Evaluating the translational value of postmortem brain reperfusion technology

2.1 Hypothermia

The study has been described many times as occurring “at normal body temperature” [e.g., 2, p. 304]. Vrselja and colleagues stated that “functions in the large mammalian brain can be restored under ex vivo normothermic conditions” [1, p. 342]. However, the assertion that the observed metabolic and other activities took place under normothermic conditions is inaccurate, and correcting this inaccuracy is important for evaluating the scientific and clinical implications of the study. A brief review of the methodology with an emphasis on temperature is appropriate.

The research animals used in the study, about 300 pigs, were obtained from a slaughterhouse. The researchers were present at the slaughterhouse when the pigs were killed by exsanguination and were then decapitated. After the animals were decapitated, the researchers quickly performed some basic procedures to prepare the head for subsequent study, including isolating the major arteries and attaching catheters to them. “In total, these procedures required approximately 10 min to complete, thereby subjecting brains to this length of warm anoxia” [1, p. 344].

The vasculature was then flushed with eight liters of cold (20°C) heparinized saline in a 30 min, three-step procedure involving gravity flush, then a powered closed-loop flow-driven flush, followed by a second gravity flush, to clear the vasculature while rapidly cooling the brains [1, p. 344]. Hence, blood and other fluids, and especially toxic by-products of ischemic anoxia, cytotoxic edema, and cellular necrosis, as well as thrombi – all predictable outcomes of exsanguination plus the 10 min of warm ischemia during the initial procedures – were quickly washed out of the vasculature, and the brains were rapidly cooled using the cold flush.

After the cold flush, the heads were put on ice and transported to the Virginia Tech lab nearby [4]. Apart from 30 min of exposure to room temperature during a craniectomy at the lab, the heads remained on ice for 4 h. After the craniectomy, epidural temperature was 12–15°C [1, p. 344]. When the experiment began, 4 h after decapitation, the brains were rewarmed at a rate of 6°C per hour, from 25 to 37°C [1, p. 344]. This temperature was presumably measured deeper within the brain than the epidural surface, whose temperature was 10–13°C colder than 25°C, as described above; however, the site of measurement during the experiment is not described. The brains reached maximum temperature of 37°C, 2 h into the experiment. The brains were not at any point rewarmed beyond 37°C.

Critically, normal body temperature is species-specific. While 37°C is normothermic for a human, it is hypothermic for the animals used in the study (Sus scrofa domesticus), whose normal core body temperature ranges from approximately 38 to 40°C, with the mean approximately 39°C [5,6,7].

In other words, the brains were rapidly flushed of blood and endogenous toxins and cooled with an anticoagulant fluid, then placed on ice for several hours, during which they remained profoundly hypothermic. When the study began, the brains were severely hypothermic; 2 h into the study, the brains reached maximum temperature of 37°C, which is mild hypothermia. But at no time at all did the pig brains actually reach normothermia for pigs.

Furthermore, normal core body temperature for mammals is cooler than normal brain temperature, due to the high metabolic activity in the brain. For example, one review comparing brain temperature to core temperature in humans found that brain temperature was higher on all measures of core temperature (rectal, blood, etc.) in all 15 studies reviewed; this difference ranged from a mean of 0.39 to 2.5°C [8; see also 9].

Therefore, the phenomena observed by Vrselja and colleagues [1] were all observed at different degrees of hypothermia, but never at normal core body temperature. Notably, it has been known for centuries that hypothermia protects brain tissue.

2.2 Physiological and biochemical milieu: experimental preparation versus clinical setting

The cold saline flush is scientifically and clinically important for a second reason: It renders the physiologic conditions in the animals’ brains dramatically different from a real, clinical setting of human anoxic brain injury. In a clinical setting, ischemia-hypoxia causes metabolic acidosis, as lactic acid builds up from the process of anaerobic glycolysis. In turn, brain acidosis, as well as tissue injury, stimulates release of inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), thereby triggering systemic inflammation as well as the activation of coagulation pathways, which can cause a variety of systemic coagulopathies [10,11,12,13]. Furthermore, due to decreased availability of energy (via decreased production of adenosine triphosphate, or ATP), the sodium-potassium pump becomes dysfunctional, leading to extrusion of potassium from the cell and intrusion of sodium and water, along with calcium ions, leading to cytotoxic edema [14, p. 23], cell membrane damage or rupture, and cellular necrosis; and all of these factors increase brain swelling and thereby also increase intracranial pressure [12,13]. If severe enough, this can result in brain herniation [15].

These physiologic derangements in the brain also have cascading and reciprocal effects on the rest of the organism, such as systemic inflammation which in turn can exacerbate brain injury; liver damage from coagulopathies can exacerbate brain injury via an accumulation of ammonia in the nervous system [10, p. 800–801]; “catecholamine storm” can occur, which involves unopposed release of massive amounts of sympathetic, stimulant hormones that cause tissue injury including to the myocardium [16,17]; dysfunction of hypothalamic areas results in multiple neuroendocrine derangements which also alter brain chemistry [13]; and so on.

To summarize, the physiologic consequences of lack of blood and oxygen to the brain include a large variety of endogenous toxins, which themselves cause further damage in a cascading and reciprocal fashion, both to the brain itself and in a systemic fashion to the organism as a whole. By immediately washing out these toxins from the pigs’ brains, and by washing out the blood and its products of coagulation, the biochemical, physiologic conditions of the experimental animals’ brains are radically different from a real clinical setting.

Finally, the fact that the animals were decapitated is also physiologically relevant, since clinical medicine must address the human organism as a whole, including the cascading and reciprocal effects throughout the organism of a variety of pathological conditions that can be induced by hypoxic-ischemic brain injury.

2.3 Confounds: traumatic brain injury and lifelong stress

Following standard slaughtering procedures, all of the research animals were stunned with a combination electric-mechanical stun device that strikes them on the head. The purpose of this is to render the animal unconscious before hanging it upside down for death by exsanguination by severing carotid arteries. However, the stun method is known to be not uniformly effective, and some animals remain conscious [18]. This is important from a scientific perspective for two reasons.

First, all of the research animals were struck with a device that, by design, causes traumatic brain injury. This is itself an important confound in a study of brain function. Second, since the device does not always work, or at least does not always work the same way, we do not know that the traumatic brain injury caused to the pigs was uniform across subjects, and this can be the case even when the animal is stunned to unconsciousness, because there could be different degrees of neurological trauma that cause unconsciousness, and there could be small but meaningful differences in the angle at which the stunner hits the animal, leading to differences in areas and degree of brain trauma.

As a scientific principle, all the animals should be as similar to each other as possible, and especially, relevant confounds need to be avoided or addressed in some way. In a study of brain function, traumatic brain injury in all the research animals is an important confound; compounding this problem, different degrees or manifestations of that brain injury may render the animals’ brains relevantly different from each other; and there is no way to rule this out. This violates the basic scientific principle of using relevantly similar animal subjects to make generalizable conclusions.

Finally, as livestock, the animals were subject to intense physical and psychological stress throughout their brief lives, including being removed early from their mothers, extreme confinement, and cutting off tails and pulling out teeth without anesthesia or other pain management; highly confined and stressful transportation to the slaughterhouse; and the environment of the slaughterhouse itself, with sights, sounds, and smells of blood and death [18,19,20]. This intense stress has well-known impacts on physiology, including on the hypothalamic-pituitary-adrenal axis, the hypothalamic-pituitary-gonadal axis, sympathetic upregulation with consequences for cardiovascular and other functions, immune system dysregulation, and others [20,21]. These stressful conditions, again, were uncontrolled, and thus generate further confounding conditions.