X-Message-Number: 33103
Date: Mon, 06 Dec 2010 03:45:05 +0100
From: "Hal Tindale" <>
Subject: Re: CryoNet #33098 - #33099


how are the fly experiments going - hal

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From: CryoNet
Sent: 12/05/10 09:00 PM
Subject: CryoNet #33098 - #33099

CryoNet - Sun 5 Dec 2010 #33098: Re: could argon/xenon improve vitrification 
solutions? [benbest] #33099: parallel histories theory [Hal Tindale] Rate This 
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#33098 Date: Sat, 04 Dec 2010 16:52:58 -0500 From:  Subject:
Re: could argon/xenon improve vitrification solutions? Sorry if I am always 
raining on your parade, Doug, but when I see misconceptions in your postings I 
feel the need to set the record straight if there is to be hope of progress. I 
do greatly appreciate the interest you show in the possibility of reversible 
cryopreservation and the research that you do toward this end. 
http://www.cryonet.org/cgi-bin/dsp.cgi?msg=33097 All of the studies you cite 
except the xenon clathrate biostasis paper deal with the subject of reducing 
ischemic damage by substituting noble gas for oxygen. Despite the fact that a 
couple of these papers involve hypothermia, this has no relevance to improving 
vitrification solutions. I also doubt that xenon clathrate cryostasis is of any 
benefit to vitrification, although it could be of value in cryopreservation 
methods classically associated with freezing. The debate still rages within the 
cryobiology community as to whether freezing or vitrification is the best method
of cryopreservation. Many single-cell and small tissue specimens can be 
successfully cryopreserved in liquid nitrogen using small amounts of 
cryoprotectant that reduce the ice formation that occurs outside of cells. The 
small amounts of ice formed can be tolerated in small tissue samples and cell 
suspensions, and using only small amounts of cryoprotectant does not involve 
much toxicity. Until less toxic cryoprotectants can be found, this is the best 
approach for certain cell types. For more detail on classical cryogenic 
cryopreservation using freezing, see 
http://www.benbest.com/cryonics/cooling.html#classical These methods have no 
hope of ever being applicable to large organs. Those ignorant of cryobiology 
often suggest that cells burst upon freezing because ice has 9% greater volume 
than water. That claim is false, because water first freezes extracellularly, 
but it is nonetheless true that a 9% greater volume of ice in the extracellular 
space will nonetheless mechanically crush cells. Clathrate hydrates occupy a 
larger volume than ice, so any attempt to form these clathrates in an organ 
would result in even greater mechanical crushing. As I said in my analysis of 
clathrate hydrates: "The volume of a clathrate is greater than that of hexagonal
ice containing the same number of molecules ? even excluding the guest molecule
... clathrate formation in biological tissues is not an alternative to 
vitrification, and would actually be expected to cause more damage than 
freezing." http://www.benbest.com/cryonics/viable.html#clathrates Freezing 
techniques cannot complement vitrification, and neither can clathrate formation.
These techniques are mutually exclusive. I note that in the xenon clathrate 
cryostasis paper you cite http://www.ncbi.nlm.nih.gov/pubmed/18787624 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2480575/ that 100 psi (6.8 
atmospheres) pressure is used. High pressure methods can be used to reduce ice 
formation due to the 9% greater volume of ice over water, but in this case high 
pressure is probably mostly to induce the xenon to form the clathrate. It was 
once necessary to use 1,000 atmospheres of pressure with VS4 vitrification 
solution (higher pressures were too damaging), until the development of VS41A 
(VS4 for 1 atmosphere) vitrification solution. The bottom line is that although 
xenon clathrates may be useful for cryogenic temperature cryopreservation of 
cell suspensions and small tissue samples, it cannot be applied to cryogenic 
organ cryopreservation or cryonics. Reduction of cryoprotectant toxicity remains
the number one priority. There are methods that complement vitrification, such 
as ice blockers and high pressures, but clathrate formation is not one of these 
methods. -- Ben Best Rate This Message: 
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#33099 Date: Sun, 05 Dec 2010 09:06:00 +0100 From: "Hal Tindale" 
<> Subject: parallel histories theory 
--========GMXBoundary106701291536360921414 imagine a long beach with a hundred 
houses full of surfers. they have never met but they all go surfing at the same 
time. someone is going to get a surfboard in the back of the head - hal ----- 
Original Message ----- From: CryoNet Sent: 12/04/10 09:00 PM To: 
 Subject: CryoNet #33097 CryoNet - Sat 4 Dec 2010 #33097: 
could argon/xenon improve vitrification solutions? [oberon] Rate This Digest: 
http://www.cryonet.org/cgi-bin/rate.cgi?msg=33097%2D33097 Administrivia To 
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line: unsubscribe To post a message to CryoNet, send your message to: 
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questions, comments, or feedback to  with "CryoNet" or "cryonics"
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#33097 Date: Fri, 3 Dec 2010 09:28:32 -0800 (PST) From:  
Subject: could argon/xenon improve vitrification solutions? [Argon and xenon, 
offer powerful neuroprotectation, and act as novel cryoprotectants. Could 
argon/xenon reduce the toxicity of vitrification solutions? By preforming an 
intracellular clathrate, cell dehydration and membrane rupture are largely 
avoided during both freezing and rewarming. Clathrate would also presumably 
reduce vitrification solution toxicity, by limiting intracellular egress of 
toxic solvents like DMSO, and ethylene glycol. IMHO, there are enough unused 
novel technologies around such as directional freezing, variable magnetic field 
freezing, flavonoid additives, and here clathrate formation for cryonics to 
develop fully reversible whole body cryopreservation protocols within 5 years. 
As far as I am aware, the only limiting factor here preventing this is a lack of
vision, and more importantly alas a lack of money. It is hard to say how to get
around the funding desert that surrounds both cryonics, and organ 
cryopreservation. A vastly more effective sales strategy is here indicated than 
I have seen to date. Perhaps "paper" research into novel technologies, and 
development of a shared library of all the relevant research papers published on
t hese novel technologies would be a start. Perhaps a think tank with input 
from both professional salesman and skeptical scientists could then be used to 
develop an effective sales campaign. Otherwise it could easily take over 50 
years before the technologies available now are finally used to engineer fully 
reversible whole organ (and whole body) cryopreservation.] J Huazhong Univ Sci 
Technolog Med Sci. 2007 Aug;27(4):426-8. Experimental study on the 
cryopreservation of LLC-PK1 epithelial cells with hypoxic UW solution. Wan C, 
Wang C, Liu T, Wang H, Yang Z. Department of General Surgery, Union Hospital, 
Tongji Medical College, Huazhong University of Science and Technology, Wuhan 
430030, China. Abstract The effects of oxygen partial pressure on 
cryopreservation of the cells with organ preservation solution were explored. 
Hypoxic UW solution was made by purging the UW solution with argon. The pig 
proximal tubule epithelial cells (LLC-PK1 cells) were cryopreserved in hypoxic 
UW solution (Ar-UW group) or standard UW solution (UW group) at 4 degrees C for 
48 h. Trypan blue staining and LDH detection were performed to evaluate the 
injury of the cells. The results showed that the oxygen partial pressure in 
Ar-UW group was significantly declined from 242+/-6 mmHg to 83+/-10 mmHg. After 
cryopreservation at 4 degrees C for 48 h, LDH leakage rate and Trypan 
blue-stained rate in Ar-UW group were (11.3+/-3.4)% and (10.5+/-4.7)%, 
respectively, which were significantly lower than in UW group [(49.5+/-6.9)% and
(47.6+/-9.3)% respectively, both P<0.01]. It was concluded that lower oxygen 
partial pressure of UW solution was more beneficial to the cryopreservation of 
LLC. PMID: 17828502 [Here xenon forms an intracellular clathrate at above 
freezing temperatures, and thereby prevents most cell dehydration during water 
ice formation at sub-zero temperatures. This proved to be highly effective at 
preserving cell viability by preventing membrane rupture by large ice c rystals,
particularly during rewarming. Unlike pure water ice, the xenon/water clathrate
itself is apparently non-damaging.] Int J Clin Exp Pathol. 2008 Jan 
1;1(5):440-7. Cardiac mitochondrial membrane stability after deep hypothermia 
using a xenon clathrate cryostasis protocol - an electron microscopy study. 
Sheleg S, Hixon H, Cohen B, Lowry D, Nedzved M. Innovative Biological 
Preservation Technologies LLC Scottsdale, AZ, USA. Sergey. Abstract We 
investigated a new cryopreservation method using xenon, a clathrate-forming gas,
under medium pressure (100psi). The objective of the study was to determine 
whether this cryostasis protocol could protect cardiac mitochondria at cryogenic
temperatures (below 100 degrees Celsius).We analyzed transmission electron 
microscopy images to obtain information about changes in mitochondrial 
morphology induced by cryopreservation of the hearts. Our data showed absence of
mitochondrial swelling, rupture of inner and outer membranes, and leakag e of 
mitochondrial matrix into the cytoplasm after applying this cryostasis protocol.
The electron microscopy results provided the first evidence that a cryostasis 
protocol using xenon as a clathrate-forming gas under pressure may have 
protective effects on intracellular membranes. This cryostasis technology may 
find applications in developing new approaches for long-term cryopreservation 
protocols. PMID: 18787624 Free text> 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2480575/pdf/ijcep0001-0440.pdf Ann 
Neurol. 2010 Mar 8. [Epub ahead of print] Xenon enhances hypothermic 
neuroprotection in asphyxiated newborn pigs. Chakkarapani E, Dingley J, Liu X, 
Hoque N, Aquilina K, Porter H, Thoresen M. Department of Clinical Sciences at 
South Bristol, University of Bristol, Bristol, United Kingdom. Abstract 
OBJECTIVE: To investigate whether inhaling 50% xenon during hypothermia (HT) 
offers better neuroprotection than xenon or HT alone. METHODS: Ninety-eight 
newborn pigs underwent a 45-mi nute global hypoxic-ischemic insult severe enough
to cause permanent brain injury, and 12 pigs underwent sham protocol. Pigs then
received intravenous anesthesia and were randomized to 6 treatment groups: (1) 
normothermia (NT; rectal temperature 38.5 degrees C, n = 18); (2) 18 hours 50% 
xenon with NT (n = 12); (3) 12 hours HT (rectal temperature 33.5 degrees C, n = 
18); (4) 24 hours HT (rectal temperature 33.5 degrees C, n = 17); (5) 18 hours 
50% xenon with 12 hours HT (n = 18); and (6) 18 hours 50% xenon with 24 hours HT
(n = 17). Fifty percent xenon was administered via a closed circle with 30% 
oxygen and 20% nitrogen. After 10 hours rewarming, cooled pigs remained 
normothermic until terminal perfusion fixation at 72 hours. Global and regional 
brain neuropathology and clinical neurological scor es were performed. RESULTS: 
Xenon (p = 0.011) and 12 or 24 hours HT (p = 0.003) treatments offered 
significant histological global, and regional neuroprotection. Combining xenon 
wit h HT yielded an additive neuroprotective effect, as there was no interaction
effect (p = 0.54). Combining Xenon with 24 hours HT offered 75% global 
histological neuroprotection with similarly improved regional neuroprotection: 
thalamus (100%), brainstem (100%), white matter (86%), basal ganglia (76%), 
cortical gray matter (74%), cerebellum (73%), and hippocampus (72%). Neurology 
scores improved in the 24-hour HT and combined xenon HT groups at 72 hours. 
INTERPRETATION: Combining xenon with HT is a promising therapy for severely 
encephalopathic infants, doubling the neuroprotection offered by HT alone. ANN 
NEUROL 2010. PMID: 20658563 [PubMed - as supplied by publisher] Stroke. 2008 
Apr;39(4):1307-13. Epub 2008 Feb 28. Xenon and hypothermia combine additively, 
offering long-term functional and histopathologic neuroprotection after neonatal
hypoxia/ischemia. Hobbs C, Thoresen M, Tucker A, Aquilina K, Chakkarapani E, 
Dingley J. Department of Clinical Sciences at South Bristol, U niversity of 
Bristol, Bristol, UK. Abstract BACKGROUND AND PURPOSE: Hypoxic/ischemic (HI) 
brain injury affects 1 to 6 per 1000 live human births, with a mortality of 15% 
to 20%. A quarter of survivors have permanent disabilities. Hypothermia is the 
only intervention that improves outcome; however, further improvements might be 
obtained by combining hypothermia with additional treatments. Xenon is a noble 
anesthetic gas with an excellent safety profile, showing great promise in vitro 
and in vivo as a neuroprotectant. We investigated combinations of 50% xenon 
(Xe(50%)) and hypothermia of 32 degrees C (HT(32 degrees C)) as a post-HI 
therapy. METHODS: An established neonatal rat HI model was used. Serial 
functional neurologic testing into adulthood 10 weeks after injury was 
performed, followed by global and regional brain histopathology evaluation. 
RESULTS: In the combination Xe(50%)HT(32 degrees C) group, complete restoration 
of long-term functional outcomes was seen. Hypotherm ia produced improvement on 
short- (P<0.00 1) and long- (P<0.001) term functional testing, whereas Xe(50%) 
alone predominantly improved long-term function (P<0.05), suggesting that 
short-term testing does not always predict eventual outcome. Similarly, the 
Xe(50%)HT(32 degrees C) combination produced the greatest (71%) improvement in 
global histopathology scores, a pattern mirrored in the regional scores, whereas
Xe(50%) and HT(32 degrees C) individually produced smaller improvements (P<0.05
and P<0.001, respectively). The interaction between the 2 treatments was 
additive. CONCLUSIONS: The xenon/hypothermia combination additively confers 
greater protection after HI than either treatment alone. The functional 
improvement is almost complete, is sustained long term, and is accompanied by 
greatly improved histopathology. The unique safety profile differentiates xenon 
as an attractive combination therapy with hypothermia to improve the otherwise 
bleak outcome from neonatal HI. PMI D: 18309163 Free text> 
http://stroke.ahajournals.org/cgi/reprint/39/4/1307 Crit Care. 2009;13(6):R206. 
Epub 2009 Dec 17. Argon: neuroprotection in in vitro models of cerebral ischemia
and traumatic brain injury. Loetscher PD, Rossaint J, Rossaint R, Weis J, Fries
M, Fahlenkamp A, Ryang YM, Grottke O, Coburn M. Department of Anesthesiology, 
University Hospital of the RWTH Aachen, Aachen, Germany. Comment in: Crit Care. 
2010;14(1):117. Abstract INTRODUCTION: Recently, it has been shown in several 
experimental settings that the noble gases xenon and helium have neuroprotective
properties. In this study we tested the hypothesis that the noble gas argon has
a neuroprotective potential as well. Since traumatic brain injury and stroke 
are widespread and generate an enormous economic and social burden, we 
investigated the possible neuroprotective effect in in vitro models of traumatic
brain injury and cerebral ischemia. METHODS: Organotypic hippocampal slice 
cultures from mice pups were subjected to either oxygen-glucose deprivation or 
to a focal mechanical trauma and subsequently treated with three different 
concentrations (25, 50 and 74%) of argon immediately after trauma or with a 
two-or-three-hour delay. After 72 hours of incubation tissue injury assessment 
was performed using propidium iodide, a staining agent that becomes fluorescent 
when it diffuses into damaged cells via disintegrated cell membranes. RESULTS: 
We could show argon's neuroprotective effects at different concentrations when 
applied directly after oxygen-glucose deprivation or trauma. Even three hours 
after application, argon was still neuroprotective. CONCLUSIONS: Argon showed a 
neuroprotective effect in both in vitro models of oxygen-glucose deprivation and
traumatic brain injury. Our promising results justify further in vivo animal 
research. PMID: 20017934 Free text> 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2811924/pdf/cc8214.pdf Neurosci 
Lett. 2009 Sep 4;460(3):232-6. Epub 20 09 Jun 7. Neuroprotection (and lack of 
neuroprotection) afforded by a series of noble gases in an in vitro model of 
neuronal injury. Jawad N, Rizvi M, Gu J, Adeyi O, Tao G, Maze M, Ma D. 
Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College 
London, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, 
United Kingdom. Abstract Xenon-induced neuroprotection has been well studied 
both in vivo and in vitro. In this study, the neuroprotective properties of the 
other noble gases, namely, krypton, argon, neon and helium, were explored in an 
in vitro model of neuronal injury. Pure neuronal cultures, derived from foetal 
BALB/c mice cortices, were provoked into injury by oxygen and glucose 
deprivation (OGD). Cultures were exposed to either nitrogen hypoxia or noble gas
hypoxia in balanced salt solution devoid of glucose for 90min. The cultures 
were allowed to recover in normal culture medium for a further 24h in nitrogen 
or noble gas. The effect of noble gases on cell reducing ability in the absence 
of OGD was also investigated. Cell reducing ability was quantified via an MTT 
assay and expressed as a ratio of the control. The OGD caused a reduction in 
cell reducing ability to 0.56+/-0.04 of the control in the absence of noble gas 
(p<0.001). Like xenon (0.92+/-0.10; p<0.001), neuroprotection was afforded by 
argon (0.71+/-0.05; p<0.01). Neon and krypton did not have a protective effect 
under our experimental conditions. Helium had a detrimental effect on the cells.
In the absence of OGD, krypton reduced the reducing ability of uninjured cells 
to 0.84+/-0.09 (p<0.01), but argon showed an improvement in reducing ability to 
1.15+/-0.11 (p<0.05). Our data suggest that the cheap and widely available noble
gas argon may have potential as a neuroprotectant for the future. PMID: 
19500647 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=33097 
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