X-Message-Number: 4953
Date:  Sun, 08 Oct 95 21:01:12 
From: mike <>
Subject: dendritic spines

Dendritic Spines:
A Possible Problem for Cryonics.
by Mike Perry, Alcor

In message #4871 (Sep. 13) Doug Skrecky reprints his 
article "Biostasis" from the Jan/Feb 1991 Mensa Canada 
Communications. This article makes reference to research 
that, while not connected specifically with cryonics, calls 
into question whether current cryonic practices achieve an 
adequate preservation for the goal of eventual reanimation 
[1]. More specifically, results are cited suggesting that 
substantial degradation of important brain structures, the 
dendritic spines, occurs under conditions that are often 
found in cryonic suspensions. Dendritic spines are tiny 
outgrowths of the dendrites of a neuron in the brain that 
form the synaptic junctions with axons. The axons in turn 
carry signals from other neurons, so the synapses are the 
vital communication link between neurons that enable the 
brain to function. Loss of dendritic spines has been 
observed in senile dementias such as Alzheimer's disease 
[2] and appears to be a factor in the substantial loss of 
memory and other mental deterioration that is seen. 

In the research cited [1], "Twenty eight adult guinea-pigs 
were killed with 0.4 ml Nembutal intravenously. At 
different intervals the brains of 4 animals were removed (5 
min., 45 min., 1.5, 4, 8, 16, 24h post-mortem respectively). 
The brains were prefixed in 10% buffered formol. Between 
death and prefixation the cadavers were kept at 4 [degrees] 
C. After a prefixation for 6 days, the brains were sectioned, 
and 3 mm slices of the frontal, parietal and occipital area of 
the neocortex were washed for 24 h in NaCl 0.9%; they 
were transferred to the Golgi fixative [K2CrO4] 10g, OsO4 
1g in 300 ml Aqua Dist. for 4 days, and impregnated in 
AgNO3 0.75% for 3 days. Subsequently, the tissue blocks 
were embedded in paraffin and cut on a sliding microtome 
at a thickness of 110 [micrometers], dehydrated, cleared in 
methyl salicylate and mounted in malinol."

Neurons of the layer V pyramidal type were chosen in the 
above-mentioned cortical areas, and spines were counted 
on 50 micrometer segments of apical and basal dendrites 
(which start at different distances from the neuron), and on 
oblique branches of dendrites. "In each animal 30 segments 
were counted in this way." While results varied somewhat 
with the type of dendritic segment, overall the results 
clearly show a substantial drop in the number of spines of 
each type beginning 1 1/2 hours postmortem. Assuming 
that 100 percent of the dendritic spines survived in the 
group with the shortest (5 min.) delay before fixation, the 
results allow an estimate of the percentage survival for 
other time delays. Mean survival rates for the apical 
dendritic spines are as follows: 3/4 hr: 97%; 1 1/2 hr: 67%; 
4 hr: 57%; 8 hr: 36%; 16 hr: 36%; 24 hr: 19%. There is a 
fairly large standard deviation of about 15-30 associated 
with these percentages, but the trend is unmistakable; 
results for the other types of dendritic spines are similar. By 
way of comparison, [2] reports a dendritic spine density in 
demented (mainly Alzheimer's) patients of 53% the normal 
group of similar age using a similar preparation protocol. 
(Another finding of [1] is that the dendrites themselves 
maintain their shape and general appearance up to four 
hours postmortem, despite the spine attrition, but then 
begin to shrink.)

The guinea-pig results are disturbing, particularly because 
they involve storage of the brain at 4 C, which might be 
considered good pre-suspension conditions for cryonic 
patients. (The guinea-pigs, however, were not given 
metabolic support or perfused, an important consideration.) 
In most cases under current cryonic practice there is 
probably *at least* an 8-16 hr. delay at around 4 C before 
freezing of the brain can *start*, let alone go to completion. 
In the *absence* of a pre-freezing protocol (metabolic 
support during the initial cooling, glycerol perfusion, etc. 
that are applied during a good suspension) this would seem 
consistent with worse conditions than were found in the 
living, demented patients [2]. Such results, it may be 
pointed out, are not incompatible with other findings of 
apparently good preservation of larger-scale brain structure 
such as the neurons themselves, or with Suda's cat-brain 
experiments in which brain waves were detected on 
warming of partially frozen brains that had been promptly 
glycerolized and cooled[3]. In this vein Mike Darwin et al. 
recently reported good ultrastructural brain preservation 
with dogs that had a good postmortem suspension protocol 
(comparable to that in use by BioPreservation and Alcor on 
human patients) and a very short (5 min.) pre-suspension 
delay. Good preservation of dendritic spines was reported, 
as verified visually, though quantitative estimates were not 
obtained.[4]. 

Dendritic spines, it should be noted, are far smaller than 
neurons, axons or dendrites and thus are more apt to be 
damaged over a short time interval. On the other hand, their 
role in brain function certainly seems significant, if not yet 
fully understood.

So there is cause for concern, though the outlook is far 
from hopeless. Under good conditions, cryonic 
preservation still seems as if it could be adequate for 
reanimation, though as always we can't be sure. In the less 
favorable cases, such as those involving a substantial 
ischemic episode prior to suspension, the outlook is less 
optimistic, though even here there is some ground for hope. 
One possibility is that the important brain information such 
as longterm memory may be very redundantly encoded, 
which will allow its inference from a small fraction of the 
structures that contain it. We don't know how longterm 
memory is stored, or how important a role dendritic spines 
may play, but we do know it must be robust enough to last 
for decades. Dendritic spines and other neuronal processes 
are replenished or added throughout life (in contrast to 
neurons, which are not much replaced after early 
childhood) so their loss may be fairly straightforward to 
make up. But certainly this cannot be taken for granted. As 
ever, we must face the possibility that yesterday's and even 
today's cryonics patients are not going to be reanimated at 
all--without radical measures such as filling-in of missing 
information from other sources.

On the other hand, the findings about dendritic spines 
appear to give us a handle we didn't already have on how 
good our preservation techniques are, and how to evaluate 
our future techniques. Research *must* move forward, both 
to add to our knowledge and, most importantly, to find 
ways of resolving whatever problems exist. Meanwhile I 
would advocate taking more seriously the idea of storing 
documentary information about yourself, to assist in 
reanimation if the brain preservation isn't good enough.

The guinea-pig study appeared in 1983. It is remarkable 
that so much time has elapsed without more notice being 
taken of its implications for cryonics. There is, apparently, 
a great deal of other mainstream scientific literature with 
potential relevance. (A Medline search under "postmortem 
brain" showed over a thousand titles for the last three years 
alone. "Neuroprotective" produced over 700 entries for the 
same period.) More searching on our part is called for. We 
can't expect others to inform us, even if there are critics 
who would also be glad to know about scientific problems 
with cryonics. Science is pretty compartmentalized, and the 
number who take seriously what we are interested in 
(survival beyond the biological limits) is still astoundingly 
small.

I thank Hugh Hixon and Thomas Donaldson for their 
assistance in preparing this article. 

References:

[1] de Ruiter, J. "The influence of post-mortem fixation on 
the reliability of the Golgi silver impregnation," *Brain 
Research*, v6, p143-147 (1983).

[2] de Ruiter, J. and H. Uylings, "Morphometric and 
dendritic analysis of fascia dentata granule cells in human 
aging and senile dementia," op. cit., v402, p217-229 
(1987).

[3] Suda, I., K. Kito and C. Adachi, "Viability of long term 
frozen cat brain in vitro," *Nature*, v212, p8 (15 Oct 
1966).

[4] Darwin, M., S. Russell, L. Wood, C. Wood, and S. 
Harris. "Canine brain cryopreservation" BPI Tech. Brief 
#16, CryoNet messages 4468 and 4474 (1-2 Jul 1995).


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