Objectives: Experimental animal models of brain death that mimic human conditions may be useful for investigating novel strategies that increase quality and quantity of organs for transplant.
Materials and Methods: Brain death was induced by increasing intracranial pressure by inflating an intracranial placed balloon catheter. Brain death was confirmed by flatline electroencephalogram, physical signs of apnea, and absence of brain stem reflexes. Donor management was done after brain death. Intracranial pressure and physiologic variables were continually monitored during 9 hours’ follow-up.
Results: Ninety percent of brain dead animals showed typical signs of brain death such as diabetes insipidus, hypertensive, and hypotensive periods. Donor care was performed for 9 hours after brain death, and the mean arterial pressure was maintained above 60 mm Hg.
Conclusions: We conclude that the rat model of brain death can be performed in a standardized, reproducible, and successful way.
Key words : Animal, Brain death, Brain injury, Intracranial pressure, Transplant
Transplants often are the only treatment for end-stage organ failure. The worldwide demand for donor organs continues to exceed the number of organs available for transplant. The prevailing shortage of organs seriously limits the extent to which this option is available. This shortage is caused by treatment of brain-dead donors that is not optimal.1,2 Management of brain dead donors is one of the most neglected areas of transplant medicine.3 The risk of kidney and liver graft dysfunction is higher in grafts from brain dead donors than in grafts from living donors.4-6 The risk of donors is higher in grafts from brain-dead donors than in grafts from living donors, which is confirmed not only from clinical practice, but also from experimental studies.7-9 Thus, brain death is a significant antigen-independent risk factor harming recovered organs before transplant.
To increase quality and quantity of organs for transplants, there is a need for clinically relevant brain death model. Brain death provokes hemodynamic, neuroendocrine, and immunologic changes.10-13 To prevent excess variations in blood pressure, inotropic support or manipulation of intracranial balloon volume is issued to maintain normotension in brain dead models. Vasopressors have known adverse events on several organs.14 Most data on brain death models arise from rats observed fewer than 6 hours. We developed a clinically relevant brain death model that imitates human conditions 9 hours before retrieval.
Materials and Methods
Twenty adult male 300-350 g Sprague-Dawley rats were used for all donor experiments. All animal care and surgical procedures were done in accordance with the guidelines of the animal care and use committee at Zhengzhou University. Rats were housed in standard cages with 5 animals per cage. The housing temperature was 22%, a relative humidity of 40% to 60%,with a 12 hour/12 hour light-dark cycle. Food and water were given ad libitum. The study was approved by the Ethics Committee for Animal Experimentation of our hospital.
The donor rats were premedicated with intra-peritoneal administration of atropine (0.25 mg). General anesthesia was induced with intraperitoneal administration of 1% pentobarbital (0.4-0.6 mL/100 g). The anterior part, limbs, and abdomen of donor animals were shaved.
Brain death was accomplished by creating intra-cranial hypertension. The scalp and the epicranial muscles and periosteum were excised, and a 2-mm hole was drilled with a 12-gauge needle through the skull, 4 mm lateral to the sagittal suture. A No. 3 Fogarty catheter balloon (Fogarty Arterial Embolectomy Catheter, Edwards Lifesciences LLC, Irvine, CA, USA) was inserted epidurally in a 2-mm burr hole and gradually inflated with 20 μL saline every 5 minutes. Brain death was induced by graded inflation of the balloon catheter under an electroencephalogram, and intracranial pressure was monitored until brain death. Brain death was confirmed by flatline electroencephalogram, physical signs of apnea, and absence of brain stem reflexes. The balloon was kept inflated for 9 hours’ follow-up.
Electroencephalographic changes were recorded by means of 3 needle electrodes attached to the epidural regions of the scalp and connected to an MP150 data acquisition system using Acknowledge 3.0 software (BIOPAC systems, Inc. Goleta, CA, USA). Recordings were taken before inflating the intracranial balloon, during inflation, and continually through the experiment. Two needle electrodes were inserted epidurally through the skull. A third needle electrode was attached to a limb for grounding. Brain death was confirmed by flatline tracings, physical signs of apnea, areflexia, and maximally dilated and fixed pupils.
All rats received tracheostomy tube insertion and were connected to the rodent respirator (Harvard Inspira Advanced Safety Ventilator, Pressure Controlled MAI 55-7059, Holliston, MA. USA). The ventilation was set to 30% oxygen in air and adapted to maintain physiologic Pco2 level between 35 and 45 mm Hg.
The saphenous artery was catheterized and connected to pressure transducers for continuous recording artery pressure. Blood gas samples were taken for acid-base balance. The catheter was placed in the tail vein for fluid administration. Basic intravenous volume substitution was done with hydroxyethyl starch solution. The mean arterial pressure was maintained above 60 mm Hg. Urine production was determined by a self-constructed catheter placed directly into the urinary bladder with a small sharp skin and bladder incision. The rate of volume substitution was modified according to the continually controlled input-output balance to maintain cardiac output. Administration of potassium chloride and sodium bicarbonate, according to levels of potassium, bicarbonate, and base excess. Hemodynamic support was maintained with intravenous fluids without inotropic or vasoactive agents. A temperature probe was inserted in the rectum for core temperature measurements. Body temperature was maintained at 37°C with a heating pad.
All values are presented as means ± the standard deviation of observations. Statistical analyse were carried out using the t test with P ≤ .05 considered significant.
The majority of the 20 brain dead animals (90%) was considered satisfactory as organ donors after 9 hours ventilation. Of these 2 rats, 1 died as a result of failing mechanical ventilation; the other died as the result of dislocation of the saphenous blood pressure cannula.
The inflated balloon catheter caused brain herniation. Starting at the moment the balloon inflation started and ending just before lack of spontaneous respiration, 50 ± 1 minutes were needed for brain death induction. All rats showed a similar pattern during the gradual onset of brain death after inflation of the Fogarty balloon (at 40 minutes mean arterial pressure = 140 ± 12 mm Hg vs mean arterial pressure = 110 ± 10 mm Hg before brain death, P < .01, Figure 1). After this storm of catecholamines, blood pressure showed a sharp decrease to a level of 55 ± 1 mm Hg followed by hypotension. The duration of hypotension was 10 ± 1 minutes and was characterized by a gradual increase. The rats were stabilized with blood pressure (average mean arterial pressure, 60-110 mm Hg), and electroencephalogram tracings were flatline in brain dead animals.
As shown in Figure 2, the balloon pressure at the time of brain death was 0.82 ± 0.1 atmospheres for a gradual increase of intracranial pressure. Slow wave activity was shown before inducing brain death owing to deep anaesthesia in all rats (Figure 3A). Faster waves were seen during balloon inflation (Figure 3B). Cerebral electrical activity ceased in all brain dead rats (Figure 3C).
Typical signs of brain death, including hyper-tensive and hypotensive periods, diabetes insipidus, and tachycardia were shown in 18 rats. Table 1 shows living donor levels and values of blood gas and acid-base balance during 9 hours of brain death. Hemodynamic changes were not significantly different during the observation period.
One limitation associated with transplant is the lack of available organs. Living-donor grafts are associated with better survival and lower rates of delayed graft function than are organs retrieved from brain death donors.4,15,16 This indicates that brain death itself, or the management of the donors, is significant. To increase the quality and quantity of organs for transplants, there must be a clinically relevant brain death model.
Two methods have been used to induce brain death in the animal experiments. The first uses models with the balloon inflated for 30 to 60 seconds. However, this model often requires inotropic or hormone replacement therapy to maintain blood pressure at a normotensive level. In previous studies, decreasing the volume of the intracranial balloon maintains acceptable blood pressure levels, and these do not mimic the clinical symptoms. In our experimental, the balloon was inflated during 9-hour follow-up. The second used models are the so-called gradual models that balloon is gradually inflated within with 40 μL saline per minute. Blood pressure consists of a hypotensive period followed by a sharp peak. The prominent blood pressure peak that occurs during induction is frequently described as a result of releasing of catecholamines.14 The increase of catecholamines leads to vasoconstriction that increases the peripheral regional vascular resistance to high levels. A significant reduction in blood flow as a result of increasing of catecholamines leads to ischemia in peripheral organs.17 The higher mortality rates of myocardial damaged and pulmonary hemorrhages is related to higher levels of catecholamines.18 The tempo and extent of the cerebral insult has an influence on the extent of catecholamines.19 In our experiments, the mean arterial pressure of the peak was 140 ± 12 mm Hg. This effect was probably because of the reduced release of catecholamines.
Previously used brain death models have been using animal rats, dogs, mice, and pigs.13,20-23 These experiments investigate hemodynamic, neuroendocrine, and immunologic changes during and after brain death. However, brain death and donor care never exceeded 6 hours in brain dead rats. To maintain stable blood pressure, inotropic support, hormone replacement therapy, or manipulating the intracranial balloon volume is often used. Vasopressors have adverse events on several organs. In addition, no further drugs are given to avoid interference with the spontaneous hemodynamic situation.14 Stable blood pressure was maintained with hydroxyethyl starch. The saphenous artery puncture method has a success rate, is minimally invasive, saves time, and can be repeated for vascular manipulation compared with a femoral artery puncture.
The widespread physiologic changes that follow brain death have a high incidence of complications jeopardizing potentially transplantable vital organs. Adverse events include cardiac arrhythmias, metabolic acidosis, hypotension, and diabetes insipidus.24-29 In acidosis, the cardiovascular system has a decreased responsiveness to catecholamines that causes a decrease in blood and dilation of the blood vessels. Mechanical ventilators can provide oxygen and remove carbon dioxide. Ventilation should be adapted to maintain physiologic Pco2 at a level between 35 and 45 mm Hg with 30% oxygen in air. Additionally, sodium bicarbonate was used to correct metabolic acidosis by analysis of blood gas. Hydroxyethyl starch was a synthetic polymer of glucose that had been used to correct intravascular volume deficits and to improve systemic hemodynamics and microcirculation.30 Fluid replacement has been considered the cornerstone for hemodynamically unstable donors because hypovolemia-related hypotension was frequently present after braindeath.26 Small volumes of hydroxyethyl starch improve cardiovascular performance and provide the same regional hemodynamic and metabolic benefits of large volumes of isotonic crystalloid solutions.31 According to the continually controlled input-output balance, hydroxyethyl starch was used to maintain donor hemodynamic stability.
We established a reproducible animal model of brain death, analogous to the clinical situation of a massive primary intracranial hemorrhage, causing herniation of the brain and subsequent brain death. Our brain death model in rats has the advantage over previously used brain death models because of donor care and therapeutic strategies.
Volume : 12
Issue : 5
Pages : 469 - 473
DOI : 10.6002/ect.2013.0229
From the 1Department of Hepatobiliary and Pancreatic Surgery; and
the 2Key Laboratory of Hepatobiliary and Pancreatic Surgery &
Digestive Organ Transplantation of Henan Province, the First Affiliated Hospital
of Zhengzhou University, Zhengzhou, Henan, China
Acknowledgements: This study was supported by the National Natural Science Foundation of China. (Grant No.81171849). The authors have no conflicts of interest to declare.
Corresponding author: Shuijun Zhang, No.1, East Jian She Road, Zhengzhou, 450052, Henan Province, China
Phone: +86 371 66862124
Fax: +86 371 66970906
Figure 1. Mean Arterial Pressure During the Entire Experiment
Figure 2. Intracranial Pressure
Figure 3. Electroencephalogram of a Rat
Table 1. Hemodynamic Changes in Arterial Blood Samples Over Time