Not realized Ref. [65] Gradual 30 or 60 min Not described Not realized Ref. [66] Gradual 8 h Not described Not realized

Ref. [67] Sudden 6, 12 and 16 h Inotropic support 27 h

**Table 2.** Experimental models in large animals used to study the effect of BD on liver graft quality.

Ventilation Not realized

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**Table 2** summarizes the experimental models in large animals used to study the effect of BD

Most organs for transplantation originate from BD donors. Although the detrimental consequences of BD have been clinically described, the underlying mechanisms and their relevance in transplantation remain poorly understood. Indeed, few studies have evaluated the effect of BD on transplantation, and, as stated along this chapter, most of the experimental studies focused in the pathophysiological changes occurring during BD without transplantation. Moreover, these studies have been performed in the different sudden and gradual BD models, so dissimilar results on pathophysiological changes and treatments have been described. Comparison of the results of animal studies and their extrapolation to human beings is feasible, but with limitations such as differences in BD and ischemia tolerance, anatomy of the liver of various species, surgical conditions used in clinical practice and those used in the experimental models. Importantly, studies performed in small animals are of limited applicability to human beings due to their different size and anatomy of the liver and their faster metabolism. Large animals exhibit greater similarity in their anatomy and physiology to humans; however, their use is restricted by serious logistical and financial difficulties, ethical concerns and limited availability of immunological tools for use in large animal species. In addition, in some cases, experimental models of BD in large animals did not mimic the clinical conditions in liver transplantation from BD. Despite the limitations of the experimental animal models, these are the best options to study hepatic I/R, especially considering that the progress of human studies is slow, the majority of human tissues are not routinely accessible for research, and there is very limited opportunity for interventional studies. The clinical application of strategies

models in which the negative effects of BD on the liver tissue have been demonstrated.

on liver graft quality.

**Large animals**

Ref. [27] Gradual 1

h

**4. Conclusions**

Further investigations will be required to investigate whether all of these experimental models of BD above mentioned are adequate to study the effect of BD on a graft that also presents I/R injury and which one best simulates the conditions present in clinical practice.

Another experimental model of BD in large animals has been reported [67], which has been previously established and neuropathologically validated [19, 67]. In this model the effect of BD on liver grafts also undergoing cold preservation and transplantation was evaluated. BD was induced suddenly by the injection of 8–17 ml of normal saline solution into the catheter balloon over a period of 5–10 min using dogs as experimental animals. This produced a sudden rise in arterial blood pressure (systolic and diastolic) and heart rate, reflecting an explosive intracranial pressure increase and defined as the "Cushing reflex" [67]. Once the diagnosis of BD was established, the dog was monitored for 16 h before the liver was removed [67]. Livers were stored floating in 1000 ml of the UW preservation solution at 4°C for 24 h [67] and at the end of the preservation time, the livers were transplanted orthotopically using a modified technique with a sutured upper caval anastomosis and cuffed anastomoses for the infrahepatic vena cava and the portal vein [67]. Although not all the clinical scenario of BD could be replicated, this experimental model simulates the sudden rise in intracranial pressure caused by acute cerebrovascular lesions or traumatic brain injury [67]. As reflected by the hepatic enzyme release during the course of BD, no evidence of deterioration of function in livers retrieved from BD donors was found. These findings are different from rodent models. In addition, it should be borne in mind that in clinical practice it is well documented that BD negatively


**Table 2.** Experimental models in large animals used to study the effect of BD on liver graft quality.

affects the function of liver grafts that are procured from a cadaveric donor and subsequently transplanted [68]. Therefore, to achieve a successful design of therapeutic strategies to protect liver grafts against harmful effects of BD, it is important to take into account only experimental models in which the negative effects of BD on the liver tissue have been demonstrated.

**Table 2** summarizes the experimental models in large animals used to study the effect of BD on liver graft quality.

#### **4. Conclusions**

of studying the effects of BD processes on organ quality and function as well as outcomes after transplantation. Furthermore, the model also may be used for explorative studies of brain injury mechanisms and for evaluating new neuromonitoring devices [27]. In another experimental model [65], BD has been induced by surgically placing an epidural balloon and gradually increasing the inflation to increase intracranial pressure to 15, 25, 35 and 45 mm Hg, maintaining each pressure level for 30 min, in piglets [65]. However, opposite results in comparison with the experimental model using a BD induction phase of 200 min [27] were observed, this means a decrease of the arterial blood pressure and tachycardia. The reason for this difference is unclear, but it is well-known from the clinical situation that a Cushing

More recently, [66] have established a clinically relevant, reproducible, large animal model of BD in pigs, based on a controlled cerebral hemorrhage. To induce intracranial hemorrhage, a needle was inserted through a burr hole over the left hemisphere, and stereotaxically placed in the internal capsule at the level of the lateral ventricle. Blood was withdrawn from the central arterial catheter in a 10 ml unheparinized syringe and then was injected in the brain with a rate of 40 ml/h. The infusion of blood continued for 60 min to maintain an intracranial pressure sufficient to ensure BD. Pigs in the control group underwent surgical preparations similar to the BD group, including burr holes and tissue glue but without injection of blood. Computerized tomographic angiography was performed 120–180 min after BD [66]. Irreversible damage to the brain stem was validated by a negative atropine test, disappearance of corneal and pupillary light reflexes, and a negative cerebral perfusion pressure sustained for more than 60 min. The disappearance of intracranial pulse pressure waves supports

Further investigations will be required to investigate whether all of these experimental models of BD above mentioned are adequate to study the effect of BD on a graft that also presents

Another experimental model of BD in large animals has been reported [67], which has been previously established and neuropathologically validated [19, 67]. In this model the effect of BD on liver grafts also undergoing cold preservation and transplantation was evaluated. BD was induced suddenly by the injection of 8–17 ml of normal saline solution into the catheter balloon over a period of 5–10 min using dogs as experimental animals. This produced a sudden rise in arterial blood pressure (systolic and diastolic) and heart rate, reflecting an explosive intracranial pressure increase and defined as the "Cushing reflex" [67]. Once the diagnosis of BD was established, the dog was monitored for 16 h before the liver was removed [67]. Livers were stored floating in 1000 ml of the UW preservation solution at 4°C for 24 h [67] and at the end of the preservation time, the livers were transplanted orthotopically using a modified technique with a sutured upper caval anastomosis and cuffed anastomoses for the infrahepatic vena cava and the portal vein [67]. Although not all the clinical scenario of BD could be replicated, this experimental model simulates the sudden rise in intracranial pressure caused by acute cerebrovascular lesions or traumatic brain injury [67]. As reflected by the hepatic enzyme release during the course of BD, no evidence of deterioration of function in livers retrieved from BD donors was found. These findings are different from rodent models. In addition, it should be borne in mind that in clinical practice it is well documented that BD negatively

I/R injury and which one best simulates the conditions present in clinical practice.

response is not always observed in patients with BD developing [27].

142 Organ Donation and Transplantation - Current Status and Future Challenges

the diagnosis, as this is a clinical parameter associated with BD [66].

Most organs for transplantation originate from BD donors. Although the detrimental consequences of BD have been clinically described, the underlying mechanisms and their relevance in transplantation remain poorly understood. Indeed, few studies have evaluated the effect of BD on transplantation, and, as stated along this chapter, most of the experimental studies focused in the pathophysiological changes occurring during BD without transplantation. Moreover, these studies have been performed in the different sudden and gradual BD models, so dissimilar results on pathophysiological changes and treatments have been described.

Comparison of the results of animal studies and their extrapolation to human beings is feasible, but with limitations such as differences in BD and ischemia tolerance, anatomy of the liver of various species, surgical conditions used in clinical practice and those used in the experimental models. Importantly, studies performed in small animals are of limited applicability to human beings due to their different size and anatomy of the liver and their faster metabolism. Large animals exhibit greater similarity in their anatomy and physiology to humans; however, their use is restricted by serious logistical and financial difficulties, ethical concerns and limited availability of immunological tools for use in large animal species. In addition, in some cases, experimental models of BD in large animals did not mimic the clinical conditions in liver transplantation from BD. Despite the limitations of the experimental animal models, these are the best options to study hepatic I/R, especially considering that the progress of human studies is slow, the majority of human tissues are not routinely accessible for research, and there is very limited opportunity for interventional studies. The clinical application of strategies


**Author details**

and Carmen Peralta1,5,6\*

Ciudad Victoria, Mexico

(CIBEREHD), Barcelona, Spain

**References**

\*Address all correspondence to: cperalta@clinic.ub.es

Universidad Autónoma de Tamaulipas, Mexico

4 Transplant Biomedicals, S.L., Barcelona, Spain

†Both authors contributed equally to this work

for 20 hr. Transplantation. 2001;**72**:1632-1636

DOI: 10.1097/01.TP.0000080983.14161.95

486. DOI: 10.1111/j.1600-6143.2005.01208.x

2010;**74**:1911-1918. DOI: 10.1212/WNL.0b013e3181e242a8

Maria Eugenia Cornide-Petronio1†, Araní Casillas-Ramírez2,3†, Mónica B. Jiménez-Castro<sup>4</sup>

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

145

1 Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

2 Hospital Regional de Alta Especialidad de Ciudad Victoria "Bicentenario 2010",

3 Facultad de Medicina e Ingeniería en Sistemas Computacionales de Matamoros,

5 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas

[1] Jiménez-Castro MB, Meroño N, Mendes-Braz M, Gracia-Sancho J, Martínez-Carreres L, Cornide-Petronio ME, Casillas-Ramirez A, Rodés J, Peralta C. The effect of brain death in rat steatotic and non-steatotic liver transplantation with previous ischemic precondi-

[2] van der Hoeven JA, Lindell S, van Schilfgaarde R, Molema G, Ter Horst GJ, Southard JH, Ploeg RJ. Donor brain death reduces survival after transplantation in rat livers preserved

[3] van der Hoeven JA, Moshage H, Schuurs T, Nijboer M, Van Schilfgaarde R, Ploeg RJ. Brain death induces apoptosis in donor liver of the rat. Transplantation. 2003;**76**:1150-1154.

[4] Kotsch K, Francuski M, Pascher A, Klemz R, Seifert M, Mittler J, Schumacher G, Buelow R, Volk HD, Tullius SG, Neuhaus P, Pratschke J. Improved long-term graft survival after HO-1 induction in brain-dead donors. American Journal of Transplantation. 2006;**6**:477-

[5] Wijdicks EF, Varelas PN, Gronseth GS, Greer DM. Evidence-based guideline update: Determining brain death in adults: Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology. American Academy of Neurology.

tioning. Journal of Hepatology. 2015;**62**:83-91. DOI: 10.1016/j.jhep.2014.07.031

6 Facultad de Medicina, Universidad International de Cataluña, Barcelona, Spain

**Table 3.** Reproducible clinical effects of brain death in the different experimental models.

designed at benchside will depend on the use of experimental models of BD that resemble as much as possible the clinical conditions that happens in BD and LT [69]. **Table 3** summarizes the reproducible clinical effects of BD that happens in the different experimental models.

We recognize the complication, but future research in similar experimental models of transplantation using BD donors is required to understand the pathophysiology of BD and elucidate the consequences of BD, especially in sub-optimal liver grafts. Thus, multidisciplinary research groups should devote additional efforts to better understand the pathophysiology of BD to ultimately develop effectual therapeutic strategies aimed at improving graft viability, and at significantly increasing the organ donor pool.
