X-Message-Number: 10840
Date: Thu, 26 Nov 1998 02:11:59 -0800 (PST)
From: Doug Skrecky <>
Subject: preserving tardigrades under pressure

 Nature 395: 853-854 October 28,1998
 "Preserving Tardigrades Under Pressure"
 By Kunihiro Seki, Masato Toyoshima

   When an animal is exposed to high hydrostatic pressure, its membranes,
 proteins and DNA are damaged. At pressures of around 30 megapascals (MPa)
 proliferation and metabolism in micro-organisms stops; at 300 MPa, most
 bacteria and multicellular organisms die. But here we show that, in
 perfluorocarbon at pressures as high as 600 MPa, small terrestrial
 animals known as tardigrades can survive in a dehydrated state.
   Terrestrial tardigrades become immobile and shrink into a form known as
 the 'tun' state when the humidity decreases. In this state, they can
 survive extreme temperatures, as low as -253 C or as high as 151 C, as
 well as exposure to a vaccuum or to X-rays. We have now tested the
 ability of the tardigrades Macrobiotus occidentalis (order Eutardigrada)
 and Echiniscus japonicus (Heterotardigrada) to survive under
 extraordinarily high hydrostatic pressures.
   We sealed M. occidentalis tardigrades in a small plastic container (6
 ml) placed inside a pressure capsule (R7K-3-10, Yamamoto Suiatu
 Kougyoucho) and compressed using water as the pressure medium. The
 outside temperature was 21 C and the water temperature inside the capsule
 was 25 C; stepped hydrostatic pressures were applied for 20 minutes at a
 time (100, 200, 300, 400, 500 and 600 MPa). Pressure was increased by 100
 MPA per minute and then increased by 100 MPa per minute and then
 decreased at the same rate. After decompression, M occidentalis was
 removed from the capsule and examined under a light microscope, which
 revealed that all organisms died at pressures over 200 MPa.
   We then investigated whether tardigrades could acquire pressure
 resistance in the tun state (a process known as anhydrobiosis) by
 dehydrating them before applying pressure. M. occidentalis and E.
 japonicus were dehydrated on filter paper in Petri dishes for more than
 24 hours, whem the relative humidity in the dishes dropped from 70-80% to
 10-30%. To prevent the tardigrades from rehydrating during compression,
 we used an inert solvent, perfluorocarbon (C8F18 Fluorinate PC77,
 Sumitomo 3M), as the pressure medium instead of water. Tardigrades were
 then removed from the pressure capsule and soaked in water to rinse off
 the perfluorocarbon. One hour later, we confirmed that they had changed
 from the tun state to the active state.
   To test whether the perfluorocarbon increased the survival of
 tardigrades exposed to high hydrostatic pressure, we subjected
 tardigrades in perfluorocarbon, which were still in the active state, to
 the same hydrostatic pressure changes. All active-state tardigrades were
 dead at pressures above 200 MPa.
   We evaluated the data at 600 MPa for the group (n = 20) in the tun
 state in perfluorocarbon and in the active state in water and
 perfluorocarbon. The survival rate of M. occidentalis was 95% at 600 MPa,
 and there was a difference between active state and tun state animals.
 The survival rate of E. japonicus was 80%, as some animals had died and
 their fluid had leaked onto the filter paper, which we attributed to
 inadequate dehydration before the experiment.
   Tardigrades are composed of about 40,000 cells, which survive not only
 high speed compression under a hydrostatic pressure of 600 MPa
 (equivalent to six times the pressure of sea water at a depth of 10,000
 meters), but also being maintained at this pressure, and high-speed
 decompression as well. With perfluorocarbon as the pressure medium, we
 have demonstrated the viability of tardigrades after keeping them in an
 anhydrobiotic state. This viability is influenced not just by pressure
 but by the absolute amount of water in the organism's body, enabling us
 to exploit its dehydration/water-absorption mechanism for preservation
 purposes.

 (the following article was previously posted on cryonet)
 New Scientist p 7 November 7, 1998
 "Putting Life on Hold"
 Weird creatures have some cute lessons for organ tranplanters
 By Jon Copley

   Taking a tip from tiny animals that can live for more than a century,
 Japanese have invented a new technique for storing human organs for
 transplant.
   The team, which has successfully revived a rat's heart after 10 days in
 storage, says the work may lead to organ banks similar to blood banks.
 These would allow doctors to avoid the frantic dash to bring suitable
 organs straight from donors to the operating table.
   Organs can usually be stored for only 30 hours before they have to be
 used. For hearts and lungs, the time limit is even less - just 4 hours.
 The main problem with keeping organs in cold storage is that water
 damages cell membranes at low temperatures. Unfortunately, removing water
 from tissues usually causes at least as much damage.
   But Kunihiro Seki and his colleagues at Kanagawa University in
 Hiratsukashi, Japan, knew that animals called tardigrades can withstand
 extreme conditions by losing most of the water in their bodies (In Brief,
 31 October, p 26). They can survive in this state for a century or more.
 When water was added to a dried-out moss kept in a museum for 120 years,
 tardigrades were later found crawling all over it.
   To achieve this feat, tardigrades use a sugar called trehalose to
 stabilise the structure of cell membranes. "This suggested that the
 physiological mechanism for preservation and resuscitation of tardigrades
 could be applied to preservation of mammalian organs," says Seki.
   To test this, the team flushed rat hearts with trehalose solution
 before packing them in silica gel to remove the water from their cells.
 The hearts were then immersed in perfluorocarbon, a biologically inert
 compound, and stored at 4 C in airtight jars. Ten days later, the team
 took the hearts out of the jars and resuscitated them. Within half an
 hour, they were beating again. Within half an hour, they were beating
 again. Measurements of their electrical activity suggested that the heart
 cells had survived intact.
   Seki believes that the trehalose and perfluorocarbon replace the water
 in the cells, preventing tissue damage. He plans to repeat the experiment
 with a complete autopsy to confirm that the tissues are preserved intact.
 The researchers also plan to demonstrate the procedure with other animal
 organs and prolong their storage for up to a year. Seki hopes that within
 a few years the technique could be used to preserve human organs.
   "The implications for transplant patients would be huge," comments
 Vanessa Morgan, who chairs the UK Transplant Co-Ordinators Association.
 "It could lead to planned, elective operations rather than emergency
 surgery." She adds that recipients could have a greater choice of donor
 organs, improving their chances of a good match. But she cautions that
 the quality of transplant organs must be maintained during long-term
 storage. "The condition of organs is crucial and I wouldn't want to
 compromise condition for time.


 Additional note by Doug Skrecky:

   Given the very poor permeation of disaccharides into tissue, I have
 some doubt that the trehalose flush had very much to do with preserving
 the rat hearts. Currently the only chemical preservation method which can
 prevent DNA and RNA from degrading quickly, is dehydration in ethanol.
 (Am J Clinical Pathology 96(1): p 144 July 1991) All attempts to preserve
 DNA and RNA in an aqueous environment have failed. Note that ethanol
 itself is not inert, and that it destroys cell membranes for example.
   What Kunihiro Seki may have discovered is the first fully reversible
 chemical preservation method - dehydration in inert perfluorocarbon.

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