INTRODUCTION

This article has been updated & replaced by my newer article Ischemia and Reperfusion Injury in Cryonics.

An ideal cryonics patient would suffer no ischemic damage. Such a patient would be in good health prior to a sudden-onset fatal event which did not affect brain tissue. The event would not be so sudden, however, as to prevent a cryonics transport team from being on site at the moment of legal declaration of death. Upon legal death, cooldown and restoration of circulation would begin immediately, followed by blood washout, perfusion with cryoprotectant solution and cooling to liquid nitrogen temperature.

A healthy experimental animal can be cryopreserved with up to 60 minutes of global ischemia and show good ultrastructure. Most signed-up cryonicists are not so lucky. 5-6% die without being frozen at all. Another 20% are autopsied. 5-6% die so suddenly that they experience long periods of ischemia, even though they are not autopsied. 20-30% experience organic brain damage due to Alzheimer's Disease, AIDS, brain cancer, etc. And another 20% experience long periods of pre-mortem shock during a slow dying process. That leaves a minority of signed-up cryonicists who get a cryopreservation that has not been preceeded by a great deal of brain tissue damage.

Ideal cryonics patients are the exception rather than the rule. Most cryonics patients are victims of some degree of ischemia, ie, tissue damage as a result of oxygen & nutrient deficiency following failure of blood circulation. This may happen during the dying process or following cessation of heartbeat -- often both. Even under the best circumstances, it is difficult to restore circulation to levels provided by a normal heartbeat.

Cryonicists have focused a great deal of attention on freezing damage and the potential of future science to repair that damage. The rationale for this argument is good, namely, that freezing damage is much like a broken window -- the pieces are still there, and molecular repair technology may be able to re-assemble them. But ischemic damage could be potentially much more serious -- leading to such degradation of structure that no future technology could possibly reconstruct. It may be that the prevention of ischemic damage may be the key critical factor toward future reconstruction of cryonics patients. Concern with freezing damage could fatally distract attention from this fact. With this in mind, it is worth discussing what happens to brain tissue during ischemia.

MECHANISMS OF BRAIN-TISSUE ISCHEMIC DAMAGE

As is shown in the flow-chart [from THE LANCET 339:533-536 (29Feb92)] ischemia results in lowered oxygen & glucose delivery to brain tissue. As a result, cells have less ATP production, ie, less available energy. Although the brain may represent only 1% of total body weight, it can account for 20% of its energy production. Most of this energy is consumed by ATP-driven ion pumps which keep Calcium (Ca²⁺) & Sodium (Na⁺) outside of cells and Potassium (K⁺) inside of cells. In ischemia, the loss of energy means that the ion gradients essential for functional neurons cannot be maintained. EEG becomes flatter and extracellular K⁺ slowly increases. Then voltage-gated ion channels open, resulting in a large increase in intracellular Na⁺ and Ca²⁺ accompanied by a large increase in extracellular K⁺.

Calcium entry into the pre-synaptic membrane of a neuron is a key factor in neurotransmitter release. In the brain, glutamate is the predominant excitatory neurotransmitter. In a normal brain, glutamate release at a synapse results in normal signalling. In ischemic brain tissue, glutamate release can result in increased calcium entry into the postsynaptic cell, creating a positive feedback loop of calcium penetration & glutamate-release known as excitotoxicity. Intracellular calcium activates phospholipase, which degrades membrane phospholipids. This not only damages the membrane, it releases the toxic free fatty acid arachidonic acid, which forms substantial amounts of oxygen free radicals. Calcium also activates enzymes which degrade protein & DNA, although this effect is less pronounced than the lipase activation.

The illustration [from JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 9:127-140 (1989)] depicting presynaptic & postsynaptic ion channels provides greater insight into the mechanisms of excitotoxicity. Calcium can enter a cell through voltage-controlled ion channels of the L-type (long-lasting) or the N-type. Calcium can also enter a cell through agonist-controlled ion channels -- the agonist in question being the neurotransmitter glutamate. Glutamate-activated receptor/channel complexes are named after their most potent agonists. The Kainate (K) and Quisqualate (Q) channels operate so similarly that they are often described as K/Q channels (also known as AMPA channels). K/Q channels pass K⁺ & Na⁺ in response to glutamate stimulation, but do not pass Ca²⁺. It is the NMDA (N-Methyl-D-Aspartate) glutamate receptors that can pass Calcium. Neurons vary in their receptor composition, so it is noteworthy that the pyramidal CA1 & CA4 neurons of the hippocampus (which have high concentrations of NMDA receptors) are especially vulnerable to being killed by ischemia. Magnesium, which blocks entry of Calcium through NMDA channels, is particularly protective of hippocampal neurons [THE JOURNAL OF NEUROSCIENCE 10(8):2493-2501 (1990)].

Normally, total brain Calcium is about twice as great extracellularly as it is intracellularly. But only about 0.1% of the intracellular Calcium is in ionized form, as compared to half of the extracellular Calcium. With the progression of ischemia, Calcium is increasingly released from endoplasmic reticulum. This release of bound Calcium from intracellular sites explains why ischemia therapies designed to limit entry of Calcium into the cell are more effective in early stages of ischemia than in the later stages.

Not all ischemic damage to neurons is Calcium-initiated. Anaerobic metabolism increases intracellular lactic acid & lowers pH. Hydrogen ions facilitate iron-mediated free-radical mechanisms. But the vast majority of free-radical damage to cells is through peroxidized unsaturated fatty acids, whereas free-radical damage to proteins & nucleic acids is relatively modest [RESUSCITATION 23:59-69 (1992)]. The notion of lysosomes as "suicide bags" is something of a myth -- lysosomes remain stable through ischemia, only bursting after cell death [AMERICAN JOURNAL OF PATHOLOGY 68:255-288 (1972)].

Brain damage in ischemia is not restricted to neurons. The glial cells known as astrocytes normally soak-up extracellular glutamate. But during ischemia, astrocytes swell-up considerably due to extracellular K⁺ -- and releasing glutamate & vasoconstrictive substances.

Neurons swell too, with the entry of Na⁺ & Cl^- , and this cellular swelling can aggravate ischemia by compressing blood vessels. Platelets release arachidonic acid, adding to that formed by cell membrane breakdown. Extracellular arachidonic acid contributes to glial cell swelling and increases permeability of the blood-brain barrier. Moreover, arachidonic acid potentiates the formation of eicosanoids, which increase aggregation of blood cells and constriction of blood vessels. Polymorphonuclear leukocytes further occlude microcirculation and release reactive metabolites.

REPERFUSION INJURY

It seems desirable to re-initiate cerebral blood flow for the purpose of perfusing brain cells with cryoprotectant. But this can result in a phenomenon known as "reperfusion injury". Indeed, in the absence of oxygen & glucose (ie, without reperfusion), no histological damage to cells is evident for more than an hour [ACTA NEUROPATHOL (BERL) 43:85-95 (1978)], and most of the structural damage seen in the first 2 hours is to mitochondria and ribosomes. Rather than resuscitating cells, the re-entry of oxygen by reperfusion may enhance free-radical damage. (Some practicing cryonicists will not do reperfusion for normothermic ischemia in excess of one hour.) An attempt to re-start the circulation may meet with a resistance known as the "no-reflow" phenomenon. Increased blood pressure is required to overcome "no-reflow" due to vasospasm, increased blood viscosity and edema [ANNALS OF EMERGENCY MEDICINE 22(pt2):324-340 (1993)].

For cryonics purposes, the increased permeability of the blood-brain barrier due to arachidonic acid and other ischemic damage may actually facilitate perfusion of cryoprotectant. Even cell membrane damage may be advantageous for an analogous reason.

CHEMICAL INTERVENTIONS IN ISCHEMIA

In the early stages of ischemia, treatment with glutamate antagonists such as dextromethorphan [an ingredient in OTC (ie, over-the-counter) cough mixtures] can be of benefit [SCIENTIFIC AMERICAN July 1991, p.56-63]. Dihydropyridine (DHP) derivatives, such as nimodipine, block L-type Calcium channels. But the main benefit of DHPs in ischemia seems to be through arteriole dilatation rather than neuron Calcium-channel blocking. Pre-treatment of dogs with nimodipine prior to ten minutes of ischemia led to an 80% normal recovery rate, as compared with an 86% death rate in untreated controls. Treatment 2 minutes post-ischemia had a negligible effect. [PHARMACOLOGY OF CEREBRAL ISCHEMIA, Joseph Krieglstein, Editor, p.65-73 (1988)].

Substances which reduce brain metabolism such as barbiturates and anti-convulsants (phenytoin) are also of benefit. Anti-clotting agents can help. Pre-treatment with anti-oxidants such as Vitamin C, Vitamin E and CuZn-SOD (SuperOxide Dismutase) in liposomes (spherical lipid droplets suspended in water) is advantageous [ANNALS OF NEUROLOGY 21:540-547 (1987)]. Deferoxamine can reduce iron-mediated free-radical damage -- and mannitol scavenges hydroxyl radical [CHEM.-BIOL. INTERACTIONS 72:229-255 (1989)]. Aminosteroids scavenge superoxide and lipid radicals, counteracting the effects of arachidonic acid [JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 14:1030-1039 (1994)].

BENEFITS OF HYPOTHERMIA

A temperature reduction from 37ºC to 26ºC completely inhibited Potassium-induced neurotransmitter release from rat astrocytes [JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 15:409-416 (1995)]. Rats reperfused after a 15-minute ischemic period had over 3 times as many hydroxyl radicals one hour later than rats subject to ischemia, but not reperfused. But rats reperfused at 30ºC rather than 36ºC had half as many hydroxyl radicals as the 36ºC reperfusion rats. [JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 16:100-106 (1996)]. Dogs showed normal brain recovery after 60 minutes at 15ºC, and even longer periods of ischemia were tolerated at 4ºC to 7ºC [THE JOURNAL OF TRAUMA 31(8):1051-1062 (1992)].

CONCLUDING REMARKS

Ischemia should definitely be minimized, but the cost/benefit tradeoffs of expensive cryonic protocols are difficult to assess. Demonstrable reduction of structural damage should be the key deciding factor, however it still must be decided which organelles are essential for reconstruction. Loss of information about water molecules and mitochondria would probably not be critical. Loss of cell membranes, however, could mean loss of information about synapses.

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