CryoNet #33098 - #33099

CryoNet - Sun 5 Dec 2010

    #33098: Re: could argon/xenon improve vitrification solutions? [benbest]
    #33099: parallel  histories  theory [Hal Tindale]

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Message #33098
Date: Sat, 04 Dec 2010 16:52:58 -0500
From: [email protected]
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

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Message #33099
Date: Sun, 05 Dec 2010 09:06:00 +0100
From: "Hal Tindale" <[email protected]>
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: [email protected]
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 subscribe to CryoNet, send email to: [email protected] with the subject line (not message _body_): subscribe To unsubscribe, use the subject line: unsubscribe To post a message to CryoNet, send your message to: [email protected] from the same address to which you are aubscribed. Send questions, comments, or feedback to [email protected] with "CryoNet" or "cryonics" somewhere in the Subject line. ---------------------------------------------------------------------- Message #33097 Date: Fri, 3 Dec 2010 09:28:32 -0800 (PST) From: [email protected] Subject: could argon/xenon improve vitrification solu
 tions? [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 prese
 rvation 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 pre
 vents 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 inform
 ation 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. Ab
 stract 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 neurologi
 cal 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, followe
 d 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 grea
 tly 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 b
 urden, 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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