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). Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4953