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.

The red blood cell as a model for cryoprotectant toxicity

Various approaches are available to investigate cryoprotectant toxicity, ranging from theoretical work in organic chemistry to  cryopreservation of complete animals. Because resuscitation of complex organisms after cryopreservation is not feasible at the moment, such investigations need to be confined to viability assays of individual cells and tissues or ultrastructural  studies.

One simple model that allows for “high throughput” investigations of cryoprotectant toxicity are red blood cells (erythrocytes). Although the toxic effects of various cryoprotective agents may differ between red blood cells, other cells, and organized tissues, positive results in a red blood cell model can be considered the first experimental hurdle that needs to be cleared before the agent is considered for testing in other models.  Because red blood cells are widely available for research, this model eliminates the need for animal experiments for initial studies. It also allows researchers to investigate human cells. Other advantages include the reduced complexity of the model  (packed red blood cells can be obtained as an off-the-shelf product) and lower costs.

Red bloods cells can be subjected to a number of different tests after exposing them to  a cryoprotective agent. The most basic test is gross observation of the red blood cells in a cryoprotectant solution. When high concentrations of a cryoprotectant are used (such as in vitrification), a stepwise approach is necessary to avoid  osmotic  injury. If a cryoprotectant solution is extremely toxic rapid hemolysis will follow, which often can be observed by a noticeable change of the color of the solution,  red cell debris sinking to the bottom of the test tube, or negligible difference between the pellet (if there is one at all) and the supernatant after centrifugation. But if the researcher is interested in agents that are not extremely toxic, or wants to compare variants of  similar agents with each other, quantitative methods and detailed observations are required using respectively spectrophotometry and light microscopy.

In 1996, Bakaltcheva et al. used the red blood cell model for an elegant and thoughtful study  of cryoprotective toxicity. The authors did not only use spectrophotometry to measure hemolysis  but also used microscopy to study the morphology of the red blood cell after exposure to various agents at different temperatures. The results of these different measurements were in turn correlated with each other in order to determine if there are general properties  affecting cryoprotectant toxicity. The authors propose that reduced toxicity can be achieved by keeping the dialectric constant of the medium and membrane close to that of an aqueous solution without solutes.  These findings can also explain why cryoprotective mixtures  of various agents (such as DMSO and formamide) can reduce toxicity.  A general rule of thumb for formulating vitrification agents with reduced toxicity seems to be to maintain most properties of water but eliminating the posibility of ice formation. It should not be a surprise that such an approach has guided the choice of solvents in areas such as cryoenzymology.