Preservation of Canine Small Bowel
by Freezing Frank M. Guttman, MD, FRCS(C), FACS; George Berdnikoff, MD, FRCP(C); Kasem Sangbhundhu, MD: Petr Braun, MD total of 133 canine small bowel segments were isolated in dogs according to a previously described technique that permits an in vivo freeze-thaw experiment to be carried out after perfusion with various cryoprotective agents. All control segments (33) survived. One hundred segments were frozen with cold intra-arterial helium and ambient cold nitrogen gas after perfusion with dimethyl sulfoxide (DMSO), inositol, or glycerol in
MATERIAL AND METHODS
\s=b\ A
51
5% and 10% concentrations, alone, or combined, and with chlorpromazine and hydrocortisone added. Inositol had no cryoprotective effect. Approximately one half of segments frozen after protection with DMSO and with glycerol alone or combined with inositol survived the freeze-thaw injury and were intact on long-term follow-up. (Arch Surg 111:260-262, 1976)
of whole organs is one of the in the field of transplantation.1 :1 Unicellular tissues may be at low temperature in a frozen state when protected with glycerol or dimethyl sulfoxide (DMSO).4 Over the past ten years, little progress has been made in our ability to preserve frozen whole organs. The importance of such progress has been under¬ scored by Starzl." "There is no way that widespread and efficient utilization of human organ homografts will ever be possible without major new developments in organ preservation which will allow banking for weeks or months. Progress in this direction has been relatively minor." We have previously shown, along with others,711 that canine small bowel may be successfully preserved when perfused with DMSO or glycerol prior to a short term freeze-thaw experiment. The object of this article is to report subsequent experiments where inositol has been used as a cryophylactic agent, alone or in combination with DMSO and glycerol. In addition, the perfusion fluids have been altered by the use of chlorpromazine and hydrocortisone. These drugs have been shown to be effective in perfusion studies due to their membrane and lysosomal stabilizing action.6·8
prolonged preservation Successfulmajor goals preserved '
Adult mongrel dogs of both sexes, weighing 14 to 18 kg, were anesthetized with pentobarbital. Segments of ileum were perfused with various solutions of cryophylactic agents for 30 minutes. The segments were then frozen, using cold helium delivered intraarterially and cold nitrogen gas intraluminally. The rate of freezing is controlled through the use of a programmer-recorder with a feedback thermistor in the circuit. Details of the technique have been previously described/ Each animal served as its own control, with one segment perfused with the same solutions and kept ischemie for the same period of time as the experimental segment. The rate of freezing previously described was modified in these experiments in that a regular rate of freezing was maintained at 1 C/min until —25 C, and then the rate was increased as rapidly as possible to -80 C. The bowel was kept at -80 C for 15 minutes and then rapidly thawed with roomtemperature helium intra-arterially and immersion in a 40 C saline bath. The segments were then implanted subcutaneously (Fig 1). The cryophylactic solutions used were:
Group
1
(A) Inositol (5%) (V/V solution of saturated inositol in
heparinized perfudex. A saturated solution of inositol is a 15.5 gm/100 ml of aqueous solution at room tempera¬
ture.) (B) Inositol (10%) (C) Inositol (5%) + chlorpromazine (CHZ) sone (HC)
+
hydrocorti-
Fig 1.—Macroscopic appearance of short bowel segments, IV2 months after freezing and thawing. Upper left segment is control. Lower left segment perfused with balanced electrolyte solution (extracellular type) with 1.4M DMSO. Segment on right perfused with 1.4M DMSO, then frozen and thawed.
Accepted for publication Oct 10, 1975. From the departments of surgery (Dr Guttman) and pathology (Dr Berdnikoff), University of Montreal, the departments of surgery (Dr Guttman) and pathology (Dr Berdnikoff), Ste-Justine Hospital for Children, the Department of Surgery (Dr Guttman), Montreal Childrens' Hospital, and the Department of Surgery (Dr Guttman), McGill University, Montreal, Canada. Dr Sangbhundhu was a fellow of the Medical Research Council of Canada, 1969-1970, and Dr Braun was a fellow of the Medical Research Council of Canada, 1973-1974. Reprint requests to Department of Surgery, Ste-Justine Hospital, 3175 Ste-Catherine Rd, Montreal H3T-1C5, Quebec. (Dr Guttman).
Downloaded From: http://archsurg.jamanetwork.com/ by a Thomas Jefferson University User on 05/14/2015
Fig 2.—Left, Microphotograph showing
normal appearance of bowel mucosa six weeks after perfusion with 10% inosi¬ tol + 10% DMSO. Slight increase in goblet cells and in cellularlty of lamina propria
100). Center, (original magnification Appearance of deep mucosal glands 15 days after freeze-thaw experiment (protec¬ tive agent, 10% Inositol+10% DMSO). Increased cellularity and some goblet cell formation (original magnification 400). Right, Photomicrograph shows Increased cellularity six weeks after freeze-thaw injury (protective agent, 10% inosi¬ tol + 10% DMSO + hydrocortisone + chlor¬ promazine) (original magnification 400).
(D) Inositol (10%) + CHZ + HC (A) Inositol (5%) + DMSO (5%) (B) Inositol (5%) + DMSO (10%) (C) Inositol (10%) + DMSO (5%) (D) Inositol (10%) + DMSO (10%) Group 3 (A) Inositol (5%) + DMSO (5%) + CHZ + HC (B) Inositol (5%) + DMSO (10%) + CHZ + HC (C) Inositol (10%) + DMSO (5%) + CHZ + HC (D) Inositol (10%) + DMSO (10%) + CHZ + HC Group 4 (A) Inositol (5%) + glycerol (5%) + CHZ + HC (B) Inositol (5%) + glycerol (10%) + CHZ + HC (C) Inositol (10%) + glycerol (5%) + CHZ + HC (D) Inositol (10%) + glycerol (10%) + CHZ + HC Fifty milligrams of chlorpromazine were added per 1,000 ml, as well as 100 mg of hydrocortisone per 1,000 ml of perfusion
Group
2
solution.
RESULTS A total of 133 segments were tested in 51 dogs. Of these, 33 segments were perfused with the same cryophylactic agents and kept ischemie for the same period of time as the frozen segments. All these segments survived and were viable many weeks after the experiment. Mild inflamma¬ tory changes were present in the microscopical investiga¬ tion of these segments perfused by all the different combination of drugs. There was no specific microscopical pattern that could be identified with any of the perfusion solutions. When inositol was perfused at 5% and 10% concentra¬ tions, none of the 24 segments survived a freeze-thaw injury. These segments, which went on to necrosis, showed early acute inflammatory change in biopsy specimens taken immediately after the reestablishment of blood flow and 48 hours later. When the damage is so severe, a prediction may be made as to nonviability on the imme¬ diate postoperative biopsy specimen. No effect was seen when chlorpromazine and hydrocortisone were added to the perfusion of inositol alone. The effect of inositol and DMSO combined at 5% and 10% levels gave survival of four out of six, two of six, two of
five, and three of five for subgroups A, B, C, and D. There was a general 50% survival rate, which was distributed fairly evenly in all the subgroups. It should be noted that in previous studies with DMSO at 10% concentration, the survival was grossly about 50%." Microscopical examination again showed the same changes as in the previous group when necrosis occurred. The microscopical changes in the surviving segments were similar to our previous studies.911 We found mild to marked edema, formation of Gruen-
hagen space, capillary congestion, and lifting of the epithe¬ lial layer from the lamina propria (Fig 2). When chlorpromazine and hydrocortisone were added to the combination of inositol and DMSO, the results improved slightly with three of six surviving in groups A and B. In the subgroup C, six of nine segments survived, and in the subgroup D, five of eight segments survived; that is, 11 of 17 segments. Although there seemed to be a slight advantage in these 10% inositol groups (C and D), the difference between their survival rate and the survival rate in subgroups A and was not significant, according to a Fisher exact test. Analysis of results comparing 5% and 10% inositol in the other three major experimental groups (2, 3, and 4) demonstrated no significant difference. When inositol was combined with glycerol, a global 50% survival rate was noted. Again, the distribution of the survival rate in the subgroups was fairly evenly spread out; that is three of six, four of six, three of six, and three of seven for subgroups A, B, C, and D. Microscopical study of these segments did not indicate any particular features with different agents. Goblet cell increase was noted, as in our previous study.11 COMMENT
Inositol is a hexahydroxyhexane that has a role in fat metabolism. It is a naturally occuring intracellular substance. Webb1" has shown that inositol affords the best cell protection after dessication due to its + binding characteristic. The properties of inositol do conform to the criteria outlined by Robertson and Jacob;i as an ideal
Downloaded From: http://archsurg.jamanetwork.com/ by a Thomas Jefferson University User on 05/14/2015
Results of
Experiment
in
important since the advent of the transplantation of single
Groups 1 Through 4* No.
(%)
of
organs." Cadavaric
Segments
_A_
1
2 3
Group (IN,t 5% and 10%) (N = 24) (IN+DMSO,* 5% and 10%) (N =22)
Necrosis
24(100) 11
(IN+DMSO+CHZ§+HC[|) (N
=
Survival
29)
(50)
12(41)
(IN+glycerol+CHZ+HC) (N 25) Total No. (%) of segments *P
by Freezing Frank M. Guttman, MD, FRCS(C), FACS; George Berdnikoff, MD, FRCP(C); Kasem Sangbhundhu, MD: Petr Braun, MD total of 133 canine small bowel segments were isolated in dogs according to a previously described technique that permits an in vivo freeze-thaw experiment to be carried out after perfusion with various cryoprotective agents. All control segments (33) survived. One hundred segments were frozen with cold intra-arterial helium and ambient cold nitrogen gas after perfusion with dimethyl sulfoxide (DMSO), inositol, or glycerol in
MATERIAL AND METHODS
\s=b\ A
51
5% and 10% concentrations, alone, or combined, and with chlorpromazine and hydrocortisone added. Inositol had no cryoprotective effect. Approximately one half of segments frozen after protection with DMSO and with glycerol alone or combined with inositol survived the freeze-thaw injury and were intact on long-term follow-up. (Arch Surg 111:260-262, 1976)
of whole organs is one of the in the field of transplantation.1 :1 Unicellular tissues may be at low temperature in a frozen state when protected with glycerol or dimethyl sulfoxide (DMSO).4 Over the past ten years, little progress has been made in our ability to preserve frozen whole organs. The importance of such progress has been under¬ scored by Starzl." "There is no way that widespread and efficient utilization of human organ homografts will ever be possible without major new developments in organ preservation which will allow banking for weeks or months. Progress in this direction has been relatively minor." We have previously shown, along with others,711 that canine small bowel may be successfully preserved when perfused with DMSO or glycerol prior to a short term freeze-thaw experiment. The object of this article is to report subsequent experiments where inositol has been used as a cryophylactic agent, alone or in combination with DMSO and glycerol. In addition, the perfusion fluids have been altered by the use of chlorpromazine and hydrocortisone. These drugs have been shown to be effective in perfusion studies due to their membrane and lysosomal stabilizing action.6·8
prolonged preservation Successfulmajor goals preserved '
Adult mongrel dogs of both sexes, weighing 14 to 18 kg, were anesthetized with pentobarbital. Segments of ileum were perfused with various solutions of cryophylactic agents for 30 minutes. The segments were then frozen, using cold helium delivered intraarterially and cold nitrogen gas intraluminally. The rate of freezing is controlled through the use of a programmer-recorder with a feedback thermistor in the circuit. Details of the technique have been previously described/ Each animal served as its own control, with one segment perfused with the same solutions and kept ischemie for the same period of time as the experimental segment. The rate of freezing previously described was modified in these experiments in that a regular rate of freezing was maintained at 1 C/min until —25 C, and then the rate was increased as rapidly as possible to -80 C. The bowel was kept at -80 C for 15 minutes and then rapidly thawed with roomtemperature helium intra-arterially and immersion in a 40 C saline bath. The segments were then implanted subcutaneously (Fig 1). The cryophylactic solutions used were:
Group
1
(A) Inositol (5%) (V/V solution of saturated inositol in
heparinized perfudex. A saturated solution of inositol is a 15.5 gm/100 ml of aqueous solution at room tempera¬
ture.) (B) Inositol (10%) (C) Inositol (5%) + chlorpromazine (CHZ) sone (HC)
+
hydrocorti-
Fig 1.—Macroscopic appearance of short bowel segments, IV2 months after freezing and thawing. Upper left segment is control. Lower left segment perfused with balanced electrolyte solution (extracellular type) with 1.4M DMSO. Segment on right perfused with 1.4M DMSO, then frozen and thawed.
Accepted for publication Oct 10, 1975. From the departments of surgery (Dr Guttman) and pathology (Dr Berdnikoff), University of Montreal, the departments of surgery (Dr Guttman) and pathology (Dr Berdnikoff), Ste-Justine Hospital for Children, the Department of Surgery (Dr Guttman), Montreal Childrens' Hospital, and the Department of Surgery (Dr Guttman), McGill University, Montreal, Canada. Dr Sangbhundhu was a fellow of the Medical Research Council of Canada, 1969-1970, and Dr Braun was a fellow of the Medical Research Council of Canada, 1973-1974. Reprint requests to Department of Surgery, Ste-Justine Hospital, 3175 Ste-Catherine Rd, Montreal H3T-1C5, Quebec. (Dr Guttman).
Downloaded From: http://archsurg.jamanetwork.com/ by a Thomas Jefferson University User on 05/14/2015
Fig 2.—Left, Microphotograph showing
normal appearance of bowel mucosa six weeks after perfusion with 10% inosi¬ tol + 10% DMSO. Slight increase in goblet cells and in cellularlty of lamina propria
100). Center, (original magnification Appearance of deep mucosal glands 15 days after freeze-thaw experiment (protec¬ tive agent, 10% Inositol+10% DMSO). Increased cellularity and some goblet cell formation (original magnification 400). Right, Photomicrograph shows Increased cellularity six weeks after freeze-thaw injury (protective agent, 10% inosi¬ tol + 10% DMSO + hydrocortisone + chlor¬ promazine) (original magnification 400).
(D) Inositol (10%) + CHZ + HC (A) Inositol (5%) + DMSO (5%) (B) Inositol (5%) + DMSO (10%) (C) Inositol (10%) + DMSO (5%) (D) Inositol (10%) + DMSO (10%) Group 3 (A) Inositol (5%) + DMSO (5%) + CHZ + HC (B) Inositol (5%) + DMSO (10%) + CHZ + HC (C) Inositol (10%) + DMSO (5%) + CHZ + HC (D) Inositol (10%) + DMSO (10%) + CHZ + HC Group 4 (A) Inositol (5%) + glycerol (5%) + CHZ + HC (B) Inositol (5%) + glycerol (10%) + CHZ + HC (C) Inositol (10%) + glycerol (5%) + CHZ + HC (D) Inositol (10%) + glycerol (10%) + CHZ + HC Fifty milligrams of chlorpromazine were added per 1,000 ml, as well as 100 mg of hydrocortisone per 1,000 ml of perfusion
Group
2
solution.
RESULTS A total of 133 segments were tested in 51 dogs. Of these, 33 segments were perfused with the same cryophylactic agents and kept ischemie for the same period of time as the frozen segments. All these segments survived and were viable many weeks after the experiment. Mild inflamma¬ tory changes were present in the microscopical investiga¬ tion of these segments perfused by all the different combination of drugs. There was no specific microscopical pattern that could be identified with any of the perfusion solutions. When inositol was perfused at 5% and 10% concentra¬ tions, none of the 24 segments survived a freeze-thaw injury. These segments, which went on to necrosis, showed early acute inflammatory change in biopsy specimens taken immediately after the reestablishment of blood flow and 48 hours later. When the damage is so severe, a prediction may be made as to nonviability on the imme¬ diate postoperative biopsy specimen. No effect was seen when chlorpromazine and hydrocortisone were added to the perfusion of inositol alone. The effect of inositol and DMSO combined at 5% and 10% levels gave survival of four out of six, two of six, two of
five, and three of five for subgroups A, B, C, and D. There was a general 50% survival rate, which was distributed fairly evenly in all the subgroups. It should be noted that in previous studies with DMSO at 10% concentration, the survival was grossly about 50%." Microscopical examination again showed the same changes as in the previous group when necrosis occurred. The microscopical changes in the surviving segments were similar to our previous studies.911 We found mild to marked edema, formation of Gruen-
hagen space, capillary congestion, and lifting of the epithe¬ lial layer from the lamina propria (Fig 2). When chlorpromazine and hydrocortisone were added to the combination of inositol and DMSO, the results improved slightly with three of six surviving in groups A and B. In the subgroup C, six of nine segments survived, and in the subgroup D, five of eight segments survived; that is, 11 of 17 segments. Although there seemed to be a slight advantage in these 10% inositol groups (C and D), the difference between their survival rate and the survival rate in subgroups A and was not significant, according to a Fisher exact test. Analysis of results comparing 5% and 10% inositol in the other three major experimental groups (2, 3, and 4) demonstrated no significant difference. When inositol was combined with glycerol, a global 50% survival rate was noted. Again, the distribution of the survival rate in the subgroups was fairly evenly spread out; that is three of six, four of six, three of six, and three of seven for subgroups A, B, C, and D. Microscopical study of these segments did not indicate any particular features with different agents. Goblet cell increase was noted, as in our previous study.11 COMMENT
Inositol is a hexahydroxyhexane that has a role in fat metabolism. It is a naturally occuring intracellular substance. Webb1" has shown that inositol affords the best cell protection after dessication due to its + binding characteristic. The properties of inositol do conform to the criteria outlined by Robertson and Jacob;i as an ideal
Downloaded From: http://archsurg.jamanetwork.com/ by a Thomas Jefferson University User on 05/14/2015
Results of
Experiment
in
important since the advent of the transplantation of single
Groups 1 Through 4* No.
(%)
of
organs." Cadavaric
Segments
_A_
1
2 3
Group (IN,t 5% and 10%) (N = 24) (IN+DMSO,* 5% and 10%) (N =22)
Necrosis
24(100) 11
(IN+DMSO+CHZ§+HC[|) (N
=
Survival
29)
(50)
12(41)
(IN+glycerol+CHZ+HC) (N 25) Total No. (%) of segments *P
