Microvasculature perfusion failure in cryonics

Under ideal circumstances cryonics patients are stabilized immediately after pronouncement of legal death by restoring  blood flow to the brain, lowering temperature, and administering medications. In most cryonics cases, however, there is a delay between pronouncement of legal death and start of cryonics procedures. In some cases there are no stabilization interventions at all. Provided that these periods of warm and cold ischemia are not too long, such patients can still be perfused with a vitrification agent. But how thorough cryoprotectant perfusion (and thus vitrification) in these cases can be remains an unresolved issue.

Since the late 1960s a number of studies have been published that document that cerebral blood flow cannot be completely restored after prolonged periods of cerebral ischemia. Brains that have been perfused with black  ink after increasing periods of ischemia have shown progressive development of no-reflow areas in the brain (as evidenced by the absence of ink). In 2002 Liu et al. used a technique that allows direct visualization of trapped erythrocytes by treating fixed brain tissue with sodium borohydride (NaBH4), which renders trapped erythrocytes fluorescent. In a rat model of focal ischemia the authors found that a significant fraction of the capillary bed (10% to 15%) in the penumbra (the area surrounding the ischemic core) is blocked by trapped erythrocytes, even after 2 hours of reperfusion.

The authors discuss a number of clinically relevant issues. They propose that the lower density of trapped erythrocytes in the ischemic core of the brain reflects hypoxia-induced lysis (which releases cytoxic hemoglobin). They further speculate that the older ink methods may have underestimated the degree of no-reflow because areas that are not accessible to red blood cells may still be accessible to other molecules. This presents an opportunity to deliver oxygen to the brain by using small oxygen carrying molecules such as perfluorocarbons.   The authors did not investigate variations in perfusion pressure or the efficacy of volume expanders to restore no-flow areas to circulation.

A focal ischemia model is not a good model for cryonics and one can only speculate what the effects of various periods of complete ischemia would be on cerebral blood flow and erythrocyte trapping. Older studies on ischemia and perfusion impairment, however, indicate that periods of 30 minutes of complete ischemia can produce substantial areas of no-flow in the brain. Unless these areas are opened to circulation during either stabilization or cryoprotectant perfusion, straight freezing of  pockets of the brain is a real possibility. It remains to be investigated if areas that are obstructed by trapped red blood cells are accessible to cryoprotectant agents and  how much of  these areas can be opened by a combination of hemodilution and non-penetrating perfusate components (through dehydration). Although cryopreservation of  ischemic brains is the norm in cryonics, our knowledge about the effects of ischemia on vitrification of the brain remains limited.

Promoting cerebral blood flow in cryonics patients

It has been shown that perfusability of the brain is significantly compromised after long-term (>5 min) ischemic events (the “no reflow” phenomenon). Improving cerebral blood flow after circulatory arrest is one of the fundamental objectives of human cryopreservation stabilization protocol.  To that end, cryonics organizations administer the resuscitation fluid Dextran-40 and the drug Streptokinase to dilute the blood (and inhibit  red cell aggregation / cold aggulination) and  break up blood clots, thereby improving macro and microvascular circulation. Research by Fischer and Ames, who investigated the effects of perfusion pressure, hemodilution, and anticoagulation (i.e., the use of heparin) on post-ischemic brain perfusion, indicated that hemodilution is the most effective component of the post-ischemic perfusion protocol for enhancing brain perfusability. However, a later study by Lin, et al. (1978) reported significant improvement of cerebral function and blood flow with combined dextran and Streptokinase administration after cardiac arrest in dogs.

In their study, the researchers measured regional cerebral blood flow and cardiac output as well as EEG (i.e., brain wave activity) during five hours of post-resuscitation physiological maintenance following 12-16 minutes of cardiac arrest. Animals were divided into three groups as follows:

Group I:   no treatment

Group II: 1 g/kg dextran 40 in 10% saline following arrest and 10 mg/kg/minute during the five hour maintenance period

Group III: combined therapy of dextran-40 and Streptokinase — same dose of dextran as Group II and 5,000 u/kg rapid infusion and 25 u/kg/minute during the five hour maintenance period

The duration of flat EEG was significantly shorter in Group III animals (20 to 45 minutes with a mean of 28.8 +/- 2.8) than in Groups I (20 to 120 minutes with a mean of 59.5 =/- 10.8) or II (20 to 62 minutes with a mean of 46.9 +/- 4.8) and showed a faster recovery pattern than in Group I (significant difference was reached at three hours). Group II also showed a faster EEG recovery than Group I, reaching significance at five hours.

Cerebral blood flow, particularly in the hippocampus and grey matter (the areas most detrimentally affected by ischemia) in Group III was significantly improved as compared to Group I as early as three hours post-arrest, and was greater than that in Group II (significantly better only in the hippocampus). There was no difference in cardiac output found between the treated and untreated groups. All groups suffered a decrease in cardiac output of nearly 50% of baseline level (measured at 3 and 5 hours post-arrest).

Hematocrit — the proportion of blood volume occupied by red blood cells — was measured in each group and was found to be significantly increased during the post-arrest period in Group I, decreased to 25% of the baseline measurement in Group III (at both 3 and 5 hours post-arrest), and unchanged in Group II.

The authors speculate that “the improvement in cerebral circulation at the microvascular level after infusion of low molecular weight dextran was thought to be 1) related to the rapid increase in plasma volume with resultant lowering of hematocrit and reduction in blood viscosity, 2) a direct effect on the RBC [red blood cell] which increases its negativity and reduces the tendency to cellular aggregation.” They also note that though some doubt had been cast by the Fischer and Ames paper on the hypothesis of vascular endothelial cell swelling as a cause of no reflow, they did observe a higher proportion of smaller diameter capillaries in ischemic brains as compared to controls, and that “if capillary narrowing does play a role in microvascular deterioration, then hemodilution and prevention of cellular aggregates such as occurs with dextran would be beneficial in minimizing poor flow in narrow capillaries.”

Taken together, these findings indicate that combined dextran-40 and Streptokinase therapy improve brain perfusion after cardiac arrest — at least for arrest periods of up to 16 minutes.– supporting the choice for these agents in cryonics. One limitation of this study, however, is that the experiments did not include a group which received only Streptokinase. Including a Streptokinase group would have given more  precise data about the individual effects of the two agents in improving post-ischemic cerebral blood flow. Recent clinical trials with clot busting agents in cardiac arrest have failed and some contemporary authors question the phenomenon of post-arrest blood clotting. Perhaps streptokinase is useful in the treatment of circulatory arrest but its efficacy is dependent upon other blood flow improving interventions such as hemodilution. The case for post-ischemic hemodilution (and interventions to reduce RBC aggregation) is strong but the case for antithrombotic therapy in cryonics (and resusctation medicine) remains to be made.

Incomplete ischemia during cardiopulmonary support

One concern about prolonged cardiopulmonary support in cryonics is that its decreasing effectiveness may not be able to meet cerebral oxygen demand, and may even become detrimental. Some investigators have  observed that severely reduced flow (cerebral blood flow less than 10% of control) to the brain may actually be more harmful than no flow at all.  Explanations of why incomplete (“trickle flow”) ischemia may be worse than complete ischemia include aggregation of slow moving blood cells,  glucose-induced excessive lactate production, and oxygen-induced free radical damage to membranes.

In contrast, a study by Steen et al. concluded that some blood flow is better than no flow at all. The authors found that dogs could sustain only 8 to 9 minutes of complete ischemia but 10 to 12 minutes of incomplete ischemia (cerebral blood flow less than 10% of control) without neurological impairment. These results are at odds with the findings of Hossmann et al. who found better electrophysiological recovery in cats and monkeys after complete ischemia, and studies by Nordstrom et al. who observed increased metabolic recovery in rats after complete ischemia.

The authors speculate that these differences may reflect the different durations of (in)complete ischemia. Hossmann et al. studied 60 minutes of ischemia and Nordstrom studied 30 minutes of ischemia. The authors note that the durations they studied (8-14 minutes) are more clinically relevant because neurological recovery with contemporary technologies is not possible after 30 or 60 minutes of cerebral ischemia. Although these findings provide support for restoration of any kind of cerebral circulation after cardiac arrest, it does not offer much guidance in evaluating the practice of prolonged cardiopulmonary support in cryonics.

The authors also draw awareness to the difficulty of correlating electrophysiological and metabolic recovery to neurological recovery. They quote a study by Salford et al. who observed some return of metabolism even though histological abnormalities had already been developed. Such studies warrant caution about using return of electrophysiological activity as an indicator of cerebral viability because it is not likely that such viability can be sustained over the long term, let alone predict functional recovery of the brain.  It is doubtful that viability in the latter, stricter, sense can be maintained during stabilization of most, if any, cryonics patients. At best, the studies that demonstrate recovery of electrophysiological and metabolic activity after prolonged cerebral ischemia offer hope that such periods of circulatory arrest do not produce acute information-theoretic death.

No metabolic or histological evidence was found to support the implication of no-reflow, lactate accumulation, and free radical damage in incomplete ischemia.  Again, the authors speculate that no-reflow may be more pronounced during longer periods of incomplete ischemia, an observation that seems to be indirectly supported by Fisher et al. who observed progressive impairment of perfusion for longer periods of ischemia.

Cryonics patients often experience shock, blood coagulation abnormalities, and decreased cerebral perfusion prior to pronouncement of legal death and cardiopulmonary support.  An additional complicating factor in cryonics is that cardiopulmonary support is often supplemented by induction of hypothermia and administration of vasopressors and neuroprotective agents. Although the paper by Steen et al. addresses a lot of issues that are important to evaluate cryonics procedures, it is clear that for real empirical guidance regarding the wisdom of prolonged cardiopulmonary support specific cryonics research models are required.