71. Canine kidney preservation by hypothermia and hyperbaria

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ANNUAL M E E T I N G ABSTRACTS

sc,gment,s. Two weeks after the reimplantation a eontralateral nephrectomy was performed. Four of six kidneys stored for 1 day, five of nine stored 2 days, and one of three kidneys stored 3 days were reimplanted successfully. Two dogs with kidneys stored 1 day, two dogs with control autotransplants, and two unoperated dogs have been followed for 2 yf, ~-~. Compensatory hyperplasia observed in the kidneys removed at eontralateral nephreetomy suggested complete failure of the reimplant. A partial failure of renal function followed contralateral nephrectomies, but marked improvement occurred during the first 2 'weeks with gradual improvement over the next 6 months. Some improvement cont.immd over 18 months, but at 2 years a deficit in function of the stored kidneys was detectable when compared by urinalysis, blood urea nitrogen, serum, creatinine, and clearante studies with the control groups. Renal storage resulted in damage and loss of function but successful reirnplantation was f01lowed by repair, although a small deficit in function was still present 2 years later. 69. Selection o f Perfusate for Preservation o f Small Intestine by Freezing. ~ RALPH HAMILTON,* H.~ZEL I. HOLST,* AND I=IEItNDON B. LF~HR. A highly specialized epithelium with a short natural life span makes small intestine a suitable organ for evahmtion of organ storage techniques. Its large surface to volume ratio permits rapid thawing by conduction. Previous work in this laboratory demonstrated survival of intestinal segments in (logs after freezing and thawing. The solution used to perfuse the segments prior to freezing was chilled, oxygenated DMSO (dimethyl sulfoxide), 10%, L M W D (low molecular weight dextran) 20%, homologous plasma 20%, BES (balanced electrolyte solution) 50%. This report deals with studies of 12 segments in 6 dogs. Paired 10-cm segments of ileum, attached only by mesenteric vessels, were delivered onto the abdominal wall and the vessels clamped. Except for use of a different perfusate, each segment of each pair was treated identically. The segments were frozen in a Dry Ice chamber at 1° to 5°C per min to --40 ° to -50°C, then thawed rapidly by immersion in warm saline solution. Circulation was established, and the segmeni, s were implanted subcutaneously. Serial biopsies sho~?ed strong similarity within each pair. The 7 surviving segments showed early hemorrhagic necrosis with regeneration to normal pattern in 3 to 5 days. Submucosal and muscular layers were preserved, although focal hemorrhages and fibro~As were sometimes present. No difference

Supported by a grant from the John A. Hartford Foundation.

in survival could be ascribed to the omission of oxygen or plasma; 10% glycerol was sul~st,itul,ed for 10% DMSO with survival. As in the former study, failures were assoeiaIed with vascular throlnbosis. In conclusion, plasma fred oxygen are not. essential components, and glycerol and DMSO al'e inter¢'hangcable in the perfusate. The histological resemblance between adjacent segments after di~'.rent perfusates was mudh closer than between segments in different dogs after the same perfusatc. More significant than composition of perfusate are control of freezing and thawing rate and careful surgi{.al technique. 70. T h e Use o f H e l i u m for Internal Freezing and Thawing o f Canine Kidneys. G. SAVARD AND R. LF,S (Canadian Liquid Air Ltd., Montreal, Quebec). The size and the he(erogenous structure of canine kidney make it impossible to obtain uniform freezing and thawing rates throughout this org'm by ordinary external thermal exchange procedures. I~ has been attemp(ed to overcome this difficulty by passing cooled or warmed helium through the blood circulatory s3'stem of the kidney. For the freezing of kidney the helium is passed through a copper coil at the bottom of a freezing chamber. Helium enters kidney through the renal artery and leaves through renal vein. ]33' means of a connected tube the rate of helium passing through the kidney can be measured outside of the freezing chamber. The chamber and its contents are cooled at controlled rates by liquid nitrogen. For thawing purposes helium is passed through anoflmr copper coil immersed, together with kidney, in a water bath "~t 40°C. The rates of freezing and thawing have been monitored by three termistor probes pieced under the "kidney capsule, and 0.5 cm and 1.5 cm deep into the tissues. It has been possible to maintain the I°C per min freezing rate at, all three horizons with no more than 2 ° to 3 ° difference. The thawing is more rapid on the surface, but even at 1.5-era level the change of t ° from --40 ° to + 4°C can be obtained in 5 to 7 rain. The histological and metabolic integrity of kidney tissue is preserved much better by this technique than by external freezing and thawing. 71. Canine Kidney Preservation By Hypotherm i a and Hyperbaria. GARY W. LYons, RONALD It. DIETZMA~, a~V R. C. LII,LmtEI (Department of Surgery, University of Minnesota, Minneapolis, Minnesota). The canine kidney may be stored successfully for 24 hrs by the combination of hypothermia (2 ° to 4°C) and hyperbaric oxy.gen ( 4 . 4 0 H P ) . It has been reported that raising the oxygen pressure to 7.9 OHP pdrmits successful preservation of the

ANNUAL MEETING ABSTRACTS canine kidney for periods up to 48 hrs. The effect, of increased oxyg(,n l)ressure and anoxic preservation on canine kidney survival was studied in the following manner. Ten mongrel (logs had their right kidney removed, stored for 48 hrs at 2 ° to 4°C anti 7.9 OHP and then autoreplanted. None of t.hese (logs survived following contralitteral nephrectomy. Ten additional (log kidneys were stored for 48 hrs at 2 ° to 4°C and 11.2 O H P prior to replantation. Again, none of tt)e,;-e dogs survived following removal of the other kidney. All dogs in these two groups died in 4 to 6 days in progressive renal failure. Contrary to previous reports, increasing the oxygen tension does not appear to permit survival of the nonperfuscd kidney

317

for 48 hrs. The effect, of anoxic preservation by the use of hyperbaric nitrogen has also been investigated. Eight canine kidneys were stored for 24 hrs at 2 ° to 40C and 4.4 atmospheres absolute nitrogen. Two of these 8 dogs are alive on a single, replanted, nitrogen-preserved kidney, 1 to 3 months after contralateral nephrectomy. The BUN, serum creatinine, and urine osmolarities are normal in 1 dog :rod returning toward normal in the second (tog. These findings indicate that the role of oxygen under pressure in preserv'ltion of the nonperfused organ for prolonged periods of time must be reevaluated and suggests a new approach to organ preservation.

Microorganisma

73. Studies on the Survival o f the Rotifer Philodina after F r e e z i n g and Thawing. "~JAMES

K. KOEItLER (Department of Biological Structure, University of Washington. Seattle, Washington). Rotifers are commonly quoted as belonging to the collection of animals exhibiting remarkable survival properties after freezing and thawing. Modern investigations exploring the effect of parameters such as cooling rate and the role of protective additives are, however, lacking. The present preliminary report represents an a t t e m p t to maximize the survival of these organisms after freezing and thawing by using appropriate combinations of additives, equilibration periods, and cooling rates. Cultures of Philodina acuticomis were maintained in the laboratory employing bacterized baked lettuce infusion as nutrient medium. Survival was scored microscopically by noting typical rotifer feeding or swimming movements in cultures 18 to 24 hrs after thawing. Slow freezing at rates of 1° per rain to 7 ° per rain were performed in a modified Linde Biological Freezer and very rapid freezing was accomplistfed by plunging samples on small copper discs into liquid freon at --150°C. The effect of glycerol addition on survival was studied at concentrations ranging from 0,1 to 10%. Glycerol concentrations at 20% were lethal to the rotifers before freezing. The pertinent results of these experiments are summarized as follows: 1) ultra rapid freezing is lethal and independent of glycerol additive concentration; 2) freezin~ in the absence of glycerol is lethal and independent of cooling rate; 3) slow cooling rates (1 ° per rain to 7 ° per min) with the presence of glycerol (0.1 to 10%) yield survival ranging from 5 to 30%; survivors of freezing and thawing are able to reproduce via the parthenogenic mode typical for these species. Some of the variability encountered

~Supported by grants from the National Science Foundation (CB-3587) and the State of

Washington Fund for Scientific Research (Initiative 171).

72. U h r a v i o l e t - l n d u c e d D a m a g e in Bacteria at Subzero T e m p e r a t u r e s . M. J. AStIW00D-SMITI-I (Medical Research Council, Radiobiological Resc'trch Unit, Harwell, Didcot, England). The enhancement of the lethal and mutagenic action of UV (2537z~,) which occurs when Escherichia colt is irradiated in the frozen state at subzero temperatures has been investigated biochemically. Damage associated primarily with the production of thymine dimens and other pyrimidine dimers, and which is normally repairable by this strain of E. coli, appears not to be involved in the low temperature lesion. Protein-DNA crosslinks induced by UV, however, are increased at low temperature. It is not known if this is biologically important. Transforming D N A (B. sublilis) does not show an increased sensitivity of UV when irradiated at - 7 9 ° compared with irradiation at +22°C. An investigation into the general applicability of this interaction between UV and low temperatures is at present in progress. Staphylococcus aureus, Aerobacter aerogenes, and Bacillus sublilis are all more sensitive to irradiation at - 7 9 ° C in phosphate buffer than at 220C. The increased sensitivity factors range from three to five. The spores of the fungus, Aspergillus nidulans, on the other hand, do not show a temperature factor. The extremely radioresistant bacterium, Micrococcus radiodurans is not more sensitive to UV at low temperatures than at 22°C. This latter fact suggests that either the new lesion induced by UV does not occur in M..radiodurans or, more likely, that it is produced but is capable of repair in this very interesting bacterium.