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Preservation of isolated organs at hypothermia and by freezing
ANNUAL
MEETING
tions when rewarmed. The thin-walled sinus venosus and atria showed significantly better recovery than the thick-walled ventricle; it was suggested that this difference may be related to the degree of penetration of the glycol into the tissue. In an attempt to achieve better penetration during perfusion at subzero temperatures, methanol was substituted for glycol in the perfusate Hearts equilibrated at room temperat.ure in nontoxic concentrations of methanol were perfused with gradually increasing concentrations as the specimen was gradually cooled to various temperatures. The Inarts were gradually rewarmed, and during the rewarming the concent,ration of methanol in the perfusntc was gradually reduced. All hearts resumed spontaneous rhythmic contractions providing they were not cooled to below -30°C or perfused with concentrations of methanol exceeding 10 31.Cooling to lower t,emperatures or exposure to higher concentrations of methanol did not permit recovery. These results show that at temperatures as IOX as -30°C methanol in concentrations up to 10 >I is comparable to et,hylene glycol in its ability to protect hearts from crpoinjury. Its failure to protect, at lower t,emperatures may be related to tho development of toxic concentrations when watcr is removed in the form of ice. (Supported by Grant HE 10981 from the Nat.ional Institutes of Health.) 76. Some Temperature ing Kidney Perfusion.
Measurements
Concern-
R. KLEN* (District Institute of National Health, Faculty Hospital, Tissue Bank, Hradec Kr&lovC, Czechoslovakia).
The temperature changes were measured during the following perfusion processes: (a) 500 ml of 20” C hot perfusion fluid (as used in the Cleveland Clinic) contained in a National Transfusion Service bottle, during cooling in a bath of thawing ice. (b) Cooled perfusion fluid (contained in the abovementioned bottle) placed in a polyurethan insulation box. (c) Short-term perfusion performed by cont,inuous flow by gravity at different speeds t,hrough an insulated or noninsulated transfusion set. (d) Perfusion through the dog kidneys (standard hot ischemic time 20 min) by the same method described at (c) above. As the balance between the temperature of the perfusion fluid in the arterial inflow and t,he venous outflow has never been observed, a study was undertaken to: (e) determine the share of the environmental temperature and the heat production of kidney metabolism at the ascertained temperaturn difference, rind (f) find out if the heat production of kidney metabolism is a useful indicator of kidney viability.
ABSTRACTS
77.
3Y!+
Paradoxical Behavior of Aspergillus and Penici&m Spores to Ultraviolet Irradiation at Lore Temperatures. M. J. ASHWOOD-SMITH AND Y. D. HORNE* (Department of Biology, University of
Victoria, Victoria, B.C.. Canada). The lethal and mutagenic effects of ubraviolet (uv) radiation at low temperat,ures in the frozen state are, for the majority of microorganisms, mucli greater than at temperatures above zero. Results of this type are best explained on the basis of changes in the quality and quantity of uv-induced photoproducts and the ability or lack of ability of organisms to repair this damage. Spores of Hacillw megaterium and Bacillus subtilis show a paradoxical response in that they become increasingly more resistant after initial hypersensitivit,y responses (change is noted about, -80°C). Studies on t,he response of the spores of P. shectrii indicafod little effect of irradiation temperature (l-23 to -196°C) although there was a tendency towards resistance a~ temperatures near -196°C (resistance iurreascd by a factor of about 2 compared to room tcmyernture irradiation). However, the spores of Aspergillus nidulans showed marked temperature effects wheu irradiated in either saline or buffer. Temperatures within the range of 0 to -80°C had littlc effect, on viability but as irradiation temperatures fell f;,on -80 to -196°C there was a progressive and ~;crv marked increase in resistance so that at -196°C thr spores were nearly 16 times as resistant as at +23”C. No change in inactivation spectra at, these two temperatures was observed (maxima 2600.9; D, at -I-23°C = 10,000 ergs mm’; D, at -196°C: = 160,000 ergs mm’). Plating of irradiated sports on media containing 0.1% caffeine demonstrated no change in any of the survival values. Thus dark repair mechanisms were not involved in differential sensitivity. The only other microorganism of many thus far investigated that shows a similar response to uv radiation with temperature is ,&ficrococcus radiodurans. (Supported by Canadian National Research Council Grant A6206.) 77a. Preservation of Isolated Organs ctt Bypothermia and by Freezing. T. I. MALISIN (Dcpt,. Surgery. School of Medicine, University of Miami. Miami, Florida 33152). Preservation by freezing of mammalian cells and tissue fragments can be accomplished with most cell and tissue types by use of cryoprotectivr agents and controlled rates of cooling. The application of the same freezing techniques to preservation of whole organs has not met with success. Therefore, the only practical method of extracorporeal storage of whole organs is hypothermia and
ANNUAL
MEETING
perfusion. Since addition of erythrocytes to perfusion media do not prolong organ survival in vitro perfusion is usually carried out with media which do not contain red blood cells. Organ perfusion with cell-free media either at normothermic or hypothermic temperatures requires the initial removal of the residual blood from the vascular tree of the perfused organ and its replacement with perfusion solutions which can deliver nutrients and oxygen to the tissues and remove the metabolic waste products. Perfusion solutions which provide for longest organ survival under the extreme conditions of the artificial environment include blood plasma devoid of lipoproteins, isotonic and hypertonic balanced salt solutions with added synthetic nutrients and sera. Under experimental conditions by perfusing at hypothermia, the storage time for kidneys has been extended to 5 days and hearts to 5-7 days. However, despite these advances with perfusion techniques, the majority of kidneys transplanted in clinical practice is still preserved by simple hypothermia, a technique which generally permits storage of about 12 hr. Although simple hypothermia for kidney preservation may be adequate for some medical centers, it was clearly demonstrated that preservation of cadaver kidneys for 72 hr has many advantages. In the final analysis, however, the organ storage will be of maximum value in clinical transplantation if the extracorporeal life-span of the graft can be extended to weeks and months, rather than hours. This can be presently accomplished only by freezing. A number of different procedures for freeze-preservation of whole complex organs have been attempted and reported in literature. None of these have so far produced organs which would sustain animals’ life on transplantation. The difficulty with freezing seems to lie not with the freezing of the cells per se but with the delivery of a required concentration of the cryoprotective agents to all parts of the organ. Therefore, the primary concern with the preservation of a functional microvascular system in organs subjected to freezing and thawing cannot be overemphasized. 78. Treatment Shocking of
of Vasomotoric the Big Toe.
Rhinitis
M. RAM* SCHARTZ* (Rothschild Hospital-Technion, Institute of Technology, Israel).
ABSTRACTS
mentioned illness. This chilling has to be carried out quickly, within a period of time from 2-5 min. The best results were obtained by the use of direct expansion of ethyl chloride directly on the large toes. Recording of the characteristic of changes of temperature in the nose showed specific response for each case. In the course of treatment and cure of the patient the reaction of the nasal mucosa changes. 79. Relationships Between Stages of Hardening, Total Protein Concentrations, Nucleic Acid Fractionations, Polysome Profiles, and Ribonuclease Activities in Mimosa Hypocotyl Sections. G. N.
J. IBERG,* F. LONGACRE,* AND G. WEDDLE* (School of Forestry, University of Missouri, Columbia, Missouri 65201). Mimosa seedlings (Albizzia julibrissin Durazzini) were cold hardened through manipulation of photoperiod and day and night temperatures. Hypocotyl sections were excised at various stages of hardening, and degree of hardiness was determined using freezing regimens with both tetrazolium red viability assays and multiple freezing point assays. At each sampling point total protein concentrations and nucleic acid concentrations were determined, while nucleic acids were fractionated into transfer RNA, 5s ribosomal RNA, DNA, light ribosomal RNA, and heavy ribosomal RNA using MAK columns. Ribosomal preparations were fractionated into monomers and various polymer fractions using sucrose gradients at each stage of hardening. Finally, ribonuclease activities were assayed at each sampling point. Relationships between degree of hardening, total protein concentrations, and the various aspects of protein synthesis will be discussed. (Supported in part by National Science Foundation Grant GB-8692 and the Missouri Agricultural Experiment Station). BROWN,
80. Paraaortal Lymph Animal Experiments.
Node
Freezing
Studied
in
K.
HOCHBERG,* L. R~HL,* CONRAD* (Department of
J. CLORIUS,* AND G. Urology, University Clinic of Surgery, Heidelberg, Germany).
by Cool
A. Israel
AND
Through following up cases of patients who suffered from vasomotoric rhinitis with a clinical story of cold as the cause of the illness, and after various treatment experiments, the following results were obtained. Local cooling to -5°C leads to the drying up of the nostrils of patients suffering from the above-
The tumors of 40 rats with foreign body sarcomas were frozen. A freezing time of 2 min, a temperature of -180°C and a probe diameter of 9.52 mm resulted in tumor necrosis of 26 mm. The freezing cone was, on average, 4-6 mm larger than the necrotic area, but the rate with which the tumor was frozen and the final temperature were insufficient to destroy all the tumor cells. Histological examination of the scar tissue 30-40 days later showed living tumor cells in the marginal zones.
MEETING
tions when rewarmed. The thin-walled sinus venosus and atria showed significantly better recovery than the thick-walled ventricle; it was suggested that this difference may be related to the degree of penetration of the glycol into the tissue. In an attempt to achieve better penetration during perfusion at subzero temperatures, methanol was substituted for glycol in the perfusate Hearts equilibrated at room temperat.ure in nontoxic concentrations of methanol were perfused with gradually increasing concentrations as the specimen was gradually cooled to various temperatures. The Inarts were gradually rewarmed, and during the rewarming the concent,ration of methanol in the perfusntc was gradually reduced. All hearts resumed spontaneous rhythmic contractions providing they were not cooled to below -30°C or perfused with concentrations of methanol exceeding 10 31.Cooling to lower t,emperatures or exposure to higher concentrations of methanol did not permit recovery. These results show that at temperatures as IOX as -30°C methanol in concentrations up to 10 >I is comparable to et,hylene glycol in its ability to protect hearts from crpoinjury. Its failure to protect, at lower t,emperatures may be related to tho development of toxic concentrations when watcr is removed in the form of ice. (Supported by Grant HE 10981 from the Nat.ional Institutes of Health.) 76. Some Temperature ing Kidney Perfusion.
Measurements
Concern-
R. KLEN* (District Institute of National Health, Faculty Hospital, Tissue Bank, Hradec Kr&lovC, Czechoslovakia).
The temperature changes were measured during the following perfusion processes: (a) 500 ml of 20” C hot perfusion fluid (as used in the Cleveland Clinic) contained in a National Transfusion Service bottle, during cooling in a bath of thawing ice. (b) Cooled perfusion fluid (contained in the abovementioned bottle) placed in a polyurethan insulation box. (c) Short-term perfusion performed by cont,inuous flow by gravity at different speeds t,hrough an insulated or noninsulated transfusion set. (d) Perfusion through the dog kidneys (standard hot ischemic time 20 min) by the same method described at (c) above. As the balance between the temperature of the perfusion fluid in the arterial inflow and t,he venous outflow has never been observed, a study was undertaken to: (e) determine the share of the environmental temperature and the heat production of kidney metabolism at the ascertained temperaturn difference, rind (f) find out if the heat production of kidney metabolism is a useful indicator of kidney viability.
ABSTRACTS
77.
3Y!+
Paradoxical Behavior of Aspergillus and Penici&m Spores to Ultraviolet Irradiation at Lore Temperatures. M. J. ASHWOOD-SMITH AND Y. D. HORNE* (Department of Biology, University of
Victoria, Victoria, B.C.. Canada). The lethal and mutagenic effects of ubraviolet (uv) radiation at low temperat,ures in the frozen state are, for the majority of microorganisms, mucli greater than at temperatures above zero. Results of this type are best explained on the basis of changes in the quality and quantity of uv-induced photoproducts and the ability or lack of ability of organisms to repair this damage. Spores of Hacillw megaterium and Bacillus subtilis show a paradoxical response in that they become increasingly more resistant after initial hypersensitivit,y responses (change is noted about, -80°C). Studies on t,he response of the spores of P. shectrii indicafod little effect of irradiation temperature (l-23 to -196°C) although there was a tendency towards resistance a~ temperatures near -196°C (resistance iurreascd by a factor of about 2 compared to room tcmyernture irradiation). However, the spores of Aspergillus nidulans showed marked temperature effects wheu irradiated in either saline or buffer. Temperatures within the range of 0 to -80°C had littlc effect, on viability but as irradiation temperatures fell f;,on -80 to -196°C there was a progressive and ~;crv marked increase in resistance so that at -196°C thr spores were nearly 16 times as resistant as at +23”C. No change in inactivation spectra at, these two temperatures was observed (maxima 2600.9; D, at -I-23°C = 10,000 ergs mm’; D, at -196°C: = 160,000 ergs mm’). Plating of irradiated sports on media containing 0.1% caffeine demonstrated no change in any of the survival values. Thus dark repair mechanisms were not involved in differential sensitivity. The only other microorganism of many thus far investigated that shows a similar response to uv radiation with temperature is ,&ficrococcus radiodurans. (Supported by Canadian National Research Council Grant A6206.) 77a. Preservation of Isolated Organs ctt Bypothermia and by Freezing. T. I. MALISIN (Dcpt,. Surgery. School of Medicine, University of Miami. Miami, Florida 33152). Preservation by freezing of mammalian cells and tissue fragments can be accomplished with most cell and tissue types by use of cryoprotectivr agents and controlled rates of cooling. The application of the same freezing techniques to preservation of whole organs has not met with success. Therefore, the only practical method of extracorporeal storage of whole organs is hypothermia and
ANNUAL
MEETING
perfusion. Since addition of erythrocytes to perfusion media do not prolong organ survival in vitro perfusion is usually carried out with media which do not contain red blood cells. Organ perfusion with cell-free media either at normothermic or hypothermic temperatures requires the initial removal of the residual blood from the vascular tree of the perfused organ and its replacement with perfusion solutions which can deliver nutrients and oxygen to the tissues and remove the metabolic waste products. Perfusion solutions which provide for longest organ survival under the extreme conditions of the artificial environment include blood plasma devoid of lipoproteins, isotonic and hypertonic balanced salt solutions with added synthetic nutrients and sera. Under experimental conditions by perfusing at hypothermia, the storage time for kidneys has been extended to 5 days and hearts to 5-7 days. However, despite these advances with perfusion techniques, the majority of kidneys transplanted in clinical practice is still preserved by simple hypothermia, a technique which generally permits storage of about 12 hr. Although simple hypothermia for kidney preservation may be adequate for some medical centers, it was clearly demonstrated that preservation of cadaver kidneys for 72 hr has many advantages. In the final analysis, however, the organ storage will be of maximum value in clinical transplantation if the extracorporeal life-span of the graft can be extended to weeks and months, rather than hours. This can be presently accomplished only by freezing. A number of different procedures for freeze-preservation of whole complex organs have been attempted and reported in literature. None of these have so far produced organs which would sustain animals’ life on transplantation. The difficulty with freezing seems to lie not with the freezing of the cells per se but with the delivery of a required concentration of the cryoprotective agents to all parts of the organ. Therefore, the primary concern with the preservation of a functional microvascular system in organs subjected to freezing and thawing cannot be overemphasized. 78. Treatment Shocking of
of Vasomotoric the Big Toe.
Rhinitis
M. RAM* SCHARTZ* (Rothschild Hospital-Technion, Institute of Technology, Israel).
ABSTRACTS
mentioned illness. This chilling has to be carried out quickly, within a period of time from 2-5 min. The best results were obtained by the use of direct expansion of ethyl chloride directly on the large toes. Recording of the characteristic of changes of temperature in the nose showed specific response for each case. In the course of treatment and cure of the patient the reaction of the nasal mucosa changes. 79. Relationships Between Stages of Hardening, Total Protein Concentrations, Nucleic Acid Fractionations, Polysome Profiles, and Ribonuclease Activities in Mimosa Hypocotyl Sections. G. N.
J. IBERG,* F. LONGACRE,* AND G. WEDDLE* (School of Forestry, University of Missouri, Columbia, Missouri 65201). Mimosa seedlings (Albizzia julibrissin Durazzini) were cold hardened through manipulation of photoperiod and day and night temperatures. Hypocotyl sections were excised at various stages of hardening, and degree of hardiness was determined using freezing regimens with both tetrazolium red viability assays and multiple freezing point assays. At each sampling point total protein concentrations and nucleic acid concentrations were determined, while nucleic acids were fractionated into transfer RNA, 5s ribosomal RNA, DNA, light ribosomal RNA, and heavy ribosomal RNA using MAK columns. Ribosomal preparations were fractionated into monomers and various polymer fractions using sucrose gradients at each stage of hardening. Finally, ribonuclease activities were assayed at each sampling point. Relationships between degree of hardening, total protein concentrations, and the various aspects of protein synthesis will be discussed. (Supported in part by National Science Foundation Grant GB-8692 and the Missouri Agricultural Experiment Station). BROWN,
80. Paraaortal Lymph Animal Experiments.
Node
Freezing
Studied
in
K.
HOCHBERG,* L. R~HL,* CONRAD* (Department of
J. CLORIUS,* AND G. Urology, University Clinic of Surgery, Heidelberg, Germany).
by Cool
A. Israel
AND
Through following up cases of patients who suffered from vasomotoric rhinitis with a clinical story of cold as the cause of the illness, and after various treatment experiments, the following results were obtained. Local cooling to -5°C leads to the drying up of the nostrils of patients suffering from the above-
The tumors of 40 rats with foreign body sarcomas were frozen. A freezing time of 2 min, a temperature of -180°C and a probe diameter of 9.52 mm resulted in tumor necrosis of 26 mm. The freezing cone was, on average, 4-6 mm larger than the necrotic area, but the rate with which the tumor was frozen and the final temperature were insufficient to destroy all the tumor cells. Histological examination of the scar tissue 30-40 days later showed living tumor cells in the marginal zones.