JOURNAL
OF SURGICAL
RESEARCH
19, 55-63 (1975)
The No-Reflow Canine
Renal
Phenomenon
Preservation
with
After Medium
199
ARNOLD G. DIETHELM, M.D.,’ JAMES DEVRIES, B.S.,2 M. W. HARTLEY, Ph.D.,3 AND S. J. PHILLIPS, B.S. Departments of Surgery and Pathology, The University of Alabama Medical Center and the Birmingham VA Hospital, Birmingham, Alabama 35294 Submitted for publication September 30, 1974
MATERIALS AND METHODS Kidney preservation achieved with cryoprecipitate of plasma and hypothermic Adult mongrel dogs, 15-25 kg in weight, pulsatile perfusion was successful in previously immunized against distemper, maintaining organ viability for 48 hr and were maintained with a general laboratory longer [2,14]. An important feature of this diet. Anesthesia was administered by perfusate has been attributed to the removal intravenous sodium pentobarbital with an of lipoproteins by freezing, thawing and endotracheal tube and a respirator. filtration, thus preventing obstruction of the Following a left nephrectomy the kidney was renal microcirculation by protein aggregates placed on the organ support unit. Renal during preservation [3]. Canine kidneys have reimplantation was accomplished by realso been successfully preserved for 24 hr vascularization to the external iliac artery using a synthetic perfusate, Medium 199, and vein with a ureterovesical anastomosis administered via pulsatile flow at 10” C [7]; performed using sterile technique. The Others [9-l 11, however, have noted edema, average ischemic interval during reimarterial injury and increased vascular re- plantation was 28 min. A contralateral sistance when isolated rabbit kidneys were nephrectomy was done at this time. During perfused with cell-free solutions including each experiment the dog received 500 ml of dextran. Ringer’s lactate by the intravenous route. A synthetic solution with a known com- No diuretics were used before nephrectomy position, relatively inexpensive and com- or after reimplantation. mercially available, would simplify the Organ support unit. This unit designed procedure of organ preservation. Because of and constructed by Sarns, Inc., Ann Arbor, this we reexamined the effectiveness of Me- MI., consisted of two identical systems cadium 199 as a perfusate in canine renal pable of sustaining two kidneys with seppreservation. Special attention was given to arate perfusate. Each system included a evaluation of the following parameters pulsatile pump, membrane oxygenator,’ during and after preservation. 1), The pa- heat exchanger, arterial bubble trap, venous tency of the intrarenal vasculature; 2), the collection reservoir and organ storage increase in renal edema after preservation; chamber. All experimental pressures were 3), the predictability of perfusion charac- maintained electively at 60/40 mm Hg with teristics during preservation compared with a temperature of 7-10°C. Perfusate flow organ viability after reimplantation; and 4), rates were dependent upon pressure and the ability of the preserved kidney to sustain intrarenal resistance. life in a contralaterally nephrectomized dog. Perfisa te. A synthetic perfusate including the following chemical properties ‘Professor and ViceChairman of Surgery. was used in all experiments: Powdered me2Product Manager, Sarns Inc., Ann Arbor, MI. 4Made by General Electric.
3Professor of Pathology.
55 Copyright @1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
JOURNAL
OF SURGICAL
RESEARCH
dium-molecular-weight dextran, 35 g; Medium 199, Earle, base 10x, 100 ml; distilled water, 900 ml; dextrose, 5 g; sodium bicarbonate, adjusted to pH 7.4, 20-30 ml; insulin, regular, 200 units; potassium penicillin G, 5 million units; neomycin sulfate, 50 mg; mycostatin, 20,000 units; mannitol 6.2512.25 g.
The osmolality ranged between 350 and 375 mosm/l with an oncotic pressure equal to 8 g of protein per 100 ml. Sodium, potassium, chloride, COz, pH, PO,, and $0, were measured before, during and at the conclusion of each period of preservation. A cryoprecipitate of plasma, prepared as outlined by Belzer [2], including the same antimicrobial agents as outlined in the synthetic solution was used as a control perfusate. Evaluation by colloidal
of the renal microcirculation carbon. Carbon black5 (pre-
pared by the Pelikan Company of Hanover, Germany, contained particles between 200 and 400 A) diluted with equal volumes of isotonic saline, pH 7.4, was injected into the renal artery at the conclusion of preservation and in some instances 4 hr after reimplantation of the kidney into the dog. All injections were performed using gravityinduced, nonpulsatile flow at 140 mm Hg. The artery and vein were simultaneously clamped and tied, and the kidney placed in 10% formalin. After complete fixation the organ was bisected and examined grossly. Specimens obtained for histologic examination were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff reagent (PAS). Additional sections, 100-200 pm thick, were imbedded in glycerin for 24 hr and examined by light microscopy. Assessment of renal edema, viability and characteris tics of preserved perfusion kidneys. Twenty-five kidneys were studied
and divided equally into five groups. Group I, 6-hr preservation (Medium 199) followed with carbon injection; Group II, 24-hr preservation (Medium 199) followed with %old By: John Henschel and Co., Inc., 141 Albertson Avenue, Albertson, Long Island, NY 11507
VOL.
19, NO. I, JULY
1975
carbon injection; Group III, 24-hr preservation (Medium 199) with reimplantation for survival; Group IV, 24-hr preservation (Medium 199) with reimplantation for 4 hr followed by carbon injection; Group V, 24-hr preservation (cryoprecipitate of plasma) with reimplantation for survival and carbon injection 2 weeks later. RESULTS Group I, 6-Hr Preservation and Carbon Injection
The average weight of five kidneys increased 15.4% during preservation (ranging from 9-24%). Flow rates increased by 25 and 66% in two kidneys, remained unchanged in two and decreased by 20% in the fifth organ. Kidneys injected with carbon and bisected appeared diffusely black without visible areas of vascular obstruction. Histological sections (H&E and PAS) confirmed the presence of carbon throughout the intrarenal vasculature. In two kidneys there were alterations in the vascular endothelium and glomeruli, suggestive of early necrosis. Glycerin sections demonstrated carbon particles throughout the cortical and medullary vasculature including glomeruli. Group II, 24-Hr Preservation and Carbon Injection
The average weight gain of five kidneys during preservation was 24% (ranging from 20-27%). Flow rates at the end of preservation were decreased by 33% in one experiment and by 40% in two other experiments. The two remaining kidneys showed no change in intrarenal resistance. All organs injected with carbon revealed a gross appearance similar to kidneys in Group I (see Fig. 1A). Microscopic glycerin sections confirmed a patent intravascular system (see Fig. 2A), and histological examination of all five kidneys after preservation demonstrated minimal evidence of intimal injury.
DIETHELM
ET AL.. CANINE
RENAL
PRESERVATION
FIG. 1. Two kidneys preserved for 24 hr with Medium 199 followed by injection with colloidal carbon, Kidney A revealed complete vascular perfusion of both cortex and medulla. Kidney B, reimplanted into the host animal for 4 hr, developed progressive obstruction of the microcirculation as demonstrated by multiple areas devoid of carbon in the cortex and medulla.
wqn.‘amol% aql q%noJql ssed 01 alqeun ale 1nq mawis Bug~allo~ snoualz pue sayll!de~ mlnqnl!lad aql u! luasald ale salD!l-reduoqle3 .uogDa!ir! uoqn3 p+aw Kq pamolloj pue ~q p ~oj pxu!ue lsoq aql ONI!paweldm!al‘661 um!paH q]!~ ~q tz ~oj pahlasald Laupy e JO uoyas uyax41f) ‘(a) .sagelpde3 relnqnlyad puE gnlauro~%aq3 u!qI!M sa[cyled uoqu23 %u!pnixy uoyqn3+3oJyu Ivual lualE?d E paleaAaJ ‘uoqJlz3 JO uogsa[u! p+ain ale!pawm! Lq pa,woiloj cgj1 mmpaw ql!m Jq pz ~oj paAIasald ‘Laupg E jo uoyas u!3a3Qt) ‘(v) ‘5 ‘f)~d
DIETHELM
ET AL.: CANINE
Group III, 24-Hr Preservation with Reimplantation
Five kidneys preserved with Medium 199 were reimplanted into the dog to determine viability by renal function. The average weight gain after preservation and before return to the host animal was 25% (ranging from 21-28%). Flow rates decreased from 12-50% in all preserved kidneys with an average of 25%. Immediately after reimplantation, the kidney became pink in color, firm to palpation with the appearance of excellent cortical blood flow. The onset of urine was observed in all animals within 15 min after completion of the vascular anastomosis. One hour after revascularization the kidney turgor was decreased to palpation, and the cortical surface appeared cyanotic. Urine output at this time was markedly diminished. Three hours after reimplantation cyanosis of the renal cortex had increased with a further diminution in cortical turgor to palpation and cessation of urine production. All dogs expired in uremia 6-10 days after reimplantation with evidence of tubular necrosis at autopsy. Group IV, 24-Hr Preservation, Reimplantation and Carbon Injection
Five kidneys preserved for 24 hr, as in Group III, were reimplanted into the dog. The average weight gain after preservation was 30% (ranging from 27-36%). Flow rates decreased 33, 18, 16 and 14% in four experiments and increased 25% in one. The appearance of the kidney during the first 3 hr after reimplantation was identical to that observed in Group III. After 4 hr of circulation through the kidney, the arterial inflow was manually occluded, the artery cannulated and the kidney injected with carbon. Carbon passed slowly and irregularly through the kidney, with large areas of the cortex excluded of carbon. After formalin fixation the bisected kidney demonstrated the cortex and medulla to have an irregular distribution of carbon with a decrease in content compared to the control kidney (see Fig. - 1B). Histologic sections
RENAL
PRESERVATION
59
(H&E and PAS) revealed the afferent arteriole and glomeruli to be engorged with intact erythrocytes and fibrin preventing passage of carbon into the glomerulus (see Fig. 3). The vasa recta showed a similar appearance of red blood cell engorgement obstructing the passage of carbon particles. There was no evidence of an acute inflammatory response characterized by polymorphonuclear leukocyte infiltration. Glycerin sections of the kidney revealed complete absenceof carbon within the glomeruli and a decrease in carbon particles within the peritubular capillaries (see Fig. 2B). Group V, 24-Hr Preservation with Cryoprecipitate of Plasma
Five kidneys preserved with plasma cryoprecipitate were reimplanted into the host animal. The average weight gain was 29% (ranging from l&35%). Flow rates remained unchanged in two experiments, decreased 50-75% in two and increased 50% in one. Immediately after reimplantation and restoration of circulation, the kidneys appeared well perfused with normal turgor to palpation. Urine flow was prompt, and all animals survived. Renal function was normal at the end of the 14th day in two dogs (Cr, 1.4 and 1.2 mg/lOO ml), borderline in two dogs (1.6 and 1.8 mg/ 100 ml) and elevated in one (2.4 mg/lOO ml). The animals were sacrificed at this time and the kidneys injected with carbon. The gross appearance was nearly identical to the control kidneys injected with carbon, as described in Group I. Glycerin sections showed both glomeruli and vasa recta to be filled with carbon including patent efferent and afferent arterioles. Histological sections demonstrated the tubules and arterial intima to be intact with carbon throughout the glomeruli (see Fig. 4). DISCUSSION Although 24-hr preservation of canine kidneys with Medium 199 delivered by pulsatile flow at 4°C was not successful in maintaining renal viability after reim-
60
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1975
FIG. 3. Obstruction to the injection of colloidal carbon particles at the glomerulus secondary to engorgement of the capillaries by trapped erythrocytes (H&E).
plantation, several important observations emerged from these experiments. First, weight gain of the kidney during preservation with a synthetic perfusate as measured by total wet weight of the organ did not provide useful criteria for predicting viability after reimplantation. In fact, weight gain was similar in those kidneys unsuccessfully preserved with Medium 199 and in kidneys maintained successfully using a cryoprecipitate of plasma. Thus, renal edema alone did not provide a means of separating viable from nonviable kidneys. Second, perfusion characteristics in Group III during renal preservation using a constant pressure
did not differentiate viable from nonviable kidneys after reimplantation. The increased intrarenal resistance, essentially the same using either the synthetic perfusate or the cryoprecipitate of plasma, did not correlate with renal viability. Third, preservation of a intrarenal microcirculation by patent pulsatile perfusion as determined by the injection of carbon particles was not maintained in the host animal after revascularization. In fact, those kidneys preserved with Medium 199 demonstrating a patent microcirculation at the conclusion of preservation (see Fig. 1A and 2A) underwent a decline in cortical blood flow shortly after reim-
DIETHELM
ET AL.. CANINE
RENAL
PRESERVATION
61
FIG. 4. Section from a kidney preserved for 24 hr using cryoprecipitate of plasma followed by successful reimplantation into the host animal with normal renal function. Injection of colloidal carbon 2 weeks later revealed completeglomerular perfusion (PAS).
plantation, as judged by a decrease in renal turgor and cyanosis of the cortex. Carbon injected via the renal artery 4 hr following restoration of blood flow in Group IV revealed obstruction to the microcirculation (see Fig. 1B) predominantly at the level of preglomerular and interlobular arteries (see Fig. 2B), demonstrating the no-reflow phenomenon to occur after 24 hr preservation with Medium 199. It is important to note that the carbon particle, 200-400 A in size, may pass through capillaries diminished in diameter which obstruct the passage of erythrocytes. Thus, intrarenal injection of carbon particles only
documented the presence of complete occlusion but did not accurately estimate partial reduction of a vessel lumen. The site of obstruction was confirmed by the inability of carbon to pass through glomeruli after reflow of blood (see Fig. 3). Canine kidneys, transplanted into presensitized developed viously dogs, hyperacute rejection [4]. Perfusion with carbon at 2, 24 and 48 hr after transplantation revealed a different histologic appearance from kidneys preserved with Medium 199. Although vascular obstruction to the passageof carbon existed, there was also a considerable number of polymorphonu-
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clear cells adherent to the endothelium and within the interstitial space. There was agglutination and hemolysis of red blood cells within the vascular spaces, including the arteries and glomerular and peritubular capillaries. The inflammatory response noted in the hyperacutely rejecting kidney with vascular obstruction was not observed in the kidneys preserved by Medium 199 and reimplanted into the dog. Thus, although the no-reflow phenomenon existed in both experiments, the mechanism appears to be different. The no-reflow phenomenon is a descriptive interpretation of the pathophysiologic events occurring after restoration of blood flow through an ischemic organ. Ames and associates recognized this phenomenon in the ischemic rabbit brain [l], and others have noted similar changes after temporary interruption of renal blood flow [5,6, 12, 131. Summers and Jamison [13] induced ischemia in the rat kidney and noted swollen capillary or endothelial cells preventing the passage of erythrocytes causing further vascular obstruction. Flores [6] confirmed these observations by an electron microscopic study of the rat kidney after ischemia noting that the effects of anoxia upon cell swelling could be obviated by administration of hypertonic mannitol but not by expansion of the extracellular spacewith isotonic saline or mannitol. The importance of the osmotic effect of the perfusing medium upon prevention and reduction of cell swelling led to the hypothesis that swollen cells secondary to anoxia may be reduced in size by a hypertonic solute, thereby maintaining the normal diameter of the capillary lumen preventing obstruction to the reflow of blood [6, 81. This explanation is pertinent to those findings noted in kidneys preserved with acellular synthetic perfusate in which vascular obstruction occurred after reimplantation and restoration of the circulation. SUMMARY Twenty-four-hr renal preservation at hypothermic temperatures with pulsatile
VOL.
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1975
flow using either Medium 199 or a cryoprecipitate of plasma maintained a patent microcirculation prior to reestablishing blood flow. However, failed reflow of blood, the no-reflow phenomenon, occurred in kidneys preserved with Medium 199 within 4 hr after reimplantation into the host animal, as determined by the intra-arterial injection of carbon. The site of vascular obstruction in the renal microcirculation was documented to be within the glomerulus or in the preafferent arteriole. Renal glomerular preservation using a cryoprecipitate of plasma maintained a patent intrarenal vasculature after restoration of blood flow providing normal or near normal function. ACKNOWLEDGMENTS The skilled technical assistance of Mr. R. Brown, Mr. J. B. Cunningham, Ms. S. L. O’Brien, and Mr. G. Soul& is gratefully acknowledged.
REFERENCES I. Ames, A., II, Wright, R. L., Kowada, M., Thurston, J. M., and Majno, G. Cerebral ischemia. II. The no-reflow phenomenon. Am. J. Pathol. 52:437, 1968. 2. Belzer, F. O., Ashby, B. S., and Dunphy, J. E. Twenty-four hour and 72 hour preservation of canine kidneys. Lancet 2~536, 1967. 3. Belzer, F. 0.. Ashby, B. S., Huang, J. S., and Dunphy, J. E. Etiology of rising perfusion pressure in isolated organ perfusion. Ann. Surg. 168:382, 1968. 4. Diethelm, A. G. Obstruction of the microcirculation in rejecting canine renal allografts. J. Surg. Res. 10:413, 1970. 5. Diethelm, A. G., and Wilson, S. J. Obstruction to the renal microcirculation after temporary ischemia.J. Surg. Res. 11:265, 1971. 6. Flares, J., DiBona, D. R., Beck, C. H., and Leaf, A. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J. Clin. Invest. 51:118, 1972. 7. Humphries, A. L., Russell, R., Stoddard, L. D., and Moretz, W. H. Perfusion of dog kidneys with cooled Medium 199 followed by auto or allotransplantation. In J. C. Norman (Ed.), Organ Perfusion and Preservation, p. 13. Appleton Century Crofts, New York, 1968. 8. Leaf, A. Cell swelling. A factor in ischemic tissue injury. Circulation 48:455, 1973. 9. Pegg, D. E. Some effects of dextran and of bovine serum albumin on the isolated perfused rabbit kidney. Cryobiology6:419, 1970.
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ET AL.:
CANINE
10. Pegg, D. E., and Farrant, J. Vascular resistance and edema in the isolated rabbit kidney perfused with a cell free solution. Cryobiology 6:200, 1969. 11. Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion using a defined perfusion fluid. Cryobiology9:420, 1972. 12. Sheehan, H. L., and Davis, J. C. Renal ischemia
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PRESERVATION
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with failed reflow. J. Pathol. Bacterial. 78:105, 1959. 13. Summers, W. K., and Jamison, R. L. Theno reflow phenomenon in renal ischemia. Lab. Invest. 25:635, 1971. 14. Woods, J. E. Successful three to seven day preservation of canine kidneys. Arch. Surg. 102:614,1971.
OF SURGICAL
RESEARCH
19, 55-63 (1975)
The No-Reflow Canine
Renal
Phenomenon
Preservation
with
After Medium
199
ARNOLD G. DIETHELM, M.D.,’ JAMES DEVRIES, B.S.,2 M. W. HARTLEY, Ph.D.,3 AND S. J. PHILLIPS, B.S. Departments of Surgery and Pathology, The University of Alabama Medical Center and the Birmingham VA Hospital, Birmingham, Alabama 35294 Submitted for publication September 30, 1974
MATERIALS AND METHODS Kidney preservation achieved with cryoprecipitate of plasma and hypothermic Adult mongrel dogs, 15-25 kg in weight, pulsatile perfusion was successful in previously immunized against distemper, maintaining organ viability for 48 hr and were maintained with a general laboratory longer [2,14]. An important feature of this diet. Anesthesia was administered by perfusate has been attributed to the removal intravenous sodium pentobarbital with an of lipoproteins by freezing, thawing and endotracheal tube and a respirator. filtration, thus preventing obstruction of the Following a left nephrectomy the kidney was renal microcirculation by protein aggregates placed on the organ support unit. Renal during preservation [3]. Canine kidneys have reimplantation was accomplished by realso been successfully preserved for 24 hr vascularization to the external iliac artery using a synthetic perfusate, Medium 199, and vein with a ureterovesical anastomosis administered via pulsatile flow at 10” C [7]; performed using sterile technique. The Others [9-l 11, however, have noted edema, average ischemic interval during reimarterial injury and increased vascular re- plantation was 28 min. A contralateral sistance when isolated rabbit kidneys were nephrectomy was done at this time. During perfused with cell-free solutions including each experiment the dog received 500 ml of dextran. Ringer’s lactate by the intravenous route. A synthetic solution with a known com- No diuretics were used before nephrectomy position, relatively inexpensive and com- or after reimplantation. mercially available, would simplify the Organ support unit. This unit designed procedure of organ preservation. Because of and constructed by Sarns, Inc., Ann Arbor, this we reexamined the effectiveness of Me- MI., consisted of two identical systems cadium 199 as a perfusate in canine renal pable of sustaining two kidneys with seppreservation. Special attention was given to arate perfusate. Each system included a evaluation of the following parameters pulsatile pump, membrane oxygenator,’ during and after preservation. 1), The pa- heat exchanger, arterial bubble trap, venous tency of the intrarenal vasculature; 2), the collection reservoir and organ storage increase in renal edema after preservation; chamber. All experimental pressures were 3), the predictability of perfusion charac- maintained electively at 60/40 mm Hg with teristics during preservation compared with a temperature of 7-10°C. Perfusate flow organ viability after reimplantation; and 4), rates were dependent upon pressure and the ability of the preserved kidney to sustain intrarenal resistance. life in a contralaterally nephrectomized dog. Perfisa te. A synthetic perfusate including the following chemical properties ‘Professor and ViceChairman of Surgery. was used in all experiments: Powdered me2Product Manager, Sarns Inc., Ann Arbor, MI. 4Made by General Electric.
3Professor of Pathology.
55 Copyright @1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
JOURNAL
OF SURGICAL
RESEARCH
dium-molecular-weight dextran, 35 g; Medium 199, Earle, base 10x, 100 ml; distilled water, 900 ml; dextrose, 5 g; sodium bicarbonate, adjusted to pH 7.4, 20-30 ml; insulin, regular, 200 units; potassium penicillin G, 5 million units; neomycin sulfate, 50 mg; mycostatin, 20,000 units; mannitol 6.2512.25 g.
The osmolality ranged between 350 and 375 mosm/l with an oncotic pressure equal to 8 g of protein per 100 ml. Sodium, potassium, chloride, COz, pH, PO,, and $0, were measured before, during and at the conclusion of each period of preservation. A cryoprecipitate of plasma, prepared as outlined by Belzer [2], including the same antimicrobial agents as outlined in the synthetic solution was used as a control perfusate. Evaluation by colloidal
of the renal microcirculation carbon. Carbon black5 (pre-
pared by the Pelikan Company of Hanover, Germany, contained particles between 200 and 400 A) diluted with equal volumes of isotonic saline, pH 7.4, was injected into the renal artery at the conclusion of preservation and in some instances 4 hr after reimplantation of the kidney into the dog. All injections were performed using gravityinduced, nonpulsatile flow at 140 mm Hg. The artery and vein were simultaneously clamped and tied, and the kidney placed in 10% formalin. After complete fixation the organ was bisected and examined grossly. Specimens obtained for histologic examination were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff reagent (PAS). Additional sections, 100-200 pm thick, were imbedded in glycerin for 24 hr and examined by light microscopy. Assessment of renal edema, viability and characteris tics of preserved perfusion kidneys. Twenty-five kidneys were studied
and divided equally into five groups. Group I, 6-hr preservation (Medium 199) followed with carbon injection; Group II, 24-hr preservation (Medium 199) followed with %old By: John Henschel and Co., Inc., 141 Albertson Avenue, Albertson, Long Island, NY 11507
VOL.
19, NO. I, JULY
1975
carbon injection; Group III, 24-hr preservation (Medium 199) with reimplantation for survival; Group IV, 24-hr preservation (Medium 199) with reimplantation for 4 hr followed by carbon injection; Group V, 24-hr preservation (cryoprecipitate of plasma) with reimplantation for survival and carbon injection 2 weeks later. RESULTS Group I, 6-Hr Preservation and Carbon Injection
The average weight of five kidneys increased 15.4% during preservation (ranging from 9-24%). Flow rates increased by 25 and 66% in two kidneys, remained unchanged in two and decreased by 20% in the fifth organ. Kidneys injected with carbon and bisected appeared diffusely black without visible areas of vascular obstruction. Histological sections (H&E and PAS) confirmed the presence of carbon throughout the intrarenal vasculature. In two kidneys there were alterations in the vascular endothelium and glomeruli, suggestive of early necrosis. Glycerin sections demonstrated carbon particles throughout the cortical and medullary vasculature including glomeruli. Group II, 24-Hr Preservation and Carbon Injection
The average weight gain of five kidneys during preservation was 24% (ranging from 20-27%). Flow rates at the end of preservation were decreased by 33% in one experiment and by 40% in two other experiments. The two remaining kidneys showed no change in intrarenal resistance. All organs injected with carbon revealed a gross appearance similar to kidneys in Group I (see Fig. 1A). Microscopic glycerin sections confirmed a patent intravascular system (see Fig. 2A), and histological examination of all five kidneys after preservation demonstrated minimal evidence of intimal injury.
DIETHELM
ET AL.. CANINE
RENAL
PRESERVATION
FIG. 1. Two kidneys preserved for 24 hr with Medium 199 followed by injection with colloidal carbon, Kidney A revealed complete vascular perfusion of both cortex and medulla. Kidney B, reimplanted into the host animal for 4 hr, developed progressive obstruction of the microcirculation as demonstrated by multiple areas devoid of carbon in the cortex and medulla.
wqn.‘amol% aql q%noJql ssed 01 alqeun ale 1nq mawis Bug~allo~ snoualz pue sayll!de~ mlnqnl!lad aql u! luasald ale salD!l-reduoqle3 .uogDa!ir! uoqn3 p+aw Kq pamolloj pue ~q p ~oj pxu!ue lsoq aql ONI!paweldm!al‘661 um!paH q]!~ ~q tz ~oj pahlasald Laupy e JO uoyas uyax41f) ‘(a) .sagelpde3 relnqnlyad puE gnlauro~%aq3 u!qI!M sa[cyled uoqu23 %u!pnixy uoyqn3+3oJyu Ivual lualE?d E paleaAaJ ‘uoqJlz3 JO uogsa[u! p+ain ale!pawm! Lq pa,woiloj cgj1 mmpaw ql!m Jq pz ~oj paAIasald ‘Laupg E jo uoyas u!3a3Qt) ‘(v) ‘5 ‘f)~d
DIETHELM
ET AL.: CANINE
Group III, 24-Hr Preservation with Reimplantation
Five kidneys preserved with Medium 199 were reimplanted into the dog to determine viability by renal function. The average weight gain after preservation and before return to the host animal was 25% (ranging from 21-28%). Flow rates decreased from 12-50% in all preserved kidneys with an average of 25%. Immediately after reimplantation, the kidney became pink in color, firm to palpation with the appearance of excellent cortical blood flow. The onset of urine was observed in all animals within 15 min after completion of the vascular anastomosis. One hour after revascularization the kidney turgor was decreased to palpation, and the cortical surface appeared cyanotic. Urine output at this time was markedly diminished. Three hours after reimplantation cyanosis of the renal cortex had increased with a further diminution in cortical turgor to palpation and cessation of urine production. All dogs expired in uremia 6-10 days after reimplantation with evidence of tubular necrosis at autopsy. Group IV, 24-Hr Preservation, Reimplantation and Carbon Injection
Five kidneys preserved for 24 hr, as in Group III, were reimplanted into the dog. The average weight gain after preservation was 30% (ranging from 27-36%). Flow rates decreased 33, 18, 16 and 14% in four experiments and increased 25% in one. The appearance of the kidney during the first 3 hr after reimplantation was identical to that observed in Group III. After 4 hr of circulation through the kidney, the arterial inflow was manually occluded, the artery cannulated and the kidney injected with carbon. Carbon passed slowly and irregularly through the kidney, with large areas of the cortex excluded of carbon. After formalin fixation the bisected kidney demonstrated the cortex and medulla to have an irregular distribution of carbon with a decrease in content compared to the control kidney (see Fig. - 1B). Histologic sections
RENAL
PRESERVATION
59
(H&E and PAS) revealed the afferent arteriole and glomeruli to be engorged with intact erythrocytes and fibrin preventing passage of carbon into the glomerulus (see Fig. 3). The vasa recta showed a similar appearance of red blood cell engorgement obstructing the passage of carbon particles. There was no evidence of an acute inflammatory response characterized by polymorphonuclear leukocyte infiltration. Glycerin sections of the kidney revealed complete absenceof carbon within the glomeruli and a decrease in carbon particles within the peritubular capillaries (see Fig. 2B). Group V, 24-Hr Preservation with Cryoprecipitate of Plasma
Five kidneys preserved with plasma cryoprecipitate were reimplanted into the host animal. The average weight gain was 29% (ranging from l&35%). Flow rates remained unchanged in two experiments, decreased 50-75% in two and increased 50% in one. Immediately after reimplantation and restoration of circulation, the kidneys appeared well perfused with normal turgor to palpation. Urine flow was prompt, and all animals survived. Renal function was normal at the end of the 14th day in two dogs (Cr, 1.4 and 1.2 mg/lOO ml), borderline in two dogs (1.6 and 1.8 mg/ 100 ml) and elevated in one (2.4 mg/lOO ml). The animals were sacrificed at this time and the kidneys injected with carbon. The gross appearance was nearly identical to the control kidneys injected with carbon, as described in Group I. Glycerin sections showed both glomeruli and vasa recta to be filled with carbon including patent efferent and afferent arterioles. Histological sections demonstrated the tubules and arterial intima to be intact with carbon throughout the glomeruli (see Fig. 4). DISCUSSION Although 24-hr preservation of canine kidneys with Medium 199 delivered by pulsatile flow at 4°C was not successful in maintaining renal viability after reim-
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FIG. 3. Obstruction to the injection of colloidal carbon particles at the glomerulus secondary to engorgement of the capillaries by trapped erythrocytes (H&E).
plantation, several important observations emerged from these experiments. First, weight gain of the kidney during preservation with a synthetic perfusate as measured by total wet weight of the organ did not provide useful criteria for predicting viability after reimplantation. In fact, weight gain was similar in those kidneys unsuccessfully preserved with Medium 199 and in kidneys maintained successfully using a cryoprecipitate of plasma. Thus, renal edema alone did not provide a means of separating viable from nonviable kidneys. Second, perfusion characteristics in Group III during renal preservation using a constant pressure
did not differentiate viable from nonviable kidneys after reimplantation. The increased intrarenal resistance, essentially the same using either the synthetic perfusate or the cryoprecipitate of plasma, did not correlate with renal viability. Third, preservation of a intrarenal microcirculation by patent pulsatile perfusion as determined by the injection of carbon particles was not maintained in the host animal after revascularization. In fact, those kidneys preserved with Medium 199 demonstrating a patent microcirculation at the conclusion of preservation (see Fig. 1A and 2A) underwent a decline in cortical blood flow shortly after reim-
DIETHELM
ET AL.. CANINE
RENAL
PRESERVATION
61
FIG. 4. Section from a kidney preserved for 24 hr using cryoprecipitate of plasma followed by successful reimplantation into the host animal with normal renal function. Injection of colloidal carbon 2 weeks later revealed completeglomerular perfusion (PAS).
plantation, as judged by a decrease in renal turgor and cyanosis of the cortex. Carbon injected via the renal artery 4 hr following restoration of blood flow in Group IV revealed obstruction to the microcirculation (see Fig. 1B) predominantly at the level of preglomerular and interlobular arteries (see Fig. 2B), demonstrating the no-reflow phenomenon to occur after 24 hr preservation with Medium 199. It is important to note that the carbon particle, 200-400 A in size, may pass through capillaries diminished in diameter which obstruct the passage of erythrocytes. Thus, intrarenal injection of carbon particles only
documented the presence of complete occlusion but did not accurately estimate partial reduction of a vessel lumen. The site of obstruction was confirmed by the inability of carbon to pass through glomeruli after reflow of blood (see Fig. 3). Canine kidneys, transplanted into presensitized developed viously dogs, hyperacute rejection [4]. Perfusion with carbon at 2, 24 and 48 hr after transplantation revealed a different histologic appearance from kidneys preserved with Medium 199. Although vascular obstruction to the passageof carbon existed, there was also a considerable number of polymorphonu-
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clear cells adherent to the endothelium and within the interstitial space. There was agglutination and hemolysis of red blood cells within the vascular spaces, including the arteries and glomerular and peritubular capillaries. The inflammatory response noted in the hyperacutely rejecting kidney with vascular obstruction was not observed in the kidneys preserved by Medium 199 and reimplanted into the dog. Thus, although the no-reflow phenomenon existed in both experiments, the mechanism appears to be different. The no-reflow phenomenon is a descriptive interpretation of the pathophysiologic events occurring after restoration of blood flow through an ischemic organ. Ames and associates recognized this phenomenon in the ischemic rabbit brain [l], and others have noted similar changes after temporary interruption of renal blood flow [5,6, 12, 131. Summers and Jamison [13] induced ischemia in the rat kidney and noted swollen capillary or endothelial cells preventing the passage of erythrocytes causing further vascular obstruction. Flores [6] confirmed these observations by an electron microscopic study of the rat kidney after ischemia noting that the effects of anoxia upon cell swelling could be obviated by administration of hypertonic mannitol but not by expansion of the extracellular spacewith isotonic saline or mannitol. The importance of the osmotic effect of the perfusing medium upon prevention and reduction of cell swelling led to the hypothesis that swollen cells secondary to anoxia may be reduced in size by a hypertonic solute, thereby maintaining the normal diameter of the capillary lumen preventing obstruction to the reflow of blood [6, 81. This explanation is pertinent to those findings noted in kidneys preserved with acellular synthetic perfusate in which vascular obstruction occurred after reimplantation and restoration of the circulation. SUMMARY Twenty-four-hr renal preservation at hypothermic temperatures with pulsatile
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1975
flow using either Medium 199 or a cryoprecipitate of plasma maintained a patent microcirculation prior to reestablishing blood flow. However, failed reflow of blood, the no-reflow phenomenon, occurred in kidneys preserved with Medium 199 within 4 hr after reimplantation into the host animal, as determined by the intra-arterial injection of carbon. The site of vascular obstruction in the renal microcirculation was documented to be within the glomerulus or in the preafferent arteriole. Renal glomerular preservation using a cryoprecipitate of plasma maintained a patent intrarenal vasculature after restoration of blood flow providing normal or near normal function. ACKNOWLEDGMENTS The skilled technical assistance of Mr. R. Brown, Mr. J. B. Cunningham, Ms. S. L. O’Brien, and Mr. G. Soul& is gratefully acknowledged.
REFERENCES I. Ames, A., II, Wright, R. L., Kowada, M., Thurston, J. M., and Majno, G. Cerebral ischemia. II. The no-reflow phenomenon. Am. J. Pathol. 52:437, 1968. 2. Belzer, F. O., Ashby, B. S., and Dunphy, J. E. Twenty-four hour and 72 hour preservation of canine kidneys. Lancet 2~536, 1967. 3. Belzer, F. 0.. Ashby, B. S., Huang, J. S., and Dunphy, J. E. Etiology of rising perfusion pressure in isolated organ perfusion. Ann. Surg. 168:382, 1968. 4. Diethelm, A. G. Obstruction of the microcirculation in rejecting canine renal allografts. J. Surg. Res. 10:413, 1970. 5. Diethelm, A. G., and Wilson, S. J. Obstruction to the renal microcirculation after temporary ischemia.J. Surg. Res. 11:265, 1971. 6. Flares, J., DiBona, D. R., Beck, C. H., and Leaf, A. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J. Clin. Invest. 51:118, 1972. 7. Humphries, A. L., Russell, R., Stoddard, L. D., and Moretz, W. H. Perfusion of dog kidneys with cooled Medium 199 followed by auto or allotransplantation. In J. C. Norman (Ed.), Organ Perfusion and Preservation, p. 13. Appleton Century Crofts, New York, 1968. 8. Leaf, A. Cell swelling. A factor in ischemic tissue injury. Circulation 48:455, 1973. 9. Pegg, D. E. Some effects of dextran and of bovine serum albumin on the isolated perfused rabbit kidney. Cryobiology6:419, 1970.
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10. Pegg, D. E., and Farrant, J. Vascular resistance and edema in the isolated rabbit kidney perfused with a cell free solution. Cryobiology 6:200, 1969. 11. Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion using a defined perfusion fluid. Cryobiology9:420, 1972. 12. Sheehan, H. L., and Davis, J. C. Renal ischemia
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with failed reflow. J. Pathol. Bacterial. 78:105, 1959. 13. Summers, W. K., and Jamison, R. L. Theno reflow phenomenon in renal ischemia. Lab. Invest. 25:635, 1971. 14. Woods, J. E. Successful three to seven day preservation of canine kidneys. Arch. Surg. 102:614,1971.
