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Destined for destruction?

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Science  04 Oct 2019:
Vol. 366, Issue 6461, pp. 46
DOI: 10.1126/science.aaz3885

One need not look beyond the stroke ward to appreciate the brain's particular vulnerability to interruptions in blood flow. Cut off from circulation, brain function falls precipitously, leaving patients in critical, and sometimes, permanently disabled, states. Indeed, this is not surprising, if we consider the monumental metabolic needs for the fatty organ to accomplish its world-altering tasks.

Brain Cells Remain Viable After Prolonged Global Anoxia

Much evidence has brought into question the inevitability of cell death after prolonged cessation of cerebral blood flow. One observation is particularly important: Human brain samples can be harvested several hours postmortem and processed for cell and tissue cultures with sufficient viability (1, 2). Indeed, cell and slice cultures have been shown to be experimentally useful for studying not only individual brain cells (3), but also brain disorders, brain damage, and neuroprotection (4). How do we reconcile these observations with what is known about the susceptibility of the brain to anoxia?

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Because tissue culture studies show that cells remain viable even hours postmortem, we hypothesized that the same might be true of the fully intact brain. My research therefore centered on restoring cellular functions in the fully intact porcine (pig) brain after prolonged anoxia. To study this phenomenon, we developed a cytoprotective solution and technology to selectively perfuse the porcine brain 4 hours after death (5).

One Person's Trash is Another's Unexpected Model

Pig brains are readily discarded by the food production industry. They are also large, complex, and highly gyrified, with a composition similar to that of human brains (6)—important factors for simulating the pathophysiology of human anoxia (7, 8). By recovering these organs during the food production process, we possess a model for helping us understand how to recover cellular functions after prolonged anoxic injury.

Why Tackle One Problem, When There are Many?

Devoid of blood flow, brain cells activate multiple deleterious mechanisms that lead to cellular death, if left unchecked (911). Many “plumbing” issues arise when reinstating blood flow, including thrombotic, vascular, and perivascular mechanisms (1214). As such, anoxic or ischemic injury is a “dirty” state.

With this in mind, we incorporated cytoprotective agents within the perfusate to promote cellular recovery and to quench excitotoxicity, free radicals, oxidative stress, and other processes invoked during cell death. To overcome the aforementioned difficulties that might accompany the use of blood, we specifically designed the perfusate to be entirely synthetic and acellular. We also incorporated microparticles to allow us to monitor perfusion dynamics by ultrasonography (5).

Because the brain cannot survive without support, we also developed a platform that mimics lungs, kidneys, and a heart. The detoxification circuit, complete with a special exchange solution, emulates renal function and ensures proper maintenance of electrolyte, metabolite, and acid-base homeostasis; the artificial lungs control dissolved gases; and the simulated heart delivers perfusates under cardiac-like pressure Waveforms (5).

Probing for Cellular Viability

We began our investigations by perfusing pig brains with a control solution, which was devoid of cytoprotective agents, starting 4 hours postmortem. After 6 hours, the brains began to assume a custard-like texture, with no observable perfusion. Microscopically, the brain cells had disintegrated, making it difficult to distinguish intracellular spaces from extracellular spaces (5).

Brains perfused with the cytoprotective solution, however, maintained low-resistance perfusion for the entire 6 hours, indicating that our perfusate prevented brain swelling. Histological and immunohistological investigation of multiple independent brain regions demonstrated structurally intact, and morphologically normal, neurons, microglia, astrocytes, oligodendrocytes, and endothelial and ependymal cells.

Although cellular integrity was preserved, these data did not provide any evidence for viability. Given that our perfusate contained antiapoptotic agents, we hypothesized that there would be a reduction in the activation of caspase-3, the major execution protein in the apoptotic pathway.

We were able to demonstrate that intervention with our technology decreased apoptosis (5). However, again, we were left without any insight into cellular functionality.

To assess whether the intact cells were indeed functional, we began by testing vascular dilatory functionality, which we confirmed by administering nimodipine, a drug used to treat vasospasm. We observed an increase in flow within the brain—exactly how functionally viable vascular cells react (15).

Next, we probed whether our technology restored glial inflammatory activity by microinjecting lipopolysaccharide, a Tolllike receptor 4 (TLR4) agonist and immunogenic agent. We observed that glial cells increased the release of inflammatory molecules in response to the injections, suggesting that glial functionality was present (5).

After the perfusion, we microdissected the hippocampus and used whole-cell patch-clamp recordings, which revealed that pyramidal neurons were still electrochemically viable and demonstrating spontaneous synaptic activity. All evidence suggesting the presence of these cellular functions was further substantiated by the presence of a robust global cerebral metabolism, as measured by arteriovenous gradients (5).

We did not, however, detect a resurgence of global electrical activity within the brain by electrocorticography. This lack of activity may be due to the various antagonists present in the perfusate, or it may simply not be possible to reestablish synchronous activity in the postmortem brain. Longer perfusion studies may provide answers to this question.

Extending Perfusion for Long-term Experimentation

Our findings indicate that cells within the large mammalian brain are not as fragile as they were once considered. Indeed, they demonstrate an observable resilience to anoxic injury under specific conditions.

Currently, we are working toward extending perfusion time, which will help us to better understand the dynamics of cellular recovery after extended anoxia. These studies will lay the groundwork for the development of a new experimental platform and may ultimately help us understand how to salvage damaged or diseased brain cells.

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FINALIST Zvonimir Vrselja

Zvonimir Vrselja received his M.D. and Ph.D. from J. J. Strossmayer University in Croatia. He completed his postdoctoral training in the laboratory of Nenad Sestan at Yale School of Medicine, where he continues to work as associate research scientist. His research focuses on understanding how brain cells react to anoxic injury following circulatory arrest, and how such cells can be structurally and functionally recovered by developing a perfusion technology.

References and Notes

Acknowledgments: I am grateful to N. Sestan for his mentorship, to co–first author S. G. Daniele for fruitful and rewarding collaboration, and to all members of the Sestan laboratory.
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