No-reflow as a post-mortem artifact

It is common medical knowledge that after 5 minutes of cardiac arrest the prospects of successful resuscitation without neurological impairment become progressively bleak. But there is less consensus on the mechanisms of such injury. One strong candidate is what is called the “no-reflow” phenomenon. No-reflow refers to the impairment of perfusion of the brain after circulation is restored. A number of researchers (e.g., Ames, Fisher) have demonstrated the existence of the no-reflow phenomenon in cerebral ischemia by perfusing the brain with carbon black after various periods of ischemia. Areas that are perfusable thus turn black, while non-perfusable areas of no-reflow do not.

In a remarkable 1992 paper by De Le Torre et al., the researchers report that the no-reflow phenomenon is only observed in the  presence of agonal or post-mortem cardiopulmonary failure.  Perfusion impairment was not observed in brains of rats that maintained stable cardiopulmonary function during, and received intravenous carbon black after, up to 30 minutes of cerebral ischemia. Clearly, these results are hard to reconcile with traditional explanations of no-reflow (e.g., blood rheology changes, edema-induced changes in the vessel lumen) and the authors offer little guidance in the discussion section of the paper on how to explain the finding that co-existence of stable cardiopulmonary function and global cerebral ischemia does not produce post-ischemic vessel filling defects after restoring blood flow to the brain.  Unless blood flow to the brain was not completely eliminated in the animals in which stable cardiopulmonary function was maintained it is difficult to imagine a mechanism of no-reflow that makes sense.

It may be that reperfusion of blood that has been circulating continuously throughout the body during the period of  cerebral ischemia accounts for improved perfusability of ischemic brains as compared to those reperfused after whole-body ischemia (i.e., cardiac arrest). Maintaining cardiopulmonary function during cerebral ischemia could improve subsequent perfusion of ischemic tissues by preventing red cell aggregation in the blood and/or inflammatory responses (e.g., neutrophil activation) initiated by reduction in, or lack of, blood flow. If this would be the case, the adverse effects of ischemia in the brain would be offset by the maintenance of physiological blood flow in the rest of the body.

The findings in this paper do not offer encouragement for the practice of human cryopreservation because cryonics patients invariably experience cardiopulmonary failure prior to stabilization intervention. Classic cryonics interventions such as administration of streptokinase and heparin do not seem to be very effective in reversing cerebral perfusion impairment,  which raises some important  questions about the phenomenon of post-mortem blood coagulation. The only effective intervention in animal research remains rapid hemodilution and high perfusion pressures, which is not easy to implement in typical cryonics casework. But why this intervention works in the light of the findings above remains something of  a mystery. Other interventions to prevent no-reflow after cardiac arrest in cryonics patients are currently being investigated and detailed reports will be forthcoming in the future.

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.