for Nanotechnological Repair
of the Frozen Human Brain
Reprinted from Cryonics: Reaching for Tommorow, Alcor Life Extension Foundation, 1991.
Guidelines are suggested for designing realistic and defensible
nanotechnological repair scenarios for the frozen human brain. A scenario is
developed which is based on a) replacing brain ice with repair networks below Tg,
b) carrying out gross structural repairs at temperatures in the range of about
-100 to -30 degrees C, and c) carrying out most intracellular repairs at more
elevated temperatures, relying in part on ordinary biological self-assembly and
self-repair for carrying out much of the work required. The presently suggested
scenario is intended as a rough outline that can facilitate rational discussion
of the feasibility of repair. No mathematical analysis is attempted in this
first specific description of "realistic" approaches to the repair of
the frozen human brain.
"Realistic" scenarios for repair are defined here as scenarios that
might actually be applied, with appropriate modifications, to the restoration of
the brains of patients in cryonic suspension. These may be distinguished from
general proofs-of-principle (1,2,3) that attempt to demonstrate general
feasibility without considering documented biological problems in detail, or
that present the limits of the possible without considering what is most
efficient, practical, and likely. No "realistic" repair scenarios have
previously been proposed to the knowledge of the author.
II. Desirable Attributes
of Repair Scenarios
Scenarios for repair of the frozen human brain should satisfy a number of
important requirements. Although the scenario proposed here is based on the
following guidelines, it does not include a self evaluation of feasibility (see
"Testability" below) and does not attempt to be a fully-developed and
fully documented work.
Factual Basis. First, realistic repair scenarios must be based on what is known
or can be inferred about the nature of the actual injury present in frozen
brains and frozen brain tissue. This is mandatory because, by the definition
above, a realistic scenario attempts to set forth specific approaches to solving
the problems of repair, which is impossible if the problems are not clearly and
accurately identified. It is essential to avoid addressing problems that do not
exist and to avoid overlooking important difficulties that are likely to arise.
Focusing on real problems is perhaps the best way to realize a technically
Parsimony. Second, the repair scenario should be parsimonious. It should not
attempt to do more than the minimum amount of work required for a satisfactory
result. The reason for this is that smaller jobs are more easily and more
credibly solved than larger ones, and the goal of a repair scenario is to
demonstrate the feasibility of repair. The fewer the number of tasks required
for repair, the more likely it is that these tasks can be accomplished. This of
course does not mean that any real problem should be minimized or ignored, but
it does mean that needless tasks (such as keeping track of individual sodium
atoms) should not be considered. Parsimony requires distinctions to be made
between what is important and what is not important.
Detail. Third, parsimony in selecting problems to solve does not necessarily
imply parsimony in describing the details of the problem solving process. Too
much detail is likely to be confused with fortune telling, which would be
inappropriate. However, when the appearance of fortune telling can be avoided,
hard detail lends reality to the scenario, particularly if it is backed up by
numbers. Thus, rather than just saying, for example, "the fractures will
[somehow!] be removed from the data base," it would be more plausible to
spell out in detail a defensible molecular plan of attack on the problem.
Testability. Fourth, of course, the repair scenario must be physically
achievable. Ideally, it should be possible to evaluate each step of the repair
scenario quantitatively to test its feasibility. Each step should also be
specific enough for a more general evaluation of its feasibility to be made. In
short, the scenario should be testable and falsifiable in all ways possible. A
recent criticism by Fahy of previous discussions of repair of frozen brains (4)
focused in part on such evaluations, e.g., evaluating the feasibility of
providing power for nanoprocedures at cryogenic temperatures. Although the
latter criticism and many similar points will not be addressed here, a
consideration of such details is important for fully-developed scenarios.
Defining The Problem:
I. What Is and Is Not Considered
The repair scenario described here deliberately ignores extraneous problems such
as postmortem damage, transport injury, circulatory obstruction, and previous
traumatic brain damage or other devastating types of cerebral deterioration
because these problems represent side issues and are not inevitable given the
level of hospital cooperation that has frequently been achieved. Furthermore,
legal changes could allow freezing to be carried out under much better
conditions, so that fewer such problems will arise. Other types of repair
scenario should if necessary (and possible) be devised to address these problems
separately. However, the damage caused by cryoprotectant perfusion prior to
freezing is discussed.
The repair scenario will assume conventional preservation, i.e., the brain is
not fixed prior to freezing: repair scenarios for fixed brains are likely to be
substantially different from what follows, for substantially different problems
will be present. It will also be assumed that the "Smith Criterion"
(5) is attained or bettered: grossly inadequate concentrations of cryoprotectant
will produce such massive mechanical damage (6) that repair, if it is possible
at all, may well depend on different principles than those described here.
The discussion that follows in the next two sections is necessarily based on
limited information, some of which may be misleading. It goes without saying
that more research is needed on every point discussed. The incompleteness of
present information, however, does not appear to be sufficient to preclude
meaningful evaluations and the development of reasonable repair scenarios. The
format of the next two sections will consist of a series of statements
describing potential problems followed in each case by an evaluation of the
problem's likely relative seriousness.
II. Perfusion Damage and
Rabbit brains fixed after perfusion with glycerol at temperatures above 10-15
degrees C and examined histologically do not show loss of ground substance or
substantial morphological alterations (6). Glycerol perfusion under low
temperature conditions can, when concentrations are high (6 M), cause extensive
shrinkage of the brain as a whole and of the component cells and processes, with
distorted histological staining (7, unpublished observations).
Evaluation. Cell shrinkage may cause problems similar to those that occur during
freezing, and will thus be considered in more detail below. Perfusion, unlike
freezing, however, may remove proteins and cellular debris from the brain.
Unless enormous concentrations of glycerol are used, however, extensive protein
loss from previously undamaged brains appears unlikely. Altered staining
implies, at worst, altered chemistry, particularly since staining is done when,
presumably, all glycerol has been removed from the tissue. However, structural
preservation of such brains is apparent (6,7), and it is quite possible that
altered staining is the result of unusual fixation in the presence of glycerol
or similar artifacts. If chemical changes have taken place as a result of
glycerol exposure, these changes should be reversible due to their stereotypical
nature and the identifiability of the chemically modified sites: actual
information loss is not likely. Moreover, these chemical changes do not appear
likely in the usual case, in which brains are perfused with lower concentrations
of glycerol than 6 M.
Some chemical alterations by glycerol are likely in even the best situations.
These alterations, however, primarily should be altered levels of ordinary
metabolic intermediates due to the actions of enzymes on glycerol (8) or due to
the differential effects of glycerol on the kinetics of different enzymes (9).
As such, they are fundamentally trivial and close to being spontaneously
reversible. Glycerol has apparently never been documented to denature any
protein under any conditions, with the possible exception of glycerol in
concentrations in excess of 95% w/w (10), a condition not remotely approached in
cryonic suspension, even during the freezing phase of the process (11).
III. Freezing Damage: Significance of Different Types of Damage
1. "Biochemical/biophysical" Freezing injury. Imagine the appearance
of a "frozen" brain cell. Approximately 60% of the volume of the brain
has been converted into extracellular ice (5). Freezing has extracted large
fractions of the intracellular water and thereby reduced cell volume (12). This
in turn has reduced cell surface area, which has the potential of forcing an
expulsion of membrane material from the plane of the membrane (see below). The
combination of cellular shrinkage, lowered temperature, and elevated glycerol
concentrations may cause the following kinds of damage.
a) Extrusion of pure lipid species from the plasma membrane, either on tethers
(13,14) or as free lipid droplets in the cytoplasm (15) or in the extracellular
space (proportional to the reduction in membrane surface area produced by
Evaluation. This is a phenomenon seen so far only in plants. Shedding of lipid
to free-floating extracellular droplets has not been seen even in plants. As
long as any lipid extrusions are intracellular or still attached to the cell of
origin, it should be clear where to redistribute the lipid on warming, if
necessary. The main difficulty arises from the inability of lipid extruded in
this way to spontaneously return to the plane of the membrane during volume
expansion on thawing: restoration of approximately isotonic volume near the
melting point causes cellular lysis in plant cells (13-15) due to inadequate
membrane surface area. This should be a relatively easy problem to address and
does not involve appreciable information loss on freezing.
b) Loss of membrane proteins (possibly including hormone receptors and potassium
channels) into the extracellular space.
Evaluation. Loss of glutamate receptors has been documented in brain tissue
frozen without cryoprotectant (16). The number, state, and precise anatomical
distribution of potassium channels in hippocampal dendrite membranes probably
encodes memory to a large degree (17), so the potential loss of these membrane
proteins is of concern. However, significant neurotransmitter receptor loss has
only been seen when no cryoprotectant was used at all, in two papers in the
literature (16). In all other cases (18), even when only low concentrations of
extracellular cryoprotectant (sucrose) were present (19), all functions tested
have been present, implying proper retention of membrane proteins. Even
intracellular organelles do not redistribute/mix proteins to a worrisome degree
after brain tissue is frozen and thawed (20). Thus, even excessive cellular
shrinkage prior to freezing (caused by inadequate penetration of glycerol)
superimposed on subsequent freeze-induced cell shrinkage should not subject
cells to greater osmotic stress than has been shown experimentally (by freezing
with only extracellular sucrose present as cryoprotectant [191) not to cause
major loss of membrane proteins.
Beyond this, however, are several other supportive observations. First,
associative learning involves not just potassium channels but also changes in
several other characteristic proteins that induce and maintain the alterations
of potassium conductance underlying memory (17). Even loss of potassium channels
should leave these remaining proteins behind, providing a clear indication of
the "trained" vs "untrained" state of given dendritic
synapses and/or perisynaptic regions. Second, altered K+ permeability probably
involves durable chemical modification of the potassium channel, so that lost
channels could be identified as "trained" or "untrained" and
counted as such; improper return of individual molecules to specific synapses
would likely be irrelevant as long as the proper total number of
"trained" potassium channels ends up at each "trained"
synapse or perisynaptic region. Finally, durability and inferrability is further
implied by the associative nature of memory, in which a given memory is stored
redundantly in several brain regions in a number of independent forms (17,21),
all of which are unlikely to be extinguished simultaneously in their entirety.
c) Denaturation of proteins.
Evaluation. Likely to apply, at most, to very few proteins (22). Furthermore,
protein denaturation is inherently reversible (23). An apparently trivial issue.
d) Improper disulfide bridge formation between some proteins (24).
Evaluation. Also an almost negligible problem, for similar reasons.
e) Leakage of concentrated extracellular solute into brain cells.
Evaluation. Like membrane lipid loss, the main problem caused by this leakage,
should it occur, would be expansion-induced lysis on thawing (25). Although the
volume of extracellular space in the brain might be considered insufficient to
permit lysis on warming, ultrastructural evidence of disruption of neuronal fine
processes in frozen-thawed brain (26) as well as ultrastructural evidence of
swelling of frozen-thawed synaptosomes (27) lends credibility to this
possibility. However, this problem can be handled in principle even more easily
than the lipid loss problem, simply by extruding the extra intracellular
osmolyte during thawing. Not a significant problem.
f) Local leakage of brain cell solute to the extracellular space. This problem
can be subdivided into leakage of small ions (primarily potassium) and small
metabolites on the one hand and large metabolites and proteins on the other.
Evaluation. The former problem should be negligible; pumping potassium back into
neurons should be straightforward (given non-leaky membranes: freeze-permeabilized
membranes evidently reseal during warming and thawing [281) and small
metabolites can be resynthesized from supplied nutrients. Leakage of proteins
and other large molecules is more serious. However, significant (e.g., 50%)
uncorrected loss of intracellular soluble protein from cell bodies could
probably be sustained without creating very serious problems, since it should be
possible to institute compensatory controls over metabolic rate and membrane
permeability consistent with the spontaneous ability of the cell to resynthesize
missing proteins on warming. Even massive protein loss from cell bodies would
not be able to erase cell identification, since cell identification will be
encoded in the types of synapses the cell makes (which will be preserved [181),
by the pattern of genetic expression readable in the nucleus (which will
probably also be preserved [291), and by membrane and perhaps non-soluble
cytoplasmic protein markers (which will be preserved well enough [201).
A different kind of potential problem could result from protein loss from torn
axon bundles (6,30). Severe losses of axoplasmic proteins at sites of tearing
could make the identification of individual nerve fibers on both sides of tears
more difficult, which may limit the ability to deduce the original connectivity
of the brain. However, it is likely that a short distance away from the tear the
axonal protein content should be largely unaffected by the tear, especially
given the gel-like nature of axoplasm (31) and the relatively rigid structures
mediating axoplasmic traffic.
Significant loss of proteins would undeniably complicate the restoration of
metabolism significantly, and should be avoided to the extent possible:
returning proteins to their proper sites would be comparatively difficult, and
simply correcting for the losses as referred to above requires considerable
metabolic "tinkering." Loss of non-proteinaceous larger metabolites
once again could significantly complicate neuronal identification and would
require considerable "Humpty Dumpty" work that would be best to avoid
if possible. During freezing, such damage will be somewhat limited by the
extracellular diffusion barriers presented by ice and high viscosity
extracellular media, but during thawing the extracellular space will be
progressively "stirred" by declining viscosity, thermal expansion,
convection, and cellular expansion. Means of blocking this "stirring"
would thus be important to deploy.
g) Precipitation of proteins and cellular buffers.
Evaluation. Little (32) direct evidence exists for this mode of injury; if it
were to occur, the result would be reduced metabolic competence secondary to
denatured or missing proteins or unfavorable pH's for normal metabolism. The
remedies--supplying replacement buffer and/or proteins and restoring
precipitated proteins and/or buffers to a soluble condition--seem fairly easy to
h) Leakage of lysosomal enzymes into the cytoplasm, predisposing to
intracellular autolysis on warming (33).
Evaluation. Not demonstrated to occur. Glycerol and low temperatures can be
expected to limit autolysis during cooling, and exogenous inhibitors should be
able to control autolysis during warming. Not a serious problem.
i) Reorganization of membrane bilayer structure into HexII forms, i.e.,
cylindrical lipid tubes (34,35). This change is spontaneously reversible, in
part, upon warming and rehydration, but will keep the membrane leaky in the
cold. Phase separation of lipid subclasses within the membrane, producing leaks
secondary to the resulting molecular packing faults in the membrane (36,37).
Evaluation. These are problems functionally comparable to osmotic or
mechanically-induced leaks noted above. No direct evidence for HexII transitions
exists for any mammalian system, and HexII forms appear unlikely in the presence
of 3-4 M glycerol before freezing, since HexII is a dehydration form (35), and
glycerol can prevent the required level of dehydration for HexII formation from
taking place (11). In both HexII formation and more conventional phase
separations, all membrane material remains in the membrane. The problem then
becomes one of preventing additional leakage from taking place during thawing,
and of redistributing solutes across membranes as needed after membrane
resealing is completed. This seems achievable in principle. In the case of HexII,
spontaneous reversal of the phase transition may lead to incorporation of lipid
and protein into incorrect leaflets of the membrane. Controlled reversal,
however, should be able to direct proper redistribution rather easily.
j) Breakdown of the structure of cytoplasm into blobs of proteinaceous material
Evaluation. This may occur if there is a breech of the cell membrane or for
other reasons that are so far poorly understood. So far, this phenomenon has
been seen only in kidney, not in brain, and does not pertain to all cells, even
in the kidney. If it should occur in brain, repair could be complicated, but it
is doubtful that any actual information loss would occur. This change might be
spontaneously reversible on warming.
2. Mechanical freezing injury. The most pressing kinds of damage are mechanical
forms of damage, not only because it is this type of injury that has actually
been observed in frozen- thawed brains, but also because the potential for
actual information loss is much more serious than is the case for the
biochemical challenges just considered. It is not yet certain that high
concentrations of glycerol will prevent such injury consistently.
Several kinds of mechanical injury could occur, including the following.
a) Memory may be encoded in part in the shapes of dendritic trees (17); these
shapes might be altered by freezing.
Evaluation. Dendritic remodeling associated with learning seems to involve
massive changes such as deletion of unused synapses and unused dendritic
branches (17). This remodeling is associated with the actions of many different
proteins and, most likely, with considerable changes in remaining synapses (17).
It is likely to be not the shape of the dendritic branchings that is important
but, instead, the specific pattern of connections, and this pattern in turn is
presumably responsible for the changes of shape of the dendritic trees (17).
Thus, the shape changes induced by freezing and thawing should be irrelevant as
long as the synapses and dendrites remain physically intact. Freezing is well
known to spare synapses (18).
Although there is evidence for axonal (6) and possibly cellular (30) tearing,
light microscopic evidence suggests that well-glycerolized hippocampal dendrites
are not broken by freezing and thawing (6). But even considerable freeze-induced
damage to dendrite branches might still leave the pattern of connections obvious
from the remaining synapses. Dendrite branching patterns and their underlying
biochemical correlates are biologically robust and dramatic and should retain a
high degree of inferrability, particularly if "stirring" is avoided
b) Disruption of non-synaptic junctions between cells and capillary separation
from the surrounding brain tissue.
Evaluation. Such problems have been observed (40), but would appear not to
involve direct information loss. Such separations could tear fine processes,
however, so the imperative to prevent extracellular diffusion on warming is
reinforced again by such observations.
c) Local (not global) ripping, twisting, and fraying of the ripped ends of nerve
tracts by contraction of the brain cells and by the push of extracellular ice,
creating debris-strewn gaps measured in microns in both length and thickness
Evaluation. A severe form of damage. Reconstruction may require a certain degree
of luck, i.e., the existence of positional relationships between nerve fibers in
a given tract that do not vary significantly from one side of the gap to the
other. Should such consistent positional relationships exist, inferring the
proper connections at the site of a gap should be straightforward. However, if
the positional relationships happen to be changing at the point of such a gap,
additional information in the form of molecular markers that might identify
individual fibers may be required for accurate inference of the pre-existing
connectivity. Electrical tests across the gap may also be required to check for
Should molecular markers be identical from one fiber to another, should
positional relationships prove unreliable, and should electrical tests prove
ambiguous, enough information for correct reconstruction may be present in the
debris pattern that exists in the frozen state, considering the limited
opportunities that exist for diffusion during freezing. In this event,
prevention of "stirring" of the debris during warming will be
critical. Finally, reconstruction might be possible based on consistent, minute
size differences between fibers. Should all of these sources of information
fail, however, the infrequency of these gaps and the generic effects of many
connections as well as the vast redundancy of the brain may make incorrect
inference of the proper connections still consistent with an adequate ultimate
clinical outcome. (Clinical observations suggest that severe local damage can be
consistent with maintenance of identity and personality.)
d) Fracture and separation of fractured halves of cells, axons, dendrites,
capillaries, and other brain elements by distances in the millimeter range after
the temperature drops to below the glass transition temperature (40,41).
(Observations of the gaps referred to in c) above might also reflect
microfractures that became "mushy" on warming and thereby resulted in
molecular blurring of the fracture faces.)
Evaluation. A catastrophic form of injury, offering perhaps the greatest
challenges for the design of molecular repair devices. However, this injury in
itself may involve little or no actual loss of information. This is the most
non-physiological type of injury and will require the most radically innovative
types of repair system for its reversal. Such repair systems do, however, seem
e) Physical disruption of capillaries due to intracapillary ice formation:
rupture of capillary wall, tearing of endothelial cells, stripping of
endothelial cells from their underlying capillary wall material, resulting in
incompetent vessels littered with emboli (40).
Evaluation. A very serious form of injury. However, no information is contained
in capillaries per se. The entire capillary network could likely be cleared out
and replaced with generic capillary "transplants" without any effect
on the identity of the patient. Repair of the existing capillaries would require
innovation on the order of what would
be required to repair fractures. Reparable in principle.
f) Stripping of myelin from axons (40): formation of gaps between the axon
membrane and the myelin, unravelling of the myelin, possible tearing of the
axolemma resulting in loss of intra-axonal material at moderately low
Evaluation. Myelin is inert, generic, non-information-containing material.
Despite the types of myelin damage described, there should be no problem in
inferring which regions of axolemma were previously covered by myelin and which
were exposed. Myelin's function is only important under physiological
conditions. Myelin repair might therefore not be necessary until the patient was
restored to normal body temperature, at which point it could probably be carried
out by ordinary or modified oligodendroglial cells, which lay down myelin under
normal conditions. Leakage of axonal material has been considered above; it may
be reduced by the presence of even a tattered myelin sheath which would act as a
Defining The Problem:
Constraints on Repair
Repair scenarios must recognize that some kinds of repair would be
extraordinarily difficult, futile, or even counterproductive to carry out at the
lowest, most protective temperatures. For example:
Extruded lipids and proteins cannot be reinserted into the membrane until the
cell volume is once again increased because there is no room for them. Restoring
cell volume while the cell is in the vitreous state would be many orders of
magnitude more difficult than performing the same process at higher
temperatures, and would be a seemingly ridiculous and possibly even impossible
task to attempt.
II. Phase Transitions
Low temperatures and membrane dehydration per se cause membrane lipid species to
crystallize or undergo HexII reorganizations. This is therefore the natural
state of these lipids at the prevailing temperatures. Any attempt to reorder the
membrane lipids into a lamellar phase will lead to spontaneous re-separation of
these phases either at the prevailing temperatures or on warming. Thus, simply
"repairing" this membrane defect at cryogenic temperatures would be
futile. Introduction of alien lipid species to prevent re-separation would be
problematic due to the absence of room in the membrane for such species and the
need to subtract native lipid to make room. These changes would all have to be
reversed later, and might create more problems than the original phase
Any denatured proteins will also prefer to be denatured under the prevailing
conditions. Renaturing them will only lead to re-denaturation as temperatures
inevitably rise later on. Preventing re-denaturation would require special
"chaperones" for each protein, whereas waiting for most denatured
proteins to spontaneously renature (23), in part or completely, during warming
would avoid most of the need for such artificial molecular folding-control
IV. Changes in Tissue
Volume: Thermal Expansion, Brittleness, & Elasticity
A fracture represents anisotropic contraction of cerebral tissue due to
temperature reduction or inhomogeneous expansion during warming. Local rips in
axons may arise for similar reasons. To fill in gaps caused by the inherent
thermal contraction of cerebral tissue may create a problem when the temperature
is raised and all of the existing structure, both the native structure and the
added structure, is inevitably forced to expand: expansion lesions such as
buckling and shearing of axons may replace the previous contraction lesions. It
may be wiser to allow thermal re-expansion during warming to at least partially
close these gaps and to effect repair only after this happens.
Likewise, many axons may be very stretched. Destretching them by adding material
to them could cause the same buckling problem when warming occurs. Finally,
tissue will be brittle below TB and may be brittle even at temperatures
moderately above this. Physically moving structures around under such conditions
may damage them. Thus, attempting to close a fracture by physically forcing the
two sides together is liable to rip structures on both sides of the gap. Thus,
some repairs made below To could induce the need for more repairs later when the
temperature is elevated.
V. Changes in Tissue
Statements have been made in the past to the effect that various cell
structures, e.g., mitochondria, will be restored to a "functional
state" while still frozen (3). This would, however, represent a nonsensical
goal for many reasons, not the least of which is that functionality requires
dilute aqueous liquid solutions, which cannot exist at low temperatures. The
correct goal is to ensure that function resumes after warming to physiological
temperatures, regardless of the repair pathway that must be followed during
warming from lower temperatures to attain this goal.
The Repair Scenario
We will assume that the repair procedures begin at a temperature slightly below
the glass transition temperature of the system.
I. Stabilizing Fractures
The first step is to stabilize existing fractures. Fractures require special
treatment, and they require it from the very beginning since, as we will see
shortly, the second repair step will obliterate non-organic components of
fracture faces and will thus make it more difficult to match fracture faces and
guide these faces together later if special precautions are not taken at the
So, the very first step is to infiltrate surface fractures with specialized
molecular devices which will form coatings or surface replicas of the fracture
faces to molecular or near-molecular resolution. (Note that the process of
fracturing releases energy that creates a very high though very brief local
elevation of temperature. The first several molecular layers on each side of a
fracture may therefore be somewhat melted or disordered. Therefore, absolute
molecular resolution may not be attainable.) It is known from standard
freeze-fracture microscopy that fracture faces can be coated below Tg with metal
films that retain their structural fidelity even after the tissue is dissolved
Thus, the formation of sufficiently stable fracture face replicas at
temperatures below Tg appears feasible and would maintain the overall geometry
of the fracture faces after dissolution of the portion of the face that is ice
and glass. Pores in the replicas of areas of pure ice or pure glass should be
included to permit outgassing during the subsequent sublimation process (see
below), which otherwise could tear holes in the replicas.
After coating of opposite fracture faces, these faces could be computationally
compared to verify complementarity. After complementarity analysis, the repair
system could build filaments between the faces. The filaments on each side of
the fracture would be complementary to each other and would connect so as to
maintain fracture face registry later when the temperature is raised. Given
sufficiently strong replicas, these "guide wires" could be attached
only to the replicas (the replicas in turn being tightly adherent to the
fracture faces themselves at all points).
The function of the wires later would be to direct each fracture face as a whole
toward the other fracture face as the gap is later closed by normal thermal
expansion in such a way as to continue to ensure perfect registry of the two
fracture faces as the gap narrows. Molecular "ratchets" along the
guide wires could apply small forces to encourage closing where this is
necessary. If the "guide wires" are built onto the replica faces at
the sites of special pores, then as the gap is closed and the faces approach
each other, the "guide wires" can be allowed to protrude into safe
regions of tissue on each side of the gap, and/or they could be disassembled at
a pace set by the narrowing of the gap.
For deeper fractures not accessible from the surface, the same process might be
accomplished by excavating the vascular compartment first, pausing for fracture
stabilization as fractures are encountered.
II. The Need for an Overall Orientation
The next thing to do is to get the big picture. The frozen brain contains highly
shrunken cells and neuronal processes compressed between sheets of ice and pools
of vitreous cryoprotectant water-solute inclusions. There may be lipid
extrusions, floating debris, ripped axons, hemorrhaged capillaries, stabilized
fractures, unraveled myelin, crystallized regions of certain surface membranes,
extruded cell contents in the extra-cellular space, and other relatively gross
alterations. We desire to identify and stabilize all of these lesions before
significant "stirring" is permitted. This is difficult to do without
large-scale cooperation of repair devices, for which a coordinate system needs
to be set up, preferably one that does not in itself cause any damage.
III. Excavating the
We approach the problem by capitalizing on the fact that about 80% of the brain
is nothing but water and cryoprotectant (42) and that most of this exists in the
form of pure ice located in the extracellular space. We first desire to remove
the ice and most of the vitrified extracellular solution. This step has two
important advantages. First, it creates room for the deployment of an
extracellular communications complex which will be used to direct subsequent
repairs. Second, it makes transmembrane diffusion in either direction
("stirring") effectively impossible when the temperature is
1. General Description of Method --Our task might best be accomplished by
a combination of direct excavation (done by relatively stupid molecular
"jackhammers"), which creates a certain amount of local warming, and
by spontaneous ice sublimation, which offsets some of the local heating due to
evaporative cooling. The rate of excavation is set so as to generate net local
temperatures of around -120 degrees C (i.e., about 5 to 15 degrees C below the
limiting To for glycerol water-solute systems).
Excavation might proceed by digging out hollow tubular "mines" through
the ice perhaps 1 micron or more in diameter. The insides of the "ice
mines" are maintained at a strong vacuum. In a vacuum simulating that of
deep space, ice evaporation rates have been shown to be sufficient near Tg to
move sublimation boundaries at rates on the order of microns per day (43)! If we
maximize the surface area available for evaporation while also maximizing the
rate of direct excavation, it might be possible to remove extracellular ice
fairly rapidly-for example, in a few months.
In doing the excavation, we are not limited to entering through the vascular
system. We can enter through ice channels wherever they may be, and they will be
everywhere, and larger in extent than most biological structures. We can also
enter through special ports built into fracture face replicas. The cerebrospinal
fluid cavities can be evacuated with bulk technologies or a combination of bulk
and molecular technologies. Since the entire brain is under a strong vacuum
during this process, pressure gradients that could cause mechanical failures
should be minimal.
In addition to the advantages of minimizing heating while maximizing water
mobilization, sublimation is also advantageous in that it is self-limiting in a
favorable way. Sublimation of water, as opposed to ice, raises the glass
transition temperature in the sublimed region and thus halts further
sublimation. Therefore, we can remove the ice by sublimation without excessively
dehydrating the glassy matrix surrounding the biomolecules of the brain, either
intra- or extracellularly. Nothing but water can evaporate off at these
Nowhere in this scheme is it necessary to pay the slightest bit of attention to
documenting the locations of water molecules or extracellular solutes such as
glycerol, sodium, or chloride, or worrying about their orientations. Other
extracellular material, such as debris, however, poses some problems. We will
return to these problems momentarily.
2. More Specific Description of Molecular Excavators -- Two or more types
of molecular "jackhammer" are envisioned. The first type is envisioned
to attack only ice. The action of this excavator is to dislodge individual water
molecules from ice and pass them to a "molecular conveyor-belt" system
analogous to the conveyors used by axons to drive axoplasmic flow. The conveyor
system transports water molecules to sites external to the brain. The second
type of excavator removes vitreous material, such as glycerol, glycerol+water,
and glycerol+salt+ water. These clumps of molecules are then passed to the
molecular conveyor for transport outside of the brain.
Small molecules operating at temperatures near -120 degrees C cannot be
self-powered. Therefore, these molecular devices must be attached to a power
distribution source. One practical means of achieving this may be to attach the
sensor-effector elements to a long mechanical rod which delivers the impulse
required to disrupt the appropriate non-covalent bond once the sensing element
identifies a proper target. This rod-tip association might be envisioned as a
sort of "molecular steamshovel" in construction, with the ability not
only to relay an impulse provided from the central power source, but also to
position the effector tip in a versatile fashion using accessory positioning
elements. The excavation could proceed with minimal, entirely local
"computations" by following a stereotyped sequence of steps little
more (and possibly less) complicated than the "computations" carried
out by a ribosome.
In order for sublimation to proceed at the highest possible rate, collisions
between sublimed ice and the ice vacuum interface should be minimized, since
sublimed water will with some probability stick to that interface and require
re-sublimation or direct dislodgement by the molecular evacuators. Without
attempting to design an efficient means of proceeding with excavation so as to
minimize this problem, it can be noted that simply designing the outgoing (but
not the incoming) portion of the conveyor to be able to adsorb free water
molecules from the vacuum would be helpful.
The energetics of both sublimation and molecular excavation should be reasonably
easy to calculate. These processes might well be the most energy-intensive part
of the repair process.
3. First Complication of Excavation: Avoiding Membrane Fracture-There is
a fundamental problem of removing the vitreous residue from the extracellular
space at temperatures just below To, namely, that the strengths of the
non-covalent bonds holding together membrane bilayers are very much lower than
the strengths of the non-covalent bonds holding together the vitreous matrix. In
ordinary freeze-fracture microscopy, fracture planes often proceed along the
plane of the middle of the membrane bilayer for this very reason (44). Applied
to the problem of excavating the vitreous residue, this means that some method
must be used to ensure that energy delivered to dislodge segments of the
vitreous residue does not accidentally dislodge lipids from cell membranes.
A variety of methods might be brought to bear on this problem. One method might
be to avoid regions that may contain nearby membranes as indicated by detection
of membrane markers protruding a considerable distance into the extracellular
maxtrix beyond the lipid bilayer proper. While this would lead to incomplete
excavation, the opportunities for transmembrane diffusion might nevertheless be
reduced sufficiently for this to be a satisfactory paradigm.
Another method might be to check obviously large dislodged chunks of residue for
the presence of lipid and, if lipid is found, to separate it from most of the
vitreous residue in which it is embedded and to reinsert it to its original site
before proceeding. It might also be possible to design the geometry of the force
application process so as to ensure that only a few molecules are dislodged at
any one time, the force being applied not to the medium at large but to a very
local and superficial area (e.g., the third vertex of a triangle).
4. Second Complication of Excavation: Extracellular Debris--There could
be a considerable amount of extracellular debris. It is essential not to remove
or damage this material, since it may be critical for inferring the undamaged
state. Fortunately, the power applied per piston cycle need only be sufficient
to break noncovalent, but not covalent, bonds, so extracellular debris should
not be degraded by the excavation process. However, it will be necessary to fix
all such debris in place, a nontrivial procedure.
Perhaps the best approach to this problem would be to erect side branches on the
molecular conveyor belts. These side branches, shaped something like trees with
their trunks originating on the conveyor belts, would possess binding sites
and/or molecular clamps selected for the encountered debris and would bind all
such debris noncovalently in place. The binding would be such as to represent
the original three-dimensional distribution of the bound debris. For this type
of molecular "book-keeping," it may not be necessary to completely
strip the debris of surrounding glassy phase. In any case, the tree-like
structure of the debris binding elements should ensure that all debris can be
more-or-less locked in place during the repair process, thus permitting
extracellular excavation to proceed without loss of information.
5. Third Complication of Excavation: Inadvertant Excavation--A further
complication arising from this process is the possible "accidental"
excavation of exposed cytoplasm/axoplasm. This can perhaps be avoided by solving
the problem of excavating extracellular debris. Sensors for biological materials
that permit immobilization of debris could similarly seal off cut axons and
As excavation concludes, the vacuum level should be reduced to ensure that
additional unwanted sublimation of water (mummification) does not take place as
temperature is later raised. The empty spaces can be filled with inert gas
and/or with water vapor in equilibrium with the tissue at ambient temperature.
IV. Establishment of
Extracellular Repair Network
As excavation/evacuation proceeds, an extracellular communication,
transportation, and coordinate system could be laid down in the space made
available. This system, penetrating throughout the extracellular portion of the
brain and in intimate physical contact with the brain everywhere, could be
thought of as a kind of "meta-brain," capable of relaying information
about the brain over long distances while potentially having a volume amounting
to more than 60% of the original volume of the entire brain (which is roughly
the volume previously occupied by ice). This volume represents about 150% of the
volume of the cellular components of the brain. The metabrain would permit all
exposed lesions to be mapped and analyzed. Undamaged structures could be passed
over without further action, except as they are needed to infer the proper
locations of abberant structures, such as debris resulting from ripped axons.
Furthermore, the metabrain could be in contact with external computers, where
most computation might occur.
V. Repair Computations for
the Extracellular Space
At this point, all labile extracellular structures have been physically
immobilized and a coordinate system is in place. No "stirring" has
taken place because all procedures have been carried out just below Tg. All
significant extracellular anatomical elements of the brain have been registered.
The "wiring diagram" of the brain can now be deduced, and all damaged
areas can be catalogued as to type and place. Where necessary, the loci of
missing structures could be deduced at this point. For example, ripped bundles
of axons are analyzed to deduce how to infer the pattern of connections between
the two ripped ends based on the direct physical remains of the ripped axons and
any other available information. The loci of missing cell membranes are deduced.
Extracellular debris are assigned to appropriate destinations.
To this point, no actual repairs have been made and the process has been
completely noninvasive as far as the actual cells of the brain are concerned.
Based on the results obtained to this point, specialized repair devices are
assigned to specific tasks and specific regions.
No tasks so far have involved the making or breaking of covalent chemical bonds.
All excavations, sensing, and computations have been based on purely physical
processes which should be able to operate at cryogenic temperatures given an
adequate external power source and power transmission system.
VI. Warming above Tg
In order to proceed with repairs, warming of the brain is slowly induced. The
advantages of warming are several. It induces changes in volume which permit
fracture healing, it induces desirable changes in tissue
pliability/deformability needed for moving structures such as cell membranes,
and it permits both diffusional transport of needed molecules and the chemical
reactions needed for repair.
The primary hazard of warming is not biochemical but diffusional. At
temperatures as high as about -50 degrees C, virtually no enzymatic activity
should be possible (22, 45). Deterioration at this temperature is likely to be
due to slow intracellular diffusional processes perhaps accompanied by slow
spontaneous breakdown of certain relatively rare labile molecules. Any enzymatic
activity that could occur is likely to be arrested in time due to lack of
available substrate or accumulated product inhibition, and thus is unlikely to
proceed very long. A special class of proteins, catabolic enzymes, may pose the
most serious problems. However, the fraction of enzymes represented by key
catabolic enzymes is small and all such activity can be blocked by specific
We can at this stage also ignore problems arising from any protein denaturation
that may exist. Denatured proteins are not likely to catalyze troublesome
reactions and are not needed for any functional role, so there is no reason to
worry about them until temperatures are brought to near-zero. At that point,
many or most of them will have spontaneously renatured, or will renature
spontaneously given additional warming. The remainder can be renatured and
disaggregated specifically at temperatures near 0 degrees C using specialized
devices for each labile enzyme. This process should be trivial enough to ignore
for the present purposes, particularly since the number of denatured proteins in
glycerolized frozen-thawed brains should be minimal as a fraction of the total.
Thus, the primary initial portion of actual repair, as opposed to simple survey
and marking of the damage, consists of coping with diffusional processes. At
temperatures between about -110 and -50 degrees C, two major types of
diffusional process can be identified: the diffusional motions that blur the
fracture interfaces we have previously marked and prepared for healing, and
diffusional motions within cells. By removing the great majority of the
extracellular space and immobilizing extracellular debris, we have precluded
transmembrane and extracellular diffusion, and by forming durable fracture
replicas and establishing the relationships between them, we have precluded
diffusional information loss at these sites. Intracellular diffusion is
relatively trivial over the short run. We therefore are able to proceed with the
extracellular repair process first, and then to turn our attention to cellular
VII. Fracture Healing
Coefficients of thermal expansion and water/glycerol diffusion coefficients
dictate the kinetics of spontaneous fracture healing in pure solutions.
Extrapolation of available data for glycerol-water solutions suggests that
spontaneous fracture healing in these solutions will first become appreciable in
the vicinity of about -80 degrees C (46). Thus, we will want to heal fractures
in cerebral tissue during warming from -100 degrees C to about -80 degrees C.
The key issues involved at this point are a) the removal of the protective
replica surfaces and b) the union of tissue on either side of the fractures.
Both a) and b) pose significant problems. Removing the replica surface will tend
to free bound species on each side of the gap for undesired diffusion. Uniting
fracture faces could be met with steric obstacles if the repair device must go
between the surfaces to repair them, since being between the two surfaces will
tend to keep the surfaces apart and thus unrepaired.
But how are fracture surfaces likely to appear? Fractured surfaces will
generally be cross-sections through various membrane-limited compartments
(cells, myelinated axons, organelles), and planar separations between membrane
bilayers. Within membrane-enclosed compartments, filamentous structures and
molecular clusters such as enzyme complexes will be cleaved. In most cases,
relatively free molecules such as cytoplasmic globular proteins should not be
fractured, and the few that might be lost in this way can be neglected.
Fractured microtubules, actin, etc. can be healed enzymatically. Steric
hindrance is not a likely problem for individual molecules. Disrupted enzyme
clusters can be reclustered (and will often recluster spontaneously when warmed
In the case of membrane-bounded compartments that have been cleaved by
fractures, one strategy would be to heal the limiting membrane first. It will
not be destabilizing to remove replica material from membranes fractured
perpendicular to the plane of the membrane because membranes can be adequately
stabilized from above and below the plane of the membrane before the replica
material is removed. As the naked membrane faces are brought together, they will
tend to fuse spontaneously (48). This is also true for bringing together
membranes fractured between leaflets. No specific chemical bonding will have to
be induced to heal the major portion of the fracture.
If membrane fluidity is too low to permit good fusion at the prevailing
temperatures, a small amount of specialty lipid can be added to the local area
to enhance fluidity sufficiently to permit fusion to occur (49). After membrane
fusion has occurred, some individual molecular species (particularly
cross-linked proteins) associated with the formerly fractured area of the
membrane might exist in a damaged (cleaved) form. These damaged components can
be examined later, at higher temperatures, where they can be enzymatically
The result would be a resealed compartment containing an internal plate of
replica material. This material can then be disassembled from the plate molecule
by molecule. As structures are uncovered by this process that require covalent
bonding, they can be rotated for access, bonded, and then rotated back into
proper position as healing proceeds. As healing proceeds, the liberated replica
material can be passed through the healed membrane and exported to the
extracellular space for removal by conveyors to outside the brain.
Some fractures are bound to penetrate through debris fields resulting from axon
tearing or from myelin unravelling. The actual fracture healing in such areas
should be relatively trivial, since there is no organized structure on either
side of the gap that must be reconstructed. The area will consist mostly of
evacuated empty space (now filled with inert gas and/or water vapor), from which
removal of replica material should be particularly easy. Since all debris have
been previously immobilized, repair of fractures through the debris will not
endanger the information content of the region.
Note that it is the cells, vascular bed, and extracellular scaffolding whose
fracture faces should be healed first. The presence of fracture replicas in gas
pockets that previously consisted of extracellular ice or glass is important for
maintaining the registry of cell surfaces and should be maintained until cell
surfaces are safely healed.
It must be recognized that even at -80 degrees C, most relevant chemical
reactions, even with the benefit of customized enzymatic catalysis, will proceed
very slowly if at all (22,45). The missing energy can be made up in a variety of
ways. First, heat could be liberated highly locally to permit reactions to
proceed. (It could be helpful in this regard that the extracellular space has
been replaced with gas, which is a good thermal insulator.) Second, exotic
chemicals (perhaps including customized free radicals ) could be used to do
the covalent bonding necessary to heal individual fractured molecules. Although
this would most likely result, in most cases, in molecules containing unnatural
structures, these foreign structures could be removed and corrected at higher
temperatures at which the proper types of chemistry are feasible. Finally, the
option exists, if all others fail, to simply hold fractured molecules together
with molecular clamps until such time as they can be chemically bonded at higher
How much time is available for these manipulations at about -80 degrees C.
Although it is not possible to be certain, the normal rule of thumb would be
that several months of storage at this temperature should be possible without
any appreciable intracellular deterioration (52). This should be more than
enough time to carry out the required extracellular repairs.
VIII. Cell Repair
1. Debris consolidation-- Having healed the fractures at about -80 degrees C,
the next major extracellular task is to redistribute cellular debris to their
proper locations. The actual transport is simplified by the absence of
extracellular diffusive barriers. Reinsertion of lipids and proteins into
membranes and into cytoplasm proceeds by means of specialized transport devices,
which could be individually powered by reactive chemicals supplied continuously
on the molecular conveyor system from outside. Once repositioned, lipids will
remain positioned through ordinary self-assembly mechanisms (given an aqueous
intracellular phase and a thin layer of aqueous extracellular fluid).
Having previously mapped and analyzed all debris down to the molecular level,
actual reconstruction of debris into tissue should be relatively
straightforward. Intracellular proteins, once deposited in the proper sites, can
be covalently bound in position or immobilized with molecular clamps. To
facilitate self- assembly, the temperature may be raised to perhaps -60 degrees
C for up to a few weeks (52,53) if need be, either early, late, or
intermittently during the reassembly process. In cases in which debris are the
result of extrusion of material from contracting membranes, their redistribution
is delayed pending cell volume re-expansion. At this stage, we repair only
debris resulting from tearing and the like.
2. Stabilization against diffusional/biochemical deterioration. While limited,
some diffusion is possible in cytoplasm at -60 degrees C. We exploit this by
introducing metabolic inhibitors at this temperature into the cytoplasm. Since
we have ready access to the external surfaces of cells, we can easily deposit
inhibitors at regular intervals along cell processes. The inhibitors are
designed to block the action of any enzymes that permit catabolism to proceed to
beyond an acceptable point. Once deposited, they can be ignored, since these
relatively low molecular weight inhibitors will reach their targets by diffusion
as rapidly as the normal substrates would otherwise reach these catabolic
With the possibility of detrimental catalyzed chemical change precluded, the
only further types of damage are diffusion (e.g., organelle swelling),
spontaneous chemical modifications (e.g., oxidation, racemization, etc.), and
structural collapse (due to declining cellular rigidity with rising temperature,
causing cellular structures to sag in the absence of extracellular supports). We
can ignore diffusional change at this stage because cells and organelles are all
highly shrunken. Random chemical damage can be ignored at this point and will be
addressed later. Minor modifications to the extracellular communications
network, which can double as a kind of extracellular "connective
tissue," are now made to ensure the prevention of sagging during continued
In order to further prepare for warming, nanocomputer-based cell repair machines
similar to those described by Drexler (1) are introduced into the cytoplasm at
this time. Although they are incapable of effecting rapid repairs at -60 degrees
C, their introduction at this time allows them to begin repairs at the first
opportunity during warming. They may well be capable of carrying out extensive
sensing and computational functions at -60 degrees C in preparation for their
actual repair activities at higher temperatures.
3. Cell Volume Restoration. As noted in the discussion on mechanisms of damage,
it is during thawing that many problems arise. In the present scenario, no ice
is present anywhere throughout the brain and, thus, the brain never has to go
through the process of thawing per se. We do,
however, ultimately have to return cell volumes and cell water contents to
normal. Our advantage is that we can do this in whatever manner is most
desirable: we can expand cell volume at temperatures lower than would normally
be associated with volume expansion during thawing (by adding both glycerol and
water to the cells, we could fully expand cell volume even at -60 degrees C if
we so chose), or we can expand cell volume at higher temperatures than would
occur during thawing (by failing to add water to the same extent as it would be
supplied by the progressive melting of ice).
The assumption we will make here is that we wish to do the former: expand the
cells at temperatures lower than would exist during thawing. The reasoning is
that there are many types of cellular injury which ultimately must be dealt
with. If we rehydrate in a manner that simulates normal thawing, we tend to have
to deal with all of these problems simultaneously. By re-expanding our cells at
temperatures in the vicinity of -60 to -30 degrees C, rather than in the normal
range of thawing (11) (i.e., -40 to -8 degrees C), we can take care of membrane
re-expansion issues more-or-less independently of metabolic issues. We may want
to favor the highest temperatures for re-expansion that do not begin to induce
appreciable metabolic problems so that we can maximize membrane fluidity and
minimize problems that may arise from unreversed membrane lipid phase
transitions during cellular and membrane re-expansion.
Before cell re-expansion can proceed, there must be sufficient extracellular
volume available for the re-expansion. We thus withdraw a portion of the
extracellular communications network, much of which has already accomplished its
purposes and is no longer needed. We leave in place conveyors for water and for
glycerol, cellular supports, and assorted other devices.
We thus begin, at about -60 degrees C, to transport glycerol and water into the
cytosol and axoplasm so as to maintain a ratio of glycerol to water that has a
freezing point of about -61 degrees C. This process is carefully coordinated
with the process of re-inserting extruded membrane material. As these two
processes proceed, we also gradually raise the temperature, adjusting our
membrane transporters to convey more and more water in comparison to glycerol so
as to maintain an intracellular freezing point just below the prevailing
temperature. Transport could again be powered by highly reactive chemicals
introduced by conveyors.
At -30 degrees C or so, most (but not all) lost volume and all formerly extruded
membrane material has been replaced. (We retain some extracellular space for the
continued presence of some supporting devices.) The extracellular machinery for
processing extruded material is withdrawn. The cells contain more than 6 M
glycerol, a higher concentration than they began with. This is a sufficient
concentration to preclude most intracellular chemistry, particularly at the
prevailing temperature. The metabolic inhibitors introduced earlier have
diffused to their targets and inactivated them. While cell volume expansion has
proceeded, similar volume control measures have been completed for intracellular
organelles. These measures have automatically included reversal of pre-existing
organelle swelling. Other membrane transporters have also had sufficient
opportunity to reverse ionic (Na+, K+, Ca+*, C1-, etc.) imbalances in both the
cytoplasm and in organelles. They will continue to be active until brain
temperature is returned to near normal values.
Volume control measures will not be entirely successful unless significant
membrane phase changes have been reversed by this point in the repair process.
This may happen spontaneously due to the elevation of temperature but, if not,
it will be induced at this time by the temporary insertion of specialty lipid or
molecules such as trehalose (54) (most likely in combination with more direct
4. Rehydration. At this point, the extracellular space can be flooded with
glycerol-water-salt-substrate-colloid solution. This is done to maintain
membrane integrity and to simplify water transport during rehydration. Colloid
will preclude cell swelling in the cold without the need for vigorous ionic
We now reverse the direction of the membrane glycerol transporters and slowly
transport glycerol to the extracellular space at the same time the glycerol
concentration in the extracellular space is similarly being reduced by transport
to the outside. By equating the proportion of cell glycerol removed to the
proportion of extracellular glycerol removed, water activity is kept identical
in the two compartments without a change in cell volume due to spontaneous
diffusion of extracellular water into the cells. (Water diffusion should be
sufficiently fast to preclude the need for specific -- and very energy intensive
transmembrane water pumping at this stage.) At all times, the glycerol
concentration within the cells is just sufficient to prevent the cytosol from
freezing. Eventually, we arrive at 0 degrees C and a glycerol concentration of
about 150mM. More of the extracellular communications and conveyance system is
Having reconstructed cellular and extracellular structures on a gross level, the
vascular system is now sufficiently intact to permit cerebral perfusion to be
reinstated. The brain vasculature should remain intact for days at 0 degrees C
even without extensive protective modifications provided it has been
sufficiently well repaired (56). The perfusate contains necessary substrates,
repair machines, and psychrophillic anabolocytes, as required. Given that
organisms have been found in nature that can grow at temperatures as low as 20
degrees C below zero (57), vigorous repairs are clearly possible at O degrees C,
despite the very low metabolic rate of the original tissue.
These new devices as well as the previously-deployed intracellular cell repair
machines therefore now proceed to correct the most critical types of continuing
damage. Their activities may include, for example: myelin synthesis and
replacement; bacterial and viral killing; protein reaggregation; cytoskeletal
reassembly; reversal of glycerol-induced biochemical reactions; de novo
synthesis of key missing proteins and other key metabolites; reversal of exotic,
unnatural chemical bonds formed in order to heal otherwise intractable lesions
at lower temperatures; removal of specialty lipid; restoration of normal
intracellular buffering and pH; repair or removal of peroxidized, racemized,
oxidized, or otherwise modified structures, resulting in their replacement with
undamaged structures. Repair is powered by the chemical energy stored in the
remaining glycerol present in the cells (precluding the need to otherwise
dispose of this glycerol and completing the return to isotonicity) as well as by
special chemical energy sources now available from the perfusate.
Almost all of the extracellular communications and general support network is
now disassembled and withdrawn.
IX. Metabolic Restoration
As temperature is elevated further, oxygen is reintroduced and many metabolic
inhibitors are degraded or inactivated. Necessary protein repairs are completed.
Successful repair is checked by examining certain key metabolite behaviors in
each significant metabolic pathway that are indicative of proper metabolic
startup. Departures from expectation are diagnostic of any lingering underlying
problems, which are then specifically corrected to the degree necessary. The
required fine-tuning adjustments could be carried out largely by de novo
synthesis of deficient proteins, by supplying inhibitory metabolites that are
normally present and needed to control the overactivity of other proteins, by
providing necessary protein cofactors that were previously lost, etc.
The synthesis of purely artificial proteins required for specialty tasks may
also be called for. Protein denaturation is reversed artificially at this point
as may be needed. [Renaturation could be accomplished by a variety of
techniques. For example: a) The protein could be completely unfolded by seizing
it at the N- or C-terminal end and passing it as a straight chain through a
molecular tunnel resembling the channel nascent polypeptide chains pass through
as they emerge from ribosomes, then allowing the emerging protein to refold
either spontaneously or in cooperation with existing intracellular folding
"chaperones" (58); or b) the protein could be attached to a
scaffolding whose shape is altered in such a way as to renature the protein or
allow the protein to complete spontaneous renaturation when released from the
scaffolding after shape alteration.] After these diagnostics and fine-tuning
tasks are completed, metabolism is released from artificial control.
X. Disease Reversal and
Brain temperature is raised to 25-37 degrees C. Cell metabolism may still be
grossly abnormal in a variety of ways: it will not have been necessary to have
previously reversed all details of the previously existing pathological state,
but only those details required for subsequent cellular self-maintenance and
self-repair. Cells "know" what their proper state is and will
spontaneously establish that state provided they are viable enough to continue
to exist and to repair themselves.
While this is happening, conventional medical nanotechnology will be at work on
specific disease processes, reversing them, establishing proper connections to
extracephalic structures, and, if need be, assisting with the provision of a new
body. Given stable physiology, these curative processes, including the partial
or even complete reversal of aging, can be allowed to proceed as long as needed.
Very few constraints on repair exist at this point. Technologies for dealing
with specific disease states will be routine and powerful and require no
Once the patient has been restored to a state approaching perfect physical
health, consciousness is restored.
Summary and Conclusion
A "realistic" scenario for the repair of the frozen brain is proposed.
It is based on the specific details of freezing injury and on the natural
resistance of most cellular constituents to freezing damage, as well as on the
natural self-assembly and self-repair of living cells. It avoids the need for
performing chemical reactions below the glass transition temperature while at
the same time avoiding the problems of diffusive information loss on warming.
Although each step has not yet been subjected to thorough analysis, each is
concrete and based on known fact. The scenario is fully open to criticism,
testing, and refinement. It thus could serve as a basis for future discussions
of the feasibility of moderate approaches to the restoration of those frozen by
This scenario is predicated on many assumptions--such as the assumption of
adequate preservation by current technology-that may be false. This scenario
does not prove that cryonics can or will succeed. It may, however, facilitate
discussion of that possibility.
References and Notes
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17. See, for example, Daniel L. Alkon's lay paper, Memory Storage and Neural
Systems, Scientific American, pp. 42-50 (July, 1989), as well as his papers in
Science (226, 1037-1045, 1984; 239, 998-1005, 1988), and Alkon's book, Memory
Traces in the Brain, Cambridge University Press, 1988.
18. The Cryobiological Case for Cryonics, booklet available from the Alcor Life
Extension Foundation; 7895 E. Acoma Dr., Scottsdale, AZ 85260. Tel: (800)
19. Hardy, J.A., P.R. Dodd, A.E. Oakley, R.H. Ferry, J.A. Edwardson, and A.M.
Kidd, Metabolically active synaptosomes can be prepared from frozen rat and
human brain, J. Neurochem., 40, 608-14 (1983).
20. Stahl, W.L., and P.D. Swanson, Effects of freezing and storage on
subcellular fractionation of guinea pig and human brain, Neurobiology, 5,
21. Kandel, E. R., and Schwartz, J. H., Principles of Neural Science, Second
Edition. Elsevier, New York, 1985.
22. Franks, Felix, Biophysics and Biochemistry at Low Temperature, Cambridge
University Press, New York, 1985
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25. Lovelock, J.E., The mechanism of the cryoprotective action of glycerol
against hemolysis by freezing and thawing, Biochem. Biophys. Acta, 11, 28-36
26. Cryonics: Reaching for Tomorrow. Booklet available from Alcor Life Extension
27. Hardy, J.A., P.R. Dodd, A.E. Oakley, R.H. Ferry, J.A. Edwardson, and A.M.
Kidd, Metabolically active synaptosomes can be prepared from frozen rat and
human brain, J. Neurochem, 40, 608-14 (1983).
28. Pegg, D.E., and M.P. Diaper, On the mechanism of the protective action of
glycerol, Biophysical Journal, 54, 471-88 (1988).
29. Houle, J.D. and G.D. Das, Cryopreservation of embryonic neural tissue and
its successful transplantation in the rat brain, Anat. Rec., 196, 81A (1980);
Houle, J.D. and G.D. Das, Freezing of embryonic neural tissue and its
transplantation in the rat brain, Brain Res., 192, 570-4 (1980); Houle, J.D. and
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T. Sorenson, A.G. Moller, and J. Zimmer, Intraocular grafts of fresh and
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histological and connective organization, J. Comp. Neurol., 227, 558-68 (1984);
Sorenson, T., S. Jensen, A.G. Moiler, and J. Zimmer, Intracephalic transplants
of freeze-stored rat hippocampal tissue, J. Comp. Neurol., 252, 468-82 (1986);
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30. Suda, I, K. Kite, and C. Adachi, Bioelectric discharges of isolated cat
brain after revival from years of frozen storage, Brain Res., 70, 527-31 (1974).
31. Baker, P.F., A.L. Hodgkin, et al, Replacement of the axoplasm of giant nerve
fibers with artificial solutions, J. Physiol. (London), 164, 330-54 (1962).
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dimethyl sulfoxide on changes in composition and pH of buffer salt solutions
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33. Persidsky, M.D., Lysosomes as primary targets of cryoinjury, Cryobiology, 8,
482-8 (1971); Persidsky, M.D., and Ellett, M.H., Lysosomes and cell cryoinjury,
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34. Steponkus, P.L., and D.V. Lynch, Freeze/thaw-induced destabilization of the
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35. Koyova, R.D., B.G. Tenchov, et al, Sugars favor formation of hexagonal (HII)
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phosphatidylethanolamines, Biochem. Biophys. Acta, 980, 377-80 (1989).
36. Lyons, J.M., Phase transitions and control of cellular metabolism at low
temperatures, Cryobiology, 9, 341-50 (1972).
37. Quinn, P.J., A lipid-phase separation model of low temperature damage to
biological membranes, Cryobiology, 22, 128-46(1985).
38. Jacobsen, I.A., Pegg, D.E., et al, Introduction and removal of
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renal cortex protected with dimethyl sulfonide, Cryobiology, 18, 550-70 (1981).
40. Unpublished experimental results of Alcor Life Extension Foundation and
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large biological systems, Cryobiology, 27, 492-510 (1990).
42. Gadea-Ciria, M., J. Gervas Camacho, et al, Water content of various regions
of the feline nervous system, Medical Biol., 53, 469-74 (1975).
43. J.G. Linner and S.A. Livesey give the following calculations in their
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Contributions, J.J. McGrath and K.R. Diller, Eds., ASME, New
44. Orci, L., and A. Parrelet, Freeze-Etch Histology. A Comparison Between Thin
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47. Both proteins and membranes exist in the form they do because of the
immiscibility of water and hydrocarbons. This immiscibility causes these
structures to self-assemble spontaneously if permitted to do so; this is the
basis of spontaneous protein renaturation and membrane assembly. Protein
clusters often involve hydrophobic associations as well, but even when other
contributions are more important, mis-clustering is, in principle, equally
spontaneously reversible. Self-assembly can happen incorrectly, but, given a
little guidance, can surely be directed to happen correctly. See also note 48
48. Fat exposed to water preferentially associates with other fat if any is
available. A fractured membrane presents two "greasy" surfaces to
water, which is entropically unfavorable; it is thermodynamically favorable for
these two cut surfaces to fuse together so as to eliminate the unfavorable
water-fat interface. This tendency is, however, reduced by low temperatures
(which reduce the energy cost of hydrating fat) and by solidification of the
membrane. A good general discussion of these issues is given in The Hydrophobic
Effect, Formation of Micelles and Biological Membranes, by Charles Tanford (2nd
Edition, 1980, John Wiley & Sons, New York). As Tanford notes (p. vii):
"The hydrophobic effect is perhaps the most important single factor in the
organization of the constituent molecules of living matter into complex
structural entities such as cell membranes and organelles."
49. "Specialty lipids" could be made by reducing the number of carbon
atoms in the fatty acid tails of ordinary membrane phospholipids, increasing the
number of double bonds (especially cis double bonds) in these tails,
fluorinating the fatty acid tails, modifying polar head groups to prevent close
association of the lipid tails (by preventing clumping of these head groups), or
by any combination of these maneuvers. These modifications are all known to
reduce the freezing points of lipids and/or hydrocarbons and hence increase
their fluidity. (See also: Small, D. M., et al, The Physical Chemistry of
lipids: From Alkanes to Phospholipids. Plenum Press, New York, 1986 [Handbook of
Lipid Research, Vol. 4].) Specialty lipids based on such modifications should,
therefore, enhance the ability of lipid phases doped with them to fuse. It does
not seem likely that the specialty lipids must reverse membrane phase
separations to effect membrane fusion. Even small, free molecules such as
pentane or its relatives could suffice: as long as the molecule is insoluble in
water and preferentially associates with hydrophobic species, it should produce
the desired effect. (The fact that membranes continue to exist at low
temperatures suggests that hydrophobic forces will remain strong enough at these
temperatures to promote self-assembly of hydrophobic entities in an aqueous
environment.) Even if a molecule such as pentane becomes volatile on warming,
the membrane will not be affected, provided it becomes sufficiently fluid before
the small species evaporates.
50. Evidence that it is permissible to heal some fracture damage at higher
temperatures is provided by the results of Dr. Luiz de Medinaceli, who found he
could regenerate rat sciatic nerves that he had first frozen and then cut
cleanly at temperatures just below 0 degrees C. The nerve ends were held
together by special tethers and extracellular potassium was elevated to keep the
cut axons alive. Only very cleanly cut nerves regenerated well. His work was
discussed in a series of papers that appeared in Experimental Neurology, Volume
81 (pages 459-468; 469-487; and 488-496) and Volume 84 (396-408), in 1983 and
1984. See particularly Vol. 81, pp. 469-496. His work is now being extended to
human patients (personal communication).
51. As Mazur discusses in reference 12, free radical reactions can proceed
relatively unabated regardless of temperature, owing to the lack of any
activation energy for these reactions.
52. Meryman, H.T., Review of biological freezing, in Cryobiology, H.T. Meryman,
Ed., Academic Press, New York, 1966, pp 1-114.
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least this long, and probably for much longer. See reference 30 and the
following reference: Suda, I., K. Kite, and C. Adachi, Viability of long-term
frozen cat brain in vitro, Nature, 212, 268-70 (1966).
54. Crowe, J.H. and L.M. Crowe, Interaction of sugars with membranes, Biochem.
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505-531 (1924); Leaf, A., Regulation of intracellular fluid volume and disease,
Am. J. Med., 49, 291-5 (1970); Mendler, N., H.J. Reulen, et al, Cold swelling
and energy metabolism in the hypothermic brain of rats and dogs, in Hibernation
and Hypothermia, Perspectives and Challenges, F.E. South, J.P. Hannon, et al,
Eds., Elsevier, New York, 1972, pp. 167-190.
56. White, R.J., M.S. Albin, et al, Prolonged whole brain refrigeration with
electrical and metabolic recovery, Nature, 209, 1320 (1966).
57. Actual growth has been confirmed at -12 degrees C, and unconfirmed reports
of cell growth at -18 degrees C, -20 degrees C, and -34 degrees C are available:
see Mazur, P., Limits to life at low temperatures and at reduced water contents
and water activities, Origin of Life, 10, 137-59 (1980). Continuing metabolism
has been documented to occur at -30 to -40 degrees C by S.M. Siegel, T. Speitel,
et al, Life in Earth extreme environments: a study of cryo-biotic
potentialities, Cryobiology, 6, 160-81 (1969).
58. For some recent references, see H. Blumberg and P.A. Silver, A homologue of
the bacterial heat-shock gene DnaJ that alters protein sorting in yeast, Nature,
349, 627-30 (1991).