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Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts
Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts James R. Elmore, M D , P e t e r Gloviczki, M D , Kelvin G. M. Brockbank, P h D , Virginia M. Miller, P h D , Rochester,Minn., and Marietta, Ga. Cryopreserved veins used as arterial grafts may be affected by both rejection and the cryopreservation process. Experiments were designed to study changes in endothelial and smooth muscle function after cryopreservation but independent of rejection. One saphenous vein from each of eight dogs was cryopreserved for subsequent use as autografrs. After 3 weeks one cryopreserved and one freshly harvested autogenous saphenous vein were implanted as bilateral femoral arterial interposition grafts. Platelet deposition was studied in vivo with indium I l l - l a b e l e d platelets. At 4 weeks the autografts were removed, and the functional characteristics of the grafts were studied in organ chambers; and the ability of nerve terminals to uptake transmitter was studied with 3H-norepinephrine. Neither patency rates, blood flows, nor platelet deposition were significantly different between freshly harvested and cryopreserved grafts. Uptake of 3H-norepinephrine was significantly reduced in both grafts as compared to unoperated veins. The smooth muscle of the cryopreserved and fresh grafts contracted comparably to alpha-adrenergic agonists and endothelin. In cryopreserved grafts, the maximal tensions that developed to KCI, prostaglandin F2e, and endothelin were greater when the endothelium was present compared to that developed by the smooth muscle alone. Calcium ionophore A23187 caused relaxations only in rings with endothelium; these were not significantly different between graft types. However, relaxations of the smooth muscle to nitric oxide were decreased in the cryopreserved grafts. These results suggest the following: (1) cryopreservation does not influence early patency rates, blood flows or platelet deposition in autogenous vein grafts; (2) functional nerve terminals are not present in fresh or cryopreserved autogenous vein grafts after 4 weeks; (3) an endothelium-derived relaxing factor is present in both fresh and cryopreserved autogenous vein grafts at 4 weeks. It remains to be determined as to whether the increased endothelial contractile responses and decreased relaxations of the smooth muscle of cryopreserved autogenous grafts would influence long-term patency of these grafts. (J VAsc StrRc 1991;13:584-92.)
Cryopreserved veins offer a potential alternative to prosthetic material for arterial reconstruction when autogenous tissue is not available. Recent improvements in the technique o f cryopreservation have made it possible to preserve vein segments with intact morphologic characteristics and with retention o f smooth muscle function."4 However, the cryoFrom the Sectionof VascularSurgery(Drs. Elmore and Gloviczki) and the Department of Physiologyand Biophysics(Dr. Miller), Mayo Cfinic and Mayo Foundation, Rochester, and CryoLife, Inc. (Dr. Brockhank), Marietta. Supported by National Institutes of Health grant HL42614 and Mayo Foundation. Presented at the Fourteenth Annual Meeting of the Midwestern Vascular Surgical Society,Toledo, Ohio, Sept. 14, 1990. Reprint requests: Virginia M. Miller, PhD, Mayo Clinic, 200 1st St. SW, Rochester, MN 55905.
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preservation process may affect functional characteristics or thrombogenicity o f these veins when used as arterial grafts. "~'~ The endothelium is able to release vasoactive substances such as nitric oxide, 7 an endotheliumderived relaxing factor and endothelin," an endothelium-derived contracting factor. These factors help maintain tone in blood vessels by modifying the underlying smooth muscle. 9 Endotheliumderived relaxing factors can also act synergistically with prostacyclin to inhibit platelet aggregation. ~o,~ The endothelium o f vein grafts interposed into the arterial circulation is exposed to altered physical and chemical environments, '2 and this can alter the production o f endothelium-derived factors? s,14 Changes in endothefial function may affect platelet deposition and therefore affect patency o f vein
Volume 13 Number 5 May 1991
grafts. ~ The specific purpose of this study was to determine the effect of cryopreservation, independent of rejection, on cryopreserved saphenous vein grafts. Platelet deposition, graft patency, and endothelial and smooth muscle function were analyzed in cryopreserved venous autografts as compared to fresh autografts after implantation as femoral artery interposition grafts in dogs. MATERIAL A N D M E T H O D S Operative procedures Eight dogs were anesthetized with sodium pentobarbital (30 mg/kg, intravenously) and received intramuscular injections of penicillin G (1 million units/kg) and gentamicin sulfate (80 mg). One saphenous vein from each dog was removed by use of sterile technique with a standard approach developed by LoGerfo et al. ~5 for harvesting saphenous veins. An 8 to 10 cm long segment of vein for cryopreservation was flushed with a sterile solution containing Dulbecco's modified Eagle's medium with 10% fetal calf serum and 3 × 10-9 mol/L papaverine. The vein segment was placed on a stent to preserve in situ length, stored in and filled with the culture medium, and shipped on ice to CryoLife, Inc. Within 24 hours the segment was cryopreserved with dimethyl sulfoxide to a temperature of - 1 9 6 ° C and stored for a minimum of 3 weeks. At the time of implantation the dog was again anesthetized with sodium pentobarbital, and both femoral arteries were exposed. Heparin sulfate (100 units/kg) was given intravenously, and the animal was volume expanded with one liter of lactated Ringer's solution. The remaining saphenous vein was harvested in the fresh state and placed in one extremity as a reversed femoral arterial interposition graft. The antogenous cryopreserved vein was thawed in a water bath at 37 ° C for 10 minutes. The cryopreservative was flushed from the thawed vein segment with serial dilutions of 0.5, 0.25, and 0.0 mol/L of mannitol in Dulbecco's modified Eagle's medium with 10% bovine serum and placed in the contralateral limb as an interposition graft. The anastomoses were performed with the aid of an operating microscope in an end-to-side fashion with continuous 8-0 Prolene sutures (Ethicon, Inc., Somerville, N.J.). The average length of grafts was 8.5 cm. The femoral artery was then ligated and transected. The order of implantation of the control graft and the cryopreserved vein was random. Blood flow through the grafts was established simultaneously. The muscle and skin incisions were closed with 2-0 Vicryl and 3-0 Vicryl (Ethicon, Inc., Somerville, N.J.). Aspirin was
Cryopreservedvein grafts 585
not administered to the animals. The animals were housed in the animal care facility for 4 weeks to allow for healing of the grafts) 6'17 The animal care complied with the "Principles of Laboratory Animal Care" (formulated by the National Society for Medical Research) and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1985). Animals received intramuscular butorphanol tartrate (10 mg/dose) as a postoperative analgesic. Platelet labeling and imaging Platelets were labeled with indium 111 (Hqn) following the technique of Dewanjee, Rao, and Didisheim) 8 Whole blood (43 ml) was collected in a sterile acid citrate dextrose solution and centrifuged at 180g for 10 minutes. The platelet-rich plasma was removed and centrifuged at 1600 g for 10 minutes resulting in a platelet pellet that was resuspended in the acid citrate dextrose solution. On average, 625 ~Ci of l~In was then combined with tropolone and incubated with the resuspended platelet pellet at room temperature for 20 minutes, and the suspension was recentrifuged. The radioactivity of the final platelet suspension was measured. The overall labeling efficiency was 68%. Labeled platelets were infused intravenously into the dogs at least 15 minur.es before release of the clamps. Because blood flow through the grafts was established simultaneously, platelet deposition occurred over an equivalent time period. Imaging of the grafts was carried out in vivo, 2 hours after release of the clamps. At this point, maximal deposition of the platelets on the grafts is expected, ~9 and images were obtained with the gamma camera (Searle Pho Gamma IV, Des Plaines, Ill). Each in vivo scan lasted for 5 minutes and was repeated three times. Results from the three scans were averaged. After 4 weeks the animals were restudied with labeled platelets. Scans at this time were obtained 24 hours after the infusion of the platelets. Two techniques were used to measure radioactivity in the grafts. The first was to compare the total activity in the cryopreserved graft to that of the fresh control graft after correcting for background activity. 2° The second method was a modification of Stratton, Thiele, and Ritchie 2~ using a graft to blood ratio. This ratio was obtained by subtracting the background counts from the graft counts and dividing this number by a whole blood count. The background counts consisted of activity in the lateral aspect of the thigh. The whole blood count was the counts present in 1 × 10 -4 ml of whole blood. This
586 Elmore et al.
was a derived number from a 5 ml sample drawn at the time of the image and counted for 60 seconds in a gamma-counter (Packard 5000, Downers Grove, Ill). Results were obtained for both the proximal and distal anastomoses, and the graft itself. Organ chamber experiments After 4 weeks the animals were anesthetized with sodium pentobarbital (30 mg/kg, intravenously) and the grafts exposed. Blood flows were measured with an ultrasonic flow probe (Transonics Inc., St. Paul, Minn.). Patency was confirmed by transecting the artery distal to the distal anastomosis and observing pulsatile bleeding. The dogs were killed with an overdose of intravenous sodium pentobarbital, and the grafts were removed. Femoral arteries and saphenous veins were obtained from six unoperated dogs to study arterial responses to acetylcholine and uptake of 3H-norepinephrine in unoperated veins. Experiments were performed in organ chambers by use of a technique previously described. 22 In brief, these organ chambers contained a modified KrebsRinger bicarbonate solution of the following millimolar composition: NaC1, 118.3; KCI, 4.7; CaCI2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; CaEDTA, 0.026; glucose, 11.1 (control solution). The tissue was cleaned of connective tissue and cut into rings each of 5 mm length. In one half of the experiments, it was necessary to deliberately remove the endothelium to study the response of the smooth muscle alone. The endothelium was removed by inserting a cotton swab wetted with control solution into the lumen of the ring and pulling the ring gently around the swab. The rings were suspended in organ chambers between a fixed base, and a force transducer by two stainless steel wires inserted into the lumen of each ring. The temperature of the organ chambers was kept constant at 37 ° C, and the solutions were aerated with a mixture of 95% 02 and 5% CO2. The tissues were placed individually at the optimal point of their length-tension relationship by use of the response to 20 mmol/L potassium chloride (KC1). After this process the rings were allowed to equilibrate at their optimal length for at least 30 minutes before responses to agonists were tested. Drugs were prepared fresh daily and dissolved in distilled water except for calcium ionophore A23187, which was dissolved in dimethyl sulfoxide. To study relaxations the rings were contracted with prostaglandin F2,~ (2 × 10 -6 tool/L) to a similar level of tension that approximated 50% of the maximal contraction to prostaglandin F2~. Responses of grafts were tested to depolarization of the smooth muscle with KCI; stimulation of
Journalof VASCULAR SURGERY
receptor-operated mechanisms was tested with phenylephrine (txl adrenergic agonist), BHT-920 (et2 adrenergic agonist), prostaglandin F2,; and endothelin, an endothelium-derived contractile peptide. ~ Relaxations were studied to receptor-operated mechanisms by use of adenosine diphosphate and acetylcholine; and with calcium ionophore A23187, which releases endothelium-derived factors through mechanisms not involving receptors. Nitric oxide was also tested to evaluate the responsiveness of the smooth muscle to this endothelimn-derived relaxing factor. 7 All drugs were obtained from Sigma Chemical (St. Louis, Mo.) except BHT-920 (Boehringer Ingelheim Pharmaceutical, Ridgefield, Conn.), endothelin (Peninsula Laboratories Inc., Belmont, Calif.) and nitric oxide (Union Carbide, Chicago, Ill.). Nitric oxide was prepared as previously described. 7 In some rings indomethacin (10- "~tool/L, combined with an equal molar concentration of Na2CO3) was used to inhibit the cyclooxygenase system and was added to the bath 30 minutes before other agonists were tested. All concentrations are reported as the final molar (tool/L) concentration in the organ chambers. Uptake o f SH-norepinephrine The ability of the nerve terminals to uptake norepinephrine was studied with 3H-norepinephrine. Strips of unoperated saphenous vein, cryopreserved graft, and fresh graft each weighing approximately 20 mg were incubated in the control solution (10 ml) and aerated for 30 minutes with 95% 02-5% CO2 at 37 ° C. Each strip was incubated for one hour with 1 × 10 -7 mol/L 3H-norepinephrine. Cocaine (5 × 10 -6 tool/L) was added to one half of the specimens to inhibit neuronal uptake of norepinephrine. The tissue was washed with control solution (10 ml for 1 hour), blotted dry, and weighed. The tissue was then extracted with 3 ml of 0.5N quaternary ammonium hydroxide in toluene, and radioactivity was measured with a Beckman LS-9800 liquid scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). Histology Rings of graft were placed in 10% buffered formaldehyde. The tissue was processed for routine light microscopy. A hematoxylin and eosin stain was used after the grafts had been sectioned (6 Ixm). Statistical analysis Only patent grafts (fresh, n = 6; cryopreserved, n = 5) were used for statistical analysis of organ chamber studies, platelet deposition, and norepi-
Volume 13 Number 5 May 1991
nephrine uptake. The role of the endothelium was determined by comparing responses of rings with and without endothelium from the same graft. The data are expressed as means + standard error of the mean (SEM); both absolute and percent changes were analyzed statistically. For contractions, an effective concentration resulting in 50% of the maximal response (EDs0) was calculated for each individual concentration-response curve, and the mean of these values was reported as the negative logarithm of the molar concentration. For relaxations an effective concentration that resulted in 50% inhibition of the maximal response (ICs0) was calculated. For organ chamber experiments performed in parallel on tissues (with and without endothelium) from the same animal, a Student's t test for paired observations was used. When more than two means were compared or differences between cryopreserved and fresh grafts were analyzed, an analysis of variance (ANOVA) was performed. For graft patency, a Fischer's exact test was used. Values were considered to be statistically different whenp < 0.05. RESULTS Graft patency and flows Five of eight (63%) of the cryopreserved grafts were patent, which was not significantly different from the fresh grafts where seven of eight (88%) grafts were patent. Blood flow through the grafts was not significantly different at implantation or harvest. At implantation, blood flow in the cryopreserved grafts was 83 _+ 17 ml/min and in the fresh grafts was 81 _+ 13 ml/min. At harvest, blood flow was 119 + 6 ml/min in the cryopreserved graft and 127 _+ 14 ml/min in the fresh graft. Platelet deposition Scans of ~In-labeled platelets obtained at implantation and before harvest revealed no significant differences in the actual radioactivity corrected for background activity between the cryopreserved and fresh grafts. Results were also analyzed by dividing the corrected counts by the number of counts in 1 × 10 -4 ml of blood collected at the time of the images to obtain a graft/blood ratio. Analysis with this method revealed no significant difference between cryopreserved and fresh grafts, or between implantation and harvest (Table I). Organ chamber experiments The basal tension of cryopreserved grafts (with endothelium: 1.9 _+ 0.1 g) and fresh grafts (with endothelium: 1.9 + 0 . 1 g ) were similar. N o difference was found in basal tension between rings with
Cryopreservedvein grafts 587
and without endothelium. The basal tension of unoperated femoral arteries, used as a control for acetylcholine, was 9.8 --- 0.5 g. N o difference was found in basal tension of arterial rings with and without endothelium. Contractions No difference was found in the maximal tension that developed to 60 mmol/L KCL in rings with or without endothelium in cryopreserved grafts (with 3.7 --_ 0.8g vs without 2.0 _-_ 0.3g) or in fresh grafts (with 1.5 --- 0.3 g vs without 1.5 +__0.4 g). The response of cryopreserved grafts with endothelium was significantly greater as compared to fresh grafts with endothelium (cryopreserved 3.7 +-- 0.8 g vs fresh 1.5 __ 0.3g). These differences were not seen in grafts without endothelium. Electrical stimulation did not result in any contraction in either the cryopreserved or fresh grafts. However, phenylephrine and BHT-960 caused contraction-dependent increases in tension in both grafts. The maximal tensions and the EDs0 for the responses to phenylephrine and BHT-960 were similar between the grafts (Table II). Endothelin caused concentration-dependent increases in tension that were not significantly different between the cryopreserved graft without endothelium or fresh grafts with and without endothelium (Fig. 1). In cryopreserved grafts with endothelium, a significant increase occurred in tension in response to endothelin as compared to the smooth muscle alone (rings without endothelium). The contractile responses to prostaglandin F2~ were significantly greater in the cryopreserved grafts with endothelium than without endothelium (Fig. 2). This response to prostaglandin F2~ was also greater than the fresh graft with or without endothelium. The EDs0 ( - log mol/L) for prostaglandin F2~ was not significantly different between cryopreserved ( - 5 . 9 + 0.10) and fresh ( - 5.7 + 0.11) grafts with endothelium. Relaxations
Acetylcholine did not cause relaxations in either graft type during contractions with prostaglandin F2~ but rather comparable concentration-dependent contractions were observed (Fig. 3). Removal of endothelium in these grafts did not significantly change the response to acetylcholine. N o significant differences were found in contractions to acetylcholine between grafts. In comparison, in the unoperated canine femoral artery a 100% relaxation was seen with intact endothelium. These relaxations in the artery were inhibited when the endothelium was removed (Fig. 3).
Journal of VASCULAR SURGERY
Elmore et al.
588
T a b l e I. Platelet deposition o n
10
cryopreserved and fresh canine autogenous vein grafts
8
=-
o
o~ (0
Background-corrected graft~blood ratios of indium ill-labeled platelets
6
•
4
(= 0 t,.
o
2
r.
o
T
;'-
10
,
,
9
,
,
8
Endothelin,
7
-log M
Fig. 1. Contractions to endothelin in cryopreserved and fresh canine autogenous saphenous vein grafts. Data are shown as mean -+ SEM. * , Cryopreserved graft with endothelium, n = 5; O, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Implantation Proximal anastomosis Graft Distal anastomosis Harvest Proximal anastomosis Graft Distal anastomosis
Cryopreserved graft
Fresh gra~
3.6 -+ 0.5 ~ 2.0 _+ 0.4 1.2 -+ 0.2
3.5 + 0.5 1.8 _+ 0.2 1.6 -+ 0.2
2.2 _+ 1.4 1.7 -+ 0.9 1.8 _+ 1.6
4.6 _+ 2.8 4.1 -+ 2.7 4.1 _+ 2.9
*Data are expressed as mean - SEM. No significant difference exists between cryopreserved and fresh autografts or between implantation and harvest.
T a b l e I I . Adrenergic responses in
cryopreserved and fresh canine autogenous vein grafts
10 8 m
"O
6
c ®
4
Phenylephrine (aa) Cryopreserved graft (n = 5) Fresh graft (n = 6) BHT-960 (a2) Cryopreserved graft (n = 5) Fresh graft (n = 6)
m o U
2 O
T 9
"I"
T
,
8 Prostaglandin
, 7
~
, 6
,
t
,
5
Mgg'imal wnsion, g
Ed so log mol/L
6.5 -+ 2.8 ~ 2.6 -+ 0.9
-6.0 ~ 0.1 -5.9 - 0.1
1.1 -+ 0.7 1.0 _+ 0.8
-6.4 _+ 0.1 -6.4 -- 0.1
*Data are expressed as mean _+ SEM. There are no significant differences in maximal tensions or EDso between the cryopreserved and fresh autografts.
F2alpha ' -log M
Fig. 2. Contractions to prostaglandin F2~ in cryopreserved and fresh canine autogenous saphenous vein grafts. Data are shown as mean + SEM. 0 , Cryopreserved graft with endothelium, n = 5; O, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Adenosine diphosphate induced concentrationdependent relaxations o f rings with and w i t h o u t e n d o t h e l i u m in b o t h the cryopreserved and fresh grafts, w h i c h were comparable in m a g n i t u d e (Fig. 4). I n the presence o f indomethacin calcium ionop h o r e A 2 3 1 8 7 caused concentration-dependent relaxations, w h i c h were significantly greater in tings with than w i t h o u t e n d o t h e l i u m in b o t h the cryopreserved and fresh grafts. N o significant difference occurred between fresh and cryopreserved grafts in their response to A 2 3 1 8 7 (Fig. 5).
I n rings w i t h o u t endothelium, in the presence o f indomethacin, nitric oxide caused concentrationdependent decreases in tension. I n cryopreserved grafts, the maximal relaxation to 3 × 10 -4 m o l / L was significantly decreased (59.8% + - 5 . 8 % and 91.5% + 4.8% in cryopreserved and fresh grafts, respectively). T h e IC50 s ( - l o g mol/L) for nitric oxide were n o t significantly different (cryopreserved 5.5 -+ 0.1 vs fresh 5.7 -+ 0.2). Uptake of 3H-norepinephrine
T h e uptake o f 3H-norepinephrine was signific a n @ reduced by cocaine in the unoperated saphenous veins. The uptake o f norepinephrine was reduced in b o t h graft types as c o m p a r e d to the saphenous veins w i t h o u t cocaine. I n the grafts, the uptake was n o t further reduced by cocaine (Table III).
Volume 13 Number 5 May 1991
Cryopreserved vein grafts 589
.o r
2 9
8
7
6
9
8
7
6
Acetylcholine, -log M
Fig. 3. Responses of canine femoral artery (left); cryopreserved and fresh canine autogenous saphenous vein grafts (right) to acetylcholine in the presence of indomethacin (10 -s mol/L). Values are expressed as a percent change in tension to a contraction with prostaglandin F2~ (2 × 10 -6 mol/L; 8.2 + 1.5g, 3.7 -+ 1.0g, and 2.1 -+ 1.0g in femoral artery, cryopreserved, and fresh grafts, respectively, with endothelium). * , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; *, fresh graft with endothelium, n -- 6; o, fresh graft without endothelium, n = 6; "%femoral artery with endothelium, n = 6; [], femoral artery without endothelium, n = 6.
0
0
20 20
E 0 X
C
40
o
<., X
w n-
60
n,.
40
80 6O 100
I
8
I
7
I
I
6
I
I
5
I
I
I
4
9
I
Adenosine diphosphate, -log M
I
8
I
I
7
I
I
I
6
A 2 3 1 8 7 , -Iog M
Fig. 4. Relaxations to adenosine diphosphate in cryopreserved and fresh canine autogenous saphenous vein grafts. Values are expressed as a percent relaxation to a contraction with prostaglandin F2~ (2 × 10 -6 mol/L; 4.3 + 1.9 g, 1.8 + 0.6g, and 7.9 -+ 1.4 in cryopreserved grafts, fresh grafts, and unoperated femoral arteries, respectively, with endothelium). 0 , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Fig. 5. Responses to calcium ionophore A23187 in cryopreserved and fresh canine saphenous vein grafts. Experiments carried out in the presence of indomethacin 10 -5 mol/L. Values are expressed as a percent relaxation to a contraction with prostaglandin F2~ (2 × 10 -6 tool/L; 5.1 -+ 2 . 3 g and 2.5 + 1 . 0 g in cryopreserved and fresh grafts, respectively, with e n d o t h e l i u m ) . . , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Histology
DISCUSSION
T h e lumen was lined with a single layer o f cells that resembled endothelium in b o t h the fresh and cryopreserved vein grafts. There was an eccentric m y o i n t i m a l hyperplasia in b o t h types o f grafts (Fig. 6). By light m i c r o s c o p y no obvious m o r p h o l o g i c differences were f o u n d between the fresh and cryopreserved grafts.
As early as 1911, Yamanouchi 23 tested different storage solutions for arterial and venous allografts including formalin, saline, and water. With improvements in the technique ofcryopreservation, this f o r m o f preservation has resulted in veins with intact m o r p h o l o g y and with retention o f s m o o t h muscle function.~4 A l t h o u g h it has been p r e s u m e d that the
Journal of VASCULAR SURGERY
590 Elmore et al.
Table III. Uptake of 3H-norepinephrine in unoperated canine saphenous veins and cryopreserved and fresh autogenous saphenous vein grafts 3H-norepinephrine uptake (disintegrations~rain~rag) Graft
Control
Unoperated vein (n = 5) 265.1 ± 20.6* Fresh graft (n = 6) 85.9 ± 15.5Cryopreserved graft 58.5 ± 8.7t
Presence of' cocaine (5 × 10-~mol/L) 40,8 +- 2.6t 72,1 -+ 14.6t 50.6 -+ 9.8t
(n = 5)
*Values shown as mean +_ SEM, tdenotes significant differences from control unoperated veins, p < 0.05.
Fig. 6. Light micrographs of cryopreserved (top) and freshly harvested (bottom) saphenous vein autografts (e, endothelium; m, media; a, adventitia). Sections were 6 ~m and stained with hematoxylin and eosin; original magnification x 160. primary cause of failure of cryopreserved vein grafts is rejection, 5"6 cryopreservation itself may affect patency by altering the endothelium and smooth muscle of these grafts. In the present study the performance of cryopreserved autogenous veins as arterial grafts was comparable to that of freshly harvested autogenous veins with regard to early graft patency and blood flows. Platelet deposition was also similar between cryopreserved and fresh autografts and was low in comparison to that observed in synthetic grafts. 2~ This suggests that the intimal surfaces of cryopreserved and fresh saphenous veins are less thrombogenetic than prosthetic grafts. 21 This inhibitory effect on thrombogenicity is probably related to the observations that the intima of vein grafts (fresh and
cryopreserved) can release relaxing factors as evidenced by relaxations to A23187 only in rings of graft with endothelium. Endothelium-derived relaxing factors, in particular nitric oxide, can act synergistically with prostacyclin to inhibit platelet aggregation.'°'~, Neither cryopreserved nor fresh autografts demonstrated functional adrenergic innervation at 4 weeks as has been suggested by anatomic studies. 24 This conclusion is supported by the lack of response to electrical stimulation and the loss of the cocainesensitive mechanism for the uptake of norepinephrine. Cocaine usually inhibits neuronal uptake of the transmitter. 2s Both alphal and alpha z receptors are present on the smooth muscle of both types of grafts as contractions to phenylephrine and BHT-960 were observed. The magnitude of these responses are reduced compared to unoperated saphenous vein. 26 Acetylcholine will cause relaxations in mammalian arteries when the endothelium is present. 9 As has been shown in other vein grafts, ~3 endotheliumdependent relaxations to acetylcholine were not observed in either the fresh or cryopreserved autogenous saphenous vein grafts. This suggests that fresh or cryopreserved autogenous vein grafts do not develop arterial-like endothelium-dependent relaxations to muscarinic agonists at least after 4 weeks of implantation. The endothelium of cryopreserved and fresh autogenous grafts can, however, release some relaxing factors as evidenced by the greater relaxations in rings with endothelium as compared to without endothelium in response to A23187. Since these experiments were conducted in the presence of indomethacin, this relaxing factor must be a substance other than a cyclooxygenase product of arachidonic acid (i.e., not prostacyclin) and is most likely nitric oxide or a nitric oxide-carried mÜ•ecu•e. 27
Volume 13 Number 5 May 1991
Differences in the responsiveness of cryopreserved and fresh autogenous grafts were observed in response to some agonists. In particular, contractions of cryopreserved grafts to KCI, PGF2~ , and endothelin were greater when the endothelium was present. In fresh grafts, contractions to these agonists were similar in the absence and presence of endothelium. These data suggest that the endothelium of cryopreserved autografts may be releasing contractile substances in response to these agonists. The reasons for this are not clear, whether this represents a direct effect of the cryopreservation process on the metabolism of endothelial cells or a general characteristic of regenerated cells remains to be determined. If the cryopreservation process makes the endothelial cells fragile and susceptible to sloughing when blood flow through the graft is established, then the cryopreserved grafts may have a greater number of regenerated cells per surface area than freshly harvested grafts. Another difference in cryopreserved and fresh autogenous grafts was observed in the response of the smooth muscle to the endothelium-derived relaxing factor nitric oxide. Cryopreserved grafts were less sensitive to its inhibitory effects than fresh grafts. The decreased sensitivity of the cryopreserved vein grafts to nitric oxide may explain the tendencies for relaxations to A23187 to be decreased in the cryopreserved grafts. Since relaxations to nitric oxide are mediated through increases in cyclic guanosine monophosphate, 27 the decreased responses in cryopreserved grafts may suggest that this system is susceptible to inactivation by the cryopreservation process. In summary, cryopreservation did not affect early patency or thrombogenicity of these autografts. The ability of the endothelium of the cryopreserved autogenous grafts to release endothelium-derived factors and of the smooth muscle to respond to them supports the concept that functional characteristics of the cryopreserved vein grafts remain after cryopreservation. However, cryopreservation increased endothelium-dependent contractions and decreased the ability of the smooth muscle to relax to an endogenous nitrovasodilator. It is not known which step(s) of the cryopreservation process is responsible for these changes of the endothelium and smooth muscle or whether such changes would affect the long-term patency of such grafts. The authors thank Kevin S. Rud for his technical assistance; Robert R. Lorenz for drawing the figures; Marcia A. Simonson for manuscript preparation; and Boerhinger-Ingelheim Pharmaceutical for the BHT-920.
Cryopreservedvein grafts
591
REFERENCES 1. Sitzmann JV, Imbembo AL, Ricotta JJ, McManama GP III, Hutchins GM. Dimethylsulfoxide-treated cryopreserved venous allografts in the arterial and venous systems. Surgery 1984;95:154-9. 2. Brockbank KGM, Donovan TJ, Ruby ST, Carpenter JF, Hagen PO, Woodley MA. Functional analysis of cryopreserved veins-preliminary report. J VASC SURG 1990;11:94102. 3. Miiller-Schweinitzer E, Tapparelli C. Pharmacological studies on frozen stored canine saphenous veins and basilar arteries. Arch Pharmacol 1986;332:74-8. 4. Thompson L, Duckworth J, Bevan J. Cryopreservation of innervation, endothelial and vascular smooth muscle function of a rabbit muscular and resistance artery. Blood Vessels 1989;26:157-64. 5. Showalter D, Durham S, Sheppeck R, et al. Cryopreserved venous homografts as vascular conduits in canine carotid arteries. Surgery 1989;106:652-9. 6. Calhoun AD, Baur GM, Porter JM, Houghton DH, Templeton JW. Fresh and cryopreserved venous allografts in genetically characterized dogs. J Surg Res 1977;22:687-96. 7. Palmer RMJ, Ferrige AG, Moncada S. The release of nitric oxide by vascular endothelial cells accounts for activity, of EDRF. Nature 1987;327:524-6. 8. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-5. 9. Furchgott RF, Vanhoutte PM. Endothelium-derived rela,'dng and contracting factors. FASEB J 1989;3:2007-18. 10. Eldor A, Hoover EL, Pett SB, Gay WA, Alonson DR, Weksler BB. Prostacyclin production by arterialized autogenous venous grafts in dogs. Prostaglandins 1981 ;22:485-98. 11. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 1987;90:687-92. 12. Henderson VJ, Mitchell RS, Kosek JC, Cohen RG, Miller DG. Biochemical (functional) adaptation of "arterialized'" vein grafts. Ann Surg 1986;203:339. 13. Miller VM, Riegal MM, Hollier LH, Vanhoutte PM. Endothelium-dependent responses in autogenous femoral veins grafted into the arterial circulation of the dog. l Clin Invest 1987;80:1350-7. 14. Miller VM, Aarhus LL, Vanhoutte PM. Modulation of endothelium-dependent responses by chronic alterations of blood flow. Am J Physiol 1986;251:H520-7. 15. LoGerfo FW, Quist WC, Crawshaw HM, Haudenschild C. An improved technique for preservation of endothelial morphology in vein grafts. Surgery 1981;90:1015-24. 16. Wyatt AP, Taylor GW. Vein grafts: changes in the endothelium of autogenous free vein grafts used as arterial replacements. Br J Surg 1966;53:943-7. 17. Reichle FA, Stewart GI, Essa N. A transmission and scanning electron microscopic study of luminal surfaces in Dacron and autogenous vein bypasses in man and dog. Surgery 1973;74: 945 -60. 18. Dewanjee MK, Rao SA, Didisheim P. Indium-III tropolone, a new high-affinity platelet label: preparation and evaluation of labeling parameters. J Nucl Med 1981;22:981-7. 19. Christenson JT, Megerman J, Hanel KC, Litalien GI, Strauss HW, Abbott WM. The effect of blood flow rates on platelet deposition in PTFE arterial bypass grafts. Trans Am Soc Artif Intern Organs 1981;27:188-91.
592
Journal of VASCULAR SURGERY
Elmore et al.
20. Pumphrey CW, Chesebro JH, Dewanjee MK, et al. In vivo quantitation of platelet deposition on human peripheral arterial bypass grafts using indium-III-labeledplatelets. Am J Cardiol 1983;51:796-801. 21. Stratton JR, Thiele BL, Ritchie JL. Platelet deposition on Dacron aortic bifurcation grafts in man: quantitation with Indium-III platelet imaging. Circulation 1982;66:128793. 22. Komori K, Gloviczki P, Bourchier RG, Miller VM, Vanhoutte PM. Endothelium-dependent vasorelaxations in response to aggregating platelets are impaired in reversed vein grafts. J VAsc SuR6 1990;22:139-47. 23. Yamanouchi H. f2ber die zirkul~irengefa~ssn~ihteund ArterienVenen-Anastomosen sowie fiber die Gef~issmeansplantationen. Dtsch Z Chir 1911;112:1-118.
24. Meagher S, McGeachie J, Prendergast F. Vein to artery grafts: an experimental study of reinnervation of the graft wall. Ann Surg 1984;200:153-8. 25. Verbeuren TJ, Vanhoutte PM. Cocaine and neuronal uptake in the canine saphenous vein. Naunyn Schmiedebergs Arch Pharmocol 1982;321:207-12. 26. Flavahan NA, Miller VM, Aarhus LL, Vanhoutte PM. Denervation augments alpha-2 but not alpha-1 adrenergic responses in canine saphenous veins. J Pharmacol Exp Ther 1987;240:589-93. 27. Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J 1989;3:31-6.
Submitted Oct. 9, 1990; accepted Dec. 21, 1990.
Cryopreserved veins offer a potential alternative to prosthetic material for arterial reconstruction when autogenous tissue is not available. Recent improvements in the technique o f cryopreservation have made it possible to preserve vein segments with intact morphologic characteristics and with retention o f smooth muscle function."4 However, the cryoFrom the Sectionof VascularSurgery(Drs. Elmore and Gloviczki) and the Department of Physiologyand Biophysics(Dr. Miller), Mayo Cfinic and Mayo Foundation, Rochester, and CryoLife, Inc. (Dr. Brockhank), Marietta. Supported by National Institutes of Health grant HL42614 and Mayo Foundation. Presented at the Fourteenth Annual Meeting of the Midwestern Vascular Surgical Society,Toledo, Ohio, Sept. 14, 1990. Reprint requests: Virginia M. Miller, PhD, Mayo Clinic, 200 1st St. SW, Rochester, MN 55905.
24/6/27527 584
preservation process may affect functional characteristics or thrombogenicity o f these veins when used as arterial grafts. "~'~ The endothelium is able to release vasoactive substances such as nitric oxide, 7 an endotheliumderived relaxing factor and endothelin," an endothelium-derived contracting factor. These factors help maintain tone in blood vessels by modifying the underlying smooth muscle. 9 Endotheliumderived relaxing factors can also act synergistically with prostacyclin to inhibit platelet aggregation. ~o,~ The endothelium o f vein grafts interposed into the arterial circulation is exposed to altered physical and chemical environments, '2 and this can alter the production o f endothelium-derived factors? s,14 Changes in endothefial function may affect platelet deposition and therefore affect patency o f vein
Volume 13 Number 5 May 1991
grafts. ~ The specific purpose of this study was to determine the effect of cryopreservation, independent of rejection, on cryopreserved saphenous vein grafts. Platelet deposition, graft patency, and endothelial and smooth muscle function were analyzed in cryopreserved venous autografts as compared to fresh autografts after implantation as femoral artery interposition grafts in dogs. MATERIAL A N D M E T H O D S Operative procedures Eight dogs were anesthetized with sodium pentobarbital (30 mg/kg, intravenously) and received intramuscular injections of penicillin G (1 million units/kg) and gentamicin sulfate (80 mg). One saphenous vein from each dog was removed by use of sterile technique with a standard approach developed by LoGerfo et al. ~5 for harvesting saphenous veins. An 8 to 10 cm long segment of vein for cryopreservation was flushed with a sterile solution containing Dulbecco's modified Eagle's medium with 10% fetal calf serum and 3 × 10-9 mol/L papaverine. The vein segment was placed on a stent to preserve in situ length, stored in and filled with the culture medium, and shipped on ice to CryoLife, Inc. Within 24 hours the segment was cryopreserved with dimethyl sulfoxide to a temperature of - 1 9 6 ° C and stored for a minimum of 3 weeks. At the time of implantation the dog was again anesthetized with sodium pentobarbital, and both femoral arteries were exposed. Heparin sulfate (100 units/kg) was given intravenously, and the animal was volume expanded with one liter of lactated Ringer's solution. The remaining saphenous vein was harvested in the fresh state and placed in one extremity as a reversed femoral arterial interposition graft. The antogenous cryopreserved vein was thawed in a water bath at 37 ° C for 10 minutes. The cryopreservative was flushed from the thawed vein segment with serial dilutions of 0.5, 0.25, and 0.0 mol/L of mannitol in Dulbecco's modified Eagle's medium with 10% bovine serum and placed in the contralateral limb as an interposition graft. The anastomoses were performed with the aid of an operating microscope in an end-to-side fashion with continuous 8-0 Prolene sutures (Ethicon, Inc., Somerville, N.J.). The average length of grafts was 8.5 cm. The femoral artery was then ligated and transected. The order of implantation of the control graft and the cryopreserved vein was random. Blood flow through the grafts was established simultaneously. The muscle and skin incisions were closed with 2-0 Vicryl and 3-0 Vicryl (Ethicon, Inc., Somerville, N.J.). Aspirin was
Cryopreservedvein grafts 585
not administered to the animals. The animals were housed in the animal care facility for 4 weeks to allow for healing of the grafts) 6'17 The animal care complied with the "Principles of Laboratory Animal Care" (formulated by the National Society for Medical Research) and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1985). Animals received intramuscular butorphanol tartrate (10 mg/dose) as a postoperative analgesic. Platelet labeling and imaging Platelets were labeled with indium 111 (Hqn) following the technique of Dewanjee, Rao, and Didisheim) 8 Whole blood (43 ml) was collected in a sterile acid citrate dextrose solution and centrifuged at 180g for 10 minutes. The platelet-rich plasma was removed and centrifuged at 1600 g for 10 minutes resulting in a platelet pellet that was resuspended in the acid citrate dextrose solution. On average, 625 ~Ci of l~In was then combined with tropolone and incubated with the resuspended platelet pellet at room temperature for 20 minutes, and the suspension was recentrifuged. The radioactivity of the final platelet suspension was measured. The overall labeling efficiency was 68%. Labeled platelets were infused intravenously into the dogs at least 15 minur.es before release of the clamps. Because blood flow through the grafts was established simultaneously, platelet deposition occurred over an equivalent time period. Imaging of the grafts was carried out in vivo, 2 hours after release of the clamps. At this point, maximal deposition of the platelets on the grafts is expected, ~9 and images were obtained with the gamma camera (Searle Pho Gamma IV, Des Plaines, Ill). Each in vivo scan lasted for 5 minutes and was repeated three times. Results from the three scans were averaged. After 4 weeks the animals were restudied with labeled platelets. Scans at this time were obtained 24 hours after the infusion of the platelets. Two techniques were used to measure radioactivity in the grafts. The first was to compare the total activity in the cryopreserved graft to that of the fresh control graft after correcting for background activity. 2° The second method was a modification of Stratton, Thiele, and Ritchie 2~ using a graft to blood ratio. This ratio was obtained by subtracting the background counts from the graft counts and dividing this number by a whole blood count. The background counts consisted of activity in the lateral aspect of the thigh. The whole blood count was the counts present in 1 × 10 -4 ml of whole blood. This
586 Elmore et al.
was a derived number from a 5 ml sample drawn at the time of the image and counted for 60 seconds in a gamma-counter (Packard 5000, Downers Grove, Ill). Results were obtained for both the proximal and distal anastomoses, and the graft itself. Organ chamber experiments After 4 weeks the animals were anesthetized with sodium pentobarbital (30 mg/kg, intravenously) and the grafts exposed. Blood flows were measured with an ultrasonic flow probe (Transonics Inc., St. Paul, Minn.). Patency was confirmed by transecting the artery distal to the distal anastomosis and observing pulsatile bleeding. The dogs were killed with an overdose of intravenous sodium pentobarbital, and the grafts were removed. Femoral arteries and saphenous veins were obtained from six unoperated dogs to study arterial responses to acetylcholine and uptake of 3H-norepinephrine in unoperated veins. Experiments were performed in organ chambers by use of a technique previously described. 22 In brief, these organ chambers contained a modified KrebsRinger bicarbonate solution of the following millimolar composition: NaC1, 118.3; KCI, 4.7; CaCI2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; CaEDTA, 0.026; glucose, 11.1 (control solution). The tissue was cleaned of connective tissue and cut into rings each of 5 mm length. In one half of the experiments, it was necessary to deliberately remove the endothelium to study the response of the smooth muscle alone. The endothelium was removed by inserting a cotton swab wetted with control solution into the lumen of the ring and pulling the ring gently around the swab. The rings were suspended in organ chambers between a fixed base, and a force transducer by two stainless steel wires inserted into the lumen of each ring. The temperature of the organ chambers was kept constant at 37 ° C, and the solutions were aerated with a mixture of 95% 02 and 5% CO2. The tissues were placed individually at the optimal point of their length-tension relationship by use of the response to 20 mmol/L potassium chloride (KC1). After this process the rings were allowed to equilibrate at their optimal length for at least 30 minutes before responses to agonists were tested. Drugs were prepared fresh daily and dissolved in distilled water except for calcium ionophore A23187, which was dissolved in dimethyl sulfoxide. To study relaxations the rings were contracted with prostaglandin F2,~ (2 × 10 -6 tool/L) to a similar level of tension that approximated 50% of the maximal contraction to prostaglandin F2~. Responses of grafts were tested to depolarization of the smooth muscle with KCI; stimulation of
Journalof VASCULAR SURGERY
receptor-operated mechanisms was tested with phenylephrine (txl adrenergic agonist), BHT-920 (et2 adrenergic agonist), prostaglandin F2,; and endothelin, an endothelium-derived contractile peptide. ~ Relaxations were studied to receptor-operated mechanisms by use of adenosine diphosphate and acetylcholine; and with calcium ionophore A23187, which releases endothelium-derived factors through mechanisms not involving receptors. Nitric oxide was also tested to evaluate the responsiveness of the smooth muscle to this endothelimn-derived relaxing factor. 7 All drugs were obtained from Sigma Chemical (St. Louis, Mo.) except BHT-920 (Boehringer Ingelheim Pharmaceutical, Ridgefield, Conn.), endothelin (Peninsula Laboratories Inc., Belmont, Calif.) and nitric oxide (Union Carbide, Chicago, Ill.). Nitric oxide was prepared as previously described. 7 In some rings indomethacin (10- "~tool/L, combined with an equal molar concentration of Na2CO3) was used to inhibit the cyclooxygenase system and was added to the bath 30 minutes before other agonists were tested. All concentrations are reported as the final molar (tool/L) concentration in the organ chambers. Uptake o f SH-norepinephrine The ability of the nerve terminals to uptake norepinephrine was studied with 3H-norepinephrine. Strips of unoperated saphenous vein, cryopreserved graft, and fresh graft each weighing approximately 20 mg were incubated in the control solution (10 ml) and aerated for 30 minutes with 95% 02-5% CO2 at 37 ° C. Each strip was incubated for one hour with 1 × 10 -7 mol/L 3H-norepinephrine. Cocaine (5 × 10 -6 tool/L) was added to one half of the specimens to inhibit neuronal uptake of norepinephrine. The tissue was washed with control solution (10 ml for 1 hour), blotted dry, and weighed. The tissue was then extracted with 3 ml of 0.5N quaternary ammonium hydroxide in toluene, and radioactivity was measured with a Beckman LS-9800 liquid scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). Histology Rings of graft were placed in 10% buffered formaldehyde. The tissue was processed for routine light microscopy. A hematoxylin and eosin stain was used after the grafts had been sectioned (6 Ixm). Statistical analysis Only patent grafts (fresh, n = 6; cryopreserved, n = 5) were used for statistical analysis of organ chamber studies, platelet deposition, and norepi-
Volume 13 Number 5 May 1991
nephrine uptake. The role of the endothelium was determined by comparing responses of rings with and without endothelium from the same graft. The data are expressed as means + standard error of the mean (SEM); both absolute and percent changes were analyzed statistically. For contractions, an effective concentration resulting in 50% of the maximal response (EDs0) was calculated for each individual concentration-response curve, and the mean of these values was reported as the negative logarithm of the molar concentration. For relaxations an effective concentration that resulted in 50% inhibition of the maximal response (ICs0) was calculated. For organ chamber experiments performed in parallel on tissues (with and without endothelium) from the same animal, a Student's t test for paired observations was used. When more than two means were compared or differences between cryopreserved and fresh grafts were analyzed, an analysis of variance (ANOVA) was performed. For graft patency, a Fischer's exact test was used. Values were considered to be statistically different whenp < 0.05. RESULTS Graft patency and flows Five of eight (63%) of the cryopreserved grafts were patent, which was not significantly different from the fresh grafts where seven of eight (88%) grafts were patent. Blood flow through the grafts was not significantly different at implantation or harvest. At implantation, blood flow in the cryopreserved grafts was 83 _+ 17 ml/min and in the fresh grafts was 81 _+ 13 ml/min. At harvest, blood flow was 119 + 6 ml/min in the cryopreserved graft and 127 _+ 14 ml/min in the fresh graft. Platelet deposition Scans of ~In-labeled platelets obtained at implantation and before harvest revealed no significant differences in the actual radioactivity corrected for background activity between the cryopreserved and fresh grafts. Results were also analyzed by dividing the corrected counts by the number of counts in 1 × 10 -4 ml of blood collected at the time of the images to obtain a graft/blood ratio. Analysis with this method revealed no significant difference between cryopreserved and fresh grafts, or between implantation and harvest (Table I). Organ chamber experiments The basal tension of cryopreserved grafts (with endothelium: 1.9 _+ 0.1 g) and fresh grafts (with endothelium: 1.9 + 0 . 1 g ) were similar. N o difference was found in basal tension between rings with
Cryopreservedvein grafts 587
and without endothelium. The basal tension of unoperated femoral arteries, used as a control for acetylcholine, was 9.8 --- 0.5 g. N o difference was found in basal tension of arterial rings with and without endothelium. Contractions No difference was found in the maximal tension that developed to 60 mmol/L KCL in rings with or without endothelium in cryopreserved grafts (with 3.7 --_ 0.8g vs without 2.0 _-_ 0.3g) or in fresh grafts (with 1.5 --- 0.3 g vs without 1.5 +__0.4 g). The response of cryopreserved grafts with endothelium was significantly greater as compared to fresh grafts with endothelium (cryopreserved 3.7 +-- 0.8 g vs fresh 1.5 __ 0.3g). These differences were not seen in grafts without endothelium. Electrical stimulation did not result in any contraction in either the cryopreserved or fresh grafts. However, phenylephrine and BHT-960 caused contraction-dependent increases in tension in both grafts. The maximal tensions and the EDs0 for the responses to phenylephrine and BHT-960 were similar between the grafts (Table II). Endothelin caused concentration-dependent increases in tension that were not significantly different between the cryopreserved graft without endothelium or fresh grafts with and without endothelium (Fig. 1). In cryopreserved grafts with endothelium, a significant increase occurred in tension in response to endothelin as compared to the smooth muscle alone (rings without endothelium). The contractile responses to prostaglandin F2~ were significantly greater in the cryopreserved grafts with endothelium than without endothelium (Fig. 2). This response to prostaglandin F2~ was also greater than the fresh graft with or without endothelium. The EDs0 ( - log mol/L) for prostaglandin F2~ was not significantly different between cryopreserved ( - 5 . 9 + 0.10) and fresh ( - 5.7 + 0.11) grafts with endothelium. Relaxations
Acetylcholine did not cause relaxations in either graft type during contractions with prostaglandin F2~ but rather comparable concentration-dependent contractions were observed (Fig. 3). Removal of endothelium in these grafts did not significantly change the response to acetylcholine. N o significant differences were found in contractions to acetylcholine between grafts. In comparison, in the unoperated canine femoral artery a 100% relaxation was seen with intact endothelium. These relaxations in the artery were inhibited when the endothelium was removed (Fig. 3).
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Elmore et al.
588
T a b l e I. Platelet deposition o n
10
cryopreserved and fresh canine autogenous vein grafts
8
=-
o
o~ (0
Background-corrected graft~blood ratios of indium ill-labeled platelets
6
•
4
(= 0 t,.
o
2
r.
o
T
;'-
10
,
,
9
,
,
8
Endothelin,
7
-log M
Fig. 1. Contractions to endothelin in cryopreserved and fresh canine autogenous saphenous vein grafts. Data are shown as mean -+ SEM. * , Cryopreserved graft with endothelium, n = 5; O, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Implantation Proximal anastomosis Graft Distal anastomosis Harvest Proximal anastomosis Graft Distal anastomosis
Cryopreserved graft
Fresh gra~
3.6 -+ 0.5 ~ 2.0 _+ 0.4 1.2 -+ 0.2
3.5 + 0.5 1.8 _+ 0.2 1.6 -+ 0.2
2.2 _+ 1.4 1.7 -+ 0.9 1.8 _+ 1.6
4.6 _+ 2.8 4.1 -+ 2.7 4.1 _+ 2.9
*Data are expressed as mean - SEM. No significant difference exists between cryopreserved and fresh autografts or between implantation and harvest.
T a b l e I I . Adrenergic responses in
cryopreserved and fresh canine autogenous vein grafts
10 8 m
"O
6
c ®
4
Phenylephrine (aa) Cryopreserved graft (n = 5) Fresh graft (n = 6) BHT-960 (a2) Cryopreserved graft (n = 5) Fresh graft (n = 6)
m o U
2 O
T 9
"I"
T
,
8 Prostaglandin
, 7
~
, 6
,
t
,
5
Mgg'imal wnsion, g
Ed so log mol/L
6.5 -+ 2.8 ~ 2.6 -+ 0.9
-6.0 ~ 0.1 -5.9 - 0.1
1.1 -+ 0.7 1.0 _+ 0.8
-6.4 _+ 0.1 -6.4 -- 0.1
*Data are expressed as mean _+ SEM. There are no significant differences in maximal tensions or EDso between the cryopreserved and fresh autografts.
F2alpha ' -log M
Fig. 2. Contractions to prostaglandin F2~ in cryopreserved and fresh canine autogenous saphenous vein grafts. Data are shown as mean + SEM. 0 , Cryopreserved graft with endothelium, n = 5; O, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Adenosine diphosphate induced concentrationdependent relaxations o f rings with and w i t h o u t e n d o t h e l i u m in b o t h the cryopreserved and fresh grafts, w h i c h were comparable in m a g n i t u d e (Fig. 4). I n the presence o f indomethacin calcium ionop h o r e A 2 3 1 8 7 caused concentration-dependent relaxations, w h i c h were significantly greater in tings with than w i t h o u t e n d o t h e l i u m in b o t h the cryopreserved and fresh grafts. N o significant difference occurred between fresh and cryopreserved grafts in their response to A 2 3 1 8 7 (Fig. 5).
I n rings w i t h o u t endothelium, in the presence o f indomethacin, nitric oxide caused concentrationdependent decreases in tension. I n cryopreserved grafts, the maximal relaxation to 3 × 10 -4 m o l / L was significantly decreased (59.8% + - 5 . 8 % and 91.5% + 4.8% in cryopreserved and fresh grafts, respectively). T h e IC50 s ( - l o g mol/L) for nitric oxide were n o t significantly different (cryopreserved 5.5 -+ 0.1 vs fresh 5.7 -+ 0.2). Uptake of 3H-norepinephrine
T h e uptake o f 3H-norepinephrine was signific a n @ reduced by cocaine in the unoperated saphenous veins. The uptake o f norepinephrine was reduced in b o t h graft types as c o m p a r e d to the saphenous veins w i t h o u t cocaine. I n the grafts, the uptake was n o t further reduced by cocaine (Table III).
Volume 13 Number 5 May 1991
Cryopreserved vein grafts 589
.o r
2 9
8
7
6
9
8
7
6
Acetylcholine, -log M
Fig. 3. Responses of canine femoral artery (left); cryopreserved and fresh canine autogenous saphenous vein grafts (right) to acetylcholine in the presence of indomethacin (10 -s mol/L). Values are expressed as a percent change in tension to a contraction with prostaglandin F2~ (2 × 10 -6 mol/L; 8.2 + 1.5g, 3.7 -+ 1.0g, and 2.1 -+ 1.0g in femoral artery, cryopreserved, and fresh grafts, respectively, with endothelium). * , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; *, fresh graft with endothelium, n -- 6; o, fresh graft without endothelium, n = 6; "%femoral artery with endothelium, n = 6; [], femoral artery without endothelium, n = 6.
0
0
20 20
E 0 X
C
40
o
<., X
w n-
60
n,.
40
80 6O 100
I
8
I
7
I
I
6
I
I
5
I
I
I
4
9
I
Adenosine diphosphate, -log M
I
8
I
I
7
I
I
I
6
A 2 3 1 8 7 , -Iog M
Fig. 4. Relaxations to adenosine diphosphate in cryopreserved and fresh canine autogenous saphenous vein grafts. Values are expressed as a percent relaxation to a contraction with prostaglandin F2~ (2 × 10 -6 mol/L; 4.3 + 1.9 g, 1.8 + 0.6g, and 7.9 -+ 1.4 in cryopreserved grafts, fresh grafts, and unoperated femoral arteries, respectively, with endothelium). 0 , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Fig. 5. Responses to calcium ionophore A23187 in cryopreserved and fresh canine saphenous vein grafts. Experiments carried out in the presence of indomethacin 10 -5 mol/L. Values are expressed as a percent relaxation to a contraction with prostaglandin F2~ (2 × 10 -6 tool/L; 5.1 -+ 2 . 3 g and 2.5 + 1 . 0 g in cryopreserved and fresh grafts, respectively, with e n d o t h e l i u m ) . . , Cryopreserved graft with endothelium, n = 5; <>, cryopreserved graft without endothelium, n = 5; e, fresh graft with endothelium, n = 6; o, fresh graft without endothelium, n = 6.
Histology
DISCUSSION
T h e lumen was lined with a single layer o f cells that resembled endothelium in b o t h the fresh and cryopreserved vein grafts. There was an eccentric m y o i n t i m a l hyperplasia in b o t h types o f grafts (Fig. 6). By light m i c r o s c o p y no obvious m o r p h o l o g i c differences were f o u n d between the fresh and cryopreserved grafts.
As early as 1911, Yamanouchi 23 tested different storage solutions for arterial and venous allografts including formalin, saline, and water. With improvements in the technique ofcryopreservation, this f o r m o f preservation has resulted in veins with intact m o r p h o l o g y and with retention o f s m o o t h muscle function.~4 A l t h o u g h it has been p r e s u m e d that the
Journal of VASCULAR SURGERY
590 Elmore et al.
Table III. Uptake of 3H-norepinephrine in unoperated canine saphenous veins and cryopreserved and fresh autogenous saphenous vein grafts 3H-norepinephrine uptake (disintegrations~rain~rag) Graft
Control
Unoperated vein (n = 5) 265.1 ± 20.6* Fresh graft (n = 6) 85.9 ± 15.5Cryopreserved graft 58.5 ± 8.7t
Presence of' cocaine (5 × 10-~mol/L) 40,8 +- 2.6t 72,1 -+ 14.6t 50.6 -+ 9.8t
(n = 5)
*Values shown as mean +_ SEM, tdenotes significant differences from control unoperated veins, p < 0.05.
Fig. 6. Light micrographs of cryopreserved (top) and freshly harvested (bottom) saphenous vein autografts (e, endothelium; m, media; a, adventitia). Sections were 6 ~m and stained with hematoxylin and eosin; original magnification x 160. primary cause of failure of cryopreserved vein grafts is rejection, 5"6 cryopreservation itself may affect patency by altering the endothelium and smooth muscle of these grafts. In the present study the performance of cryopreserved autogenous veins as arterial grafts was comparable to that of freshly harvested autogenous veins with regard to early graft patency and blood flows. Platelet deposition was also similar between cryopreserved and fresh autografts and was low in comparison to that observed in synthetic grafts. 2~ This suggests that the intimal surfaces of cryopreserved and fresh saphenous veins are less thrombogenetic than prosthetic grafts. 21 This inhibitory effect on thrombogenicity is probably related to the observations that the intima of vein grafts (fresh and
cryopreserved) can release relaxing factors as evidenced by relaxations to A23187 only in rings of graft with endothelium. Endothelium-derived relaxing factors, in particular nitric oxide, can act synergistically with prostacyclin to inhibit platelet aggregation.'°'~, Neither cryopreserved nor fresh autografts demonstrated functional adrenergic innervation at 4 weeks as has been suggested by anatomic studies. 24 This conclusion is supported by the lack of response to electrical stimulation and the loss of the cocainesensitive mechanism for the uptake of norepinephrine. Cocaine usually inhibits neuronal uptake of the transmitter. 2s Both alphal and alpha z receptors are present on the smooth muscle of both types of grafts as contractions to phenylephrine and BHT-960 were observed. The magnitude of these responses are reduced compared to unoperated saphenous vein. 26 Acetylcholine will cause relaxations in mammalian arteries when the endothelium is present. 9 As has been shown in other vein grafts, ~3 endotheliumdependent relaxations to acetylcholine were not observed in either the fresh or cryopreserved autogenous saphenous vein grafts. This suggests that fresh or cryopreserved autogenous vein grafts do not develop arterial-like endothelium-dependent relaxations to muscarinic agonists at least after 4 weeks of implantation. The endothelium of cryopreserved and fresh autogenous grafts can, however, release some relaxing factors as evidenced by the greater relaxations in rings with endothelium as compared to without endothelium in response to A23187. Since these experiments were conducted in the presence of indomethacin, this relaxing factor must be a substance other than a cyclooxygenase product of arachidonic acid (i.e., not prostacyclin) and is most likely nitric oxide or a nitric oxide-carried mÜ•ecu•e. 27
Volume 13 Number 5 May 1991
Differences in the responsiveness of cryopreserved and fresh autogenous grafts were observed in response to some agonists. In particular, contractions of cryopreserved grafts to KCI, PGF2~ , and endothelin were greater when the endothelium was present. In fresh grafts, contractions to these agonists were similar in the absence and presence of endothelium. These data suggest that the endothelium of cryopreserved autografts may be releasing contractile substances in response to these agonists. The reasons for this are not clear, whether this represents a direct effect of the cryopreservation process on the metabolism of endothelial cells or a general characteristic of regenerated cells remains to be determined. If the cryopreservation process makes the endothelial cells fragile and susceptible to sloughing when blood flow through the graft is established, then the cryopreserved grafts may have a greater number of regenerated cells per surface area than freshly harvested grafts. Another difference in cryopreserved and fresh autogenous grafts was observed in the response of the smooth muscle to the endothelium-derived relaxing factor nitric oxide. Cryopreserved grafts were less sensitive to its inhibitory effects than fresh grafts. The decreased sensitivity of the cryopreserved vein grafts to nitric oxide may explain the tendencies for relaxations to A23187 to be decreased in the cryopreserved grafts. Since relaxations to nitric oxide are mediated through increases in cyclic guanosine monophosphate, 27 the decreased responses in cryopreserved grafts may suggest that this system is susceptible to inactivation by the cryopreservation process. In summary, cryopreservation did not affect early patency or thrombogenicity of these autografts. The ability of the endothelium of the cryopreserved autogenous grafts to release endothelium-derived factors and of the smooth muscle to respond to them supports the concept that functional characteristics of the cryopreserved vein grafts remain after cryopreservation. However, cryopreservation increased endothelium-dependent contractions and decreased the ability of the smooth muscle to relax to an endogenous nitrovasodilator. It is not known which step(s) of the cryopreservation process is responsible for these changes of the endothelium and smooth muscle or whether such changes would affect the long-term patency of such grafts. The authors thank Kevin S. Rud for his technical assistance; Robert R. Lorenz for drawing the figures; Marcia A. Simonson for manuscript preparation; and Boerhinger-Ingelheim Pharmaceutical for the BHT-920.
Cryopreservedvein grafts
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Submitted Oct. 9, 1990; accepted Dec. 21, 1990.