X-Message-Number: 8943
Date: Fri, 19 Dec 1997 01:17:42 -0800
From: Paul Wakfer <>
Newsgroups: sci.cryonics
Subject: Prometheus Project Donations & Pilot Project Plans
Donations from All Organizations
The total of financial contributions to the Prometheus Project reached
$26,011 on Tuesday when I received a $1,000 donation from John Bull, a
member of the Cryonics Institute. This was also an important milestone
with respect to the unifying nature of the Prometheus Project. We now
have financial contributions from all four major cryonics organizations.
Here are the names of those who have made finanicial contributions to
this point:
Ben Best $1000
John Bull $1000
Thomas Donaldson $1000
Jim Halperin $1000
Joe Hovey $1000
Bob Krueger $2011
Mark Mugler $1000
Mike Perry $1000
John Pietrzak $1000
Ken Stone $500
Edgar Swank $8000
Roy Yowell $6500
In addition, there is another $1,500 clearly promised and "on-its-way":
$1000 from Brook Norton and another $500 from Ken Stone.
In addition, the following people have given significant amounts of time
and/or resources to benefit the Prometheus Project:
Russell Cheney
Thomas Donaldson
Peter Gouras
Allen Hardy
Loren Hardy
Saul Kent
Brian Wowk
Leonard Zubkoff
Outline of the Pilot Prometheus Project by Greg Fahy
At a special meeting in Las Vegas, Prometheus Project Research
Director Dr. Greg Fahy annouced his plans for the initial pilot
experiments designed to lead Project investigators to optimal whole
brain cryopreservation methods with maximum efficiency. The project
will use a complex brain information processing circuit, the hippocampal
slice, as a test system to determine the conditions required for optimal
cryopreservation of functioning neural networks. Such systems have
never been studied in the past, and concentrating on a brain slice model
should allow the major cryobiological variables to be explored within a
one year time frame rather than in a four year time frame while avoiding
12-hour days and needless expense in testing poor preservation methods
on whole brains.
The hippocampal slice is a standard neurobiological preparation and
can be cultured for hours to weeks, making it possible to prepare slices
on one day, expose them to cryopreservation-related conditions the
following day, and assess their viability the following day, or even
after several days of recovery. The hippocampal slice is a polysynaptic
pathway involving more than one neurotransmitter, and can be
characterized by impuse delay times across multiple or single synapses,
the response to blockers or stimulators of synaptic transmission,
conduction velocities along axon and dendrite fields, and resting
membrane potentials. The
hippocampal slice is also of clinical relevance for studies of epilepsy,
Alzheimer's disease, amnesia, ischemia/reperfusion injury, and
schizophrenia and is transplantable.
The primary means of assessing hippocampal integrity will be the
use of voltage sensitive dyes. These dyes faithfully follow changes in
membrane potential in real time, allowing the function of the slice to
be visualized directly and recorded using high speed videomicrography.
Methods have already been published by which waves of depolarization
have been directly visualized, recorded, quantitated, and modified by
pharmacological interventions. Since the passage of a wave across the
hippocampal slice requires times measured in miliseconds, even assays
requiring repetitive measurements for signal averaging could be
completed in minutes. This would allow large numbers of slices to be
assessed in a single day, so that the effects of various
cryopreservation variables can be observed rapidly and efficiently.
Voltage sensitive dyes can measure not only membrane potential, but also
intracellular calcium loading and intracellular sodium levels. Thus,
not only can functional integrity and recovery over days of post-insult
self-repair be determined, but factors associated with observed injury
can be measured as well. In the case of calcium loading, methods have
been published for measuring calcium loading and membrane potential in
the same slice at the same time.
One major advantage of the use of voltage sensitive dyes is the
ability to directly visualize the entire slice. If there are holes
caused by ice damage, they will be visible. If there are subregions of
the preparation that are damaged, they will be visible. It will not be
possible to be misled by placing microelectrodes at unrepresentative
points in the slice. All damage that is present, in terms of the ability
to maintain normal electrical functions, will be seen.
A second major advantage is ease of obtaining and visually
representing the data. Once the system is properly set up, relatively
unskilled technologists should be able to operate it effectively. And
when it comes time to convey the results of the experiment, a picture is
worth a thousand words.
A third advantage is that, when the functional tests have been
completed, the hippocampal slice can be bathed with fixative for
detailed light and electron microscopy, hopefully with nary a
microelectrode track to distort the results. Thus, the slice permits
both function and ultrastructure to be used as endpoints without
conflicts between the two.
At this writing, an unsettled question is the extent to which it
will be possible to avoid using expensive and complex methods for
electrically stimulating the slices so as to induce a signal to be
followed by the visual method. It may be possible to locally apply an
excitatory neurotransmitter (glutamate) so as to initiate a response,
wash it off, and reapply it as may be called for, rather than using
electrical stimulation. It is also known that LTP can be induced in the
hippocampus by exposing it to elevated potassium concentrations or to 10
mM glycine, so delivering a correctly tailored series of electrical
stimuli to create LTP to determine
whether LTP can be created after treatment, or sustained during and
after treatment when induced before treatment, is not necessary.
Instead, the bathing medium surrounding the slice can be temporarily
changed, something that any technologist can easily accomplish.
Nevertheless, if electrical stimulation is needed, which would be
particularly true if signal averaging of 100 or more responses to a
given stimulus is needed, then it will be used.
The most likely candidate animal for the initial studies at this
time is the rat, simply because so much work has already been done on
this model. The rat is less expensive, is well-studied, and can be used
to compare the effects of isolating slices either in the standard way
(which involves warm ischemic exposure) or after hypothermic perfusion
of the whole animal to avoid warm ischemic injury during slice
preparation. Slice preparation in the conventional way is rapid using
the rat model, being accomplished in several minutes from start to
finish.
Approximately seven slices of the dorsal hippocampus can be
obtained per rat. If three rats are worked up in a given day, which
should be easily accomplished, then about 21 slices will be available
the following day for studies. This would allow 3 replicates of each of
7 variables on the treatment day, and readout of the results the
following day. If six rats are worked up on a Monday, then half of the
slices could be treated on Tuesday and assessed on Wednesday, and the
remaining
slices could be treated on Thursday and read out on Friday. This would
allow full documentation of the effects of 7 variables in a week, or an
average of one variable per day. Using whole brains to look at the same
variables would require 21 very long and arduous treatment days as well
as other days for setup and cleanup, and more days for data analysis
than would be needed for the slice model. Based on analogous work with
kidneys, three such experiments could be done per week, requiring 7
weeks to examine the same 21 variables that could optimistically be
covered in one week using the slice model.
A further point is that the same optical methods used to
characterize injury in the slice model can be applied to the intact
brain in later experiments. Voltage sensitive dyes have been used to
visualize the visual cortical optical dominance columns in monkeys in as
little as 13 seconds, something it took Hubel and Wiesel 20 years to do
with microelectrodes. More importantly, voltage sensitive dyes
partition into nerve cell membranes and axons at a rate that depends on
the magnitude
of the membrane voltage at the time they are being administered. This
has allowed the electrical activity of subsurface brain regions to be
"frozen" in time with a resolution of 20 seconds or less by loading the
dyes over times that are not long enough to fully saturate the membranes
with dye. After the dye is administered, the brain is immediately
removed, frozen, and sectioned, and the section examined for
fluorescence. The resulting signal is stable for protracted times and
does not redistribute over the cut brain surface. Therefore, in
addition to other methods, the same method used in slices to suggest the
most promising approaches for
cryopreserving the whole brain can later be used on the whole brain to
allow a direct comparison of effects seen in slices and effects seen in
whole brains.
Although the fine details of this pilot project remain to be
finalized, the essentials of the plan are clear and compelling. The
ultimate details will depend in large part on the amount of money
available to propell the project forward. The project has seed money,
quiet laboratory space, a sheltered position under the supervision of a
very supportive department head at a major medical research center, and
unlimited access to electron microscopy that can be done directly by
project scientists (rather than through an individual who may not
provide proper representation of the condition of the slices). What is
lacking is a budget for the project. The budget will depend directly on
the degree of financial support obtained in the near future. The target
date for submission of a protocol to the medical center animal care
committee is January 19th. Support received prior to that date will be
used in estimating the budget, and therefore the amount of work, that
can be included in the submitted protocol. These contributions will
determine when a technologist can be hired, whether two technologists
can be hired to share the work and accelerate the project further, and
how much of all other required elements of the project -- equipment,
training, supplies, animals, consulting, and electron and light
micrographs -- can be marshalled for the project.
Thus, your contribution now, preferrably in time to provide a tax
write-off for fiscal year 1997, is needed. This is the beginning. With
your support, we could learn more about brain cryopreservation in 1998
than has been learned in the entire history of this field to date, and
1998 could serve as the gateway to truly promising studies on the whole
brain in 1999. Who knows? By acting today, the coming of the millenium
may become even more of an event than we expected.
Comment from Paul Wakfer:
I hope that everyone reading this is as inspired by Greg's words as I am
at this moment.
Those wishing to make a financial contribution to the Prometheus Pilot
Project should send their check to:
Paul Wakfer, trustee for FLLS
1220 E Washington St #24
Colton, CA 92324-6436
NOTE: FLLS stands for the Full Length Life Society, a charitable trust
which has been established to do the promotion, fund raising and
educational work which the Prometheus Project requires. IRS 501(c)3 tax
exempt charitable status has been applied for and it pending. The
research itself will be done by the Life On Hold Trust (LOHT) under
contract to FLLS.
Since I will be away from SoCal (home in Toronto visiting family) from
Dec 20 to Jan 10, I ask those sending financial contributions to also
send me email informing me of this. My being away also means that,
although you should date your checks in 1997, you can count on them not
being deposited until Jan 12 at the earliest.
-- Paul --
Voice/Fax: 909-481-9620 Page: 800-805-2870
The Prometheus Project -- http://prometheus.morelife.org
Research to Perfect Suspended Animation for Stabilization
of Patients Waiting for Cures for Their Terminal Diseases
Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=8943