Heart Vessels (1992) 7: 148-154

Heart

aWessels

© Springer-Verlag1992

Morphological and functional study of free arterial grafts T a d a s h i I s o m u r a , K o u i c h i H i s a t o m i , H i r o t o I n u z u k a , M a s a r u Nishimi, A k i o H i r a n o , and K i r o k u Ohishi Second Department of Surgery, Knrume University Hospital, 67 Asahi-machi, Kurume, Fukuoka, 830 Japan

S u m m a r y . Morphological and functional changes of free arterial gratis in dogs were studied for 3 weeks after implantation and the changes were compared to those in implanted free vein grafts. In the arterial grafts, endothelial cells with abundant pinocytotic vesicles and some cytoplasmic folds were observed by transmission and scanning electron microscope and cell detachment was seen only at the site of anastomosis, while most cells were detached in the vein grafts. The site of mechanical damage in the arterial grafts was covered by regeneräted endothelial cells which showed similar morphological findings to the normal arterial endothelial cells. In contrast, regenerated cells in the vein grafts started to cover the denuded area 7 days after the implantation and had completely covered it by 3 weeks. Prostacyclin was produced more abundantly in arterial grafts than in vein grafts at any phase after implantation. The level of prostacyclin production was between 30 and 40 pg/mg in any phase after implantation of free arterial grafts, while in vein grafts the level was 2.5 pg/mg at the day of implantation and increased to 13.6pg/mg at 21 days. This study showed that the endothelial cells were well preserved and the level of prostacyclin production was high in the arterial grafts, and thus the grafts seemed to show potent anti-thrombogenicity after implantation. Although late changes in arterial and vein grafts were not investigated in this experimental protocol, these results may suggest that the arterial graft is superior to the vein graft e r e n in the early period after its implantation as a free graft.

Key words: Free arterial g r a f t - Electron microscope Prostacyclin - Implantation

Introduction The arterial graft has been the conduit of choice in bypass surgery, as seen in coronary arterial bypass grafting (CABG), because the internal thoracic artery (ITA) shows a better patency rate compared to the saphenous vein graft (SVG) in the late postoperative period [1]. The arterial graft is used not only as a pedicled graft but also as a free graft. We studied the morphological and functional changes in free arterial and vein grafts after implantation in an animal experiment to disclose the superiority of the arterial graft even in the early period after its implantation as a free graft.

Materials and methods Animal experiment Mongrel dogs weighing 12-18 kg were used. After the dogs were anesthetized with a subcutaneous injection of ketamine hydrochloride (5mg/kg) and intravenous sodium pentobarbital (25mg/kg), they were intubated and placed on a positive pressure ventilator. The femoral vein and artery were exposed and approximately 6-cm lengths were harvested from one side as free grafts. The grafts were rinsed and preserved in physiological saline solution at room temperature. Approximately 4 cm of the femoral artery on the other side was exposed and was cut at the center, after which both ends of the free vein or arterial graft were anastomosed to the femoral artery with a running 7-0 Prolene suture. The grafts were collected immediately after anastomosing, as well as at i h and 7, 14, and 21 days after the implantations, and the morphological and functional changes of the implanted grafts were studied. Five patent and nonstenotic arteries and veins from different dogs were examined at each period. No anticoagulants were administered either during or after implantation. A normal femoral vein or artery was used as a control. E l e c t r o n m i c r o s c o p i c analysis

Address correspondence to: T. Isomura

Received December 10, 1991; revision received March 2, 1992; accepted March 6, 1992

The implanted graft was removed and the site of the anastomoses and the center of the graft were fixed in

T. Isomura et al.: Free arterial grafts Karnovski's fixative (2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% CaC12 in 0.1M sodium cacodylate buffer, pH 7.4) for 6 h at 20°C. The fixed tissues were then washed in cold 0.1 M sodium cacodylate buffer, pH 7.4, and processed for transmission (TEM) or scanning (SEM) electron microscopy. For TEM, a thin section was stained with lead citrate and examined with a Hitachi H-7000 electron microscope. For SEM, the tissues were dried by using the critical point of carbon dioxide, coated with gold-palladium and examined with a Hitachi S-800 scanning electron microscope. Prostacyclin production of the implanted graft

Radioimmunoassay of 6-keto-prostaglandin FI~ The removed vessels were washed with cold Hank's solution (8g NaC1, 0.4g KC1, 0.048g anhydrous Na2HPO4, 0.06g anhydrous KH2PO4, 0.049g anhydrous MgSO4, 0.047g anhydrous MgC12, 0.14 g anhydrous CaC12, and i g glucose in 11 of distilled water, pH 7.4), the surrounding tissue was removed, and the specimens were kept at -70°C until used. The samples were cut into 2.0-cm sections and incubated in Hank's solution for i h at 4°C. The specimens were incubated in 2 ml of Hank's solution at 37°C for 15 min. The release of 6-keto-prostaglandin Flor, a hydration product, into the medium, was estimated by radioimmunoassay (6-keto-PGFl~ 3H RIA kit, Amersham International Buckinghamshire, UK). The results were reported as picograms of 6-keto-PGFlù per milliliter of Hank's solution

Fig. 1A-l). Scanning electron microscopical findings of arterial (A) (×175) and venous (B) (×300) grafts l h after implantation. The arterial graft showed detachment of the endothelial cell layer (arrow heads) around the suture (arrow) but a regularly arranged endothelial cell layer was seen on most of the inner surface. At 21 days after implan-

149 for incubation or nanograms of 6-keto-PGFl~ per wet-weight of the vessel (mg) or surface area of the vessel (cm2).

Statistical analysis The significance of the differences between the mean values of arterial and venous samples were analyzed using Student's t-test. A probability value of less than 0.05 was considered significant. Unless otherwise specified, results are presented as mean _+ standard deviation.

Resuits M o r p h o l o g i c a l c h a n g e s o f the i m p l a n t e d vessels

Immediately after harvesting the vessels Arterial grafi. The

endothelial cells were arranged in the longitudinal direction of the vessel and there was no cell detachment.

Vein graft. The endothelial cells were flat and regularly arranged with little detachment or little deposition of platelets or fibrin. After immersion into saline solution at 20°C, a few endothelial cells in the vein graft became detached with exposure of the underlying layer.

tation (C) (×525), regenerated endothelial cells completely covered the denuded inner surface of the implanted graft. At 7 days after implantation (D) (x750), regenerated cells (arrows) started to be visible on the surface of the denuded layer of the implanted free vein graft

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T. Isomura et al.: Free arterial grafts

Fig. 2A,B. Transmission electron microscope findings of endothelial cells of the arterial graft. The basement membrane (BM) was thick and numerous pinocytotic vesicles (pv) were observed as well as abundant cytoplasmic organella

One hour after implantation Arterial grafi. Endothelial cells were detached around the suture line, but most of the inner surface was covered by regularly arranged endothelial cells (Fig. lA). TEM findings showed a wavy pattern of cell arrangement and the cells protruded into the lumina of the vessel (Fig. 2A). The endothelial cells contained numerous pinocytotic vesicles, rough endoplasmic reticulum, occasional cytoplasmic lipid bodies as well as numerous cytoplasmic filaments. The endothelial basement membrane was relatively thick (Fig. 2B).

Vein grafi. Most of the endothelial cells had become detached and the subendothelial layer was exposed,

accompanied by deposition of platelets and fibrin (Fig. 1B).

Seven days after implanmtion Arterial graft. Focal depositions of platelets and fibrin were seen at the site of the anastomoses, while no such depositions were found in the center of the graft. Most of the findings were similar to those for the control graft.

Vein graft. Regenerated cells forming island structures had begun to cover the denuded inner surface of the vessel. The cells were arranged irregularlyl while the protruding center part contained abundant microvilli (Fig. 1D).

T. Isomura et al. : Free arterial grafts

Fig. 3A-C. The endothelial cells of an arterial graft 21 days after implantation. Many cytoplasmic filaments (F) were observed under the scanning (A) or transmission (B) electron microscope, as well as numerous pinocytotic

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vesicles (pv) and an occasional coated vesicle (cv). The basement membrane (BM) beneath the cell layer was thick

Fig. 4. The endothelial cells which had covered the denuded area of the arterial graft contained an irregularshaped nucleus, numerous pinocytotic vesicles (pv), a few intracytoplasmic filaments, and a thin basement membrane

Fourteen days after implantation Arterial graft.

The platelet clot had started to disappear at the site of anastomosis and small cells had begun to cover the surface.

Vein graft. The platelet adhesion had disappeared and most of the internal surface of the graft was covered by regenerative endothelial cells. These cells were smaller than those seen 7 days after implantation and spindleshaped cells were frequently observed. Twenty-one days after implantation Arterial graft.

The small cells seen 14 days after the implantation now completely covered the denuded

area (Fig. 1C) but the cells were arranged irregularly. The endothelial cells were arranged regularly except for the regenerated area and showed numerous pinocytotic vesicles and occasional coated vesicles as well as numerous cytoplasmic filaments. Few cytoplasmic folds (Fig. 3A,B) were seen. The basement membrane was thick (Fig. 3B and C). These findings were similar to those of the control endothelial cells in Fig. 1. However, the endothelial cells which covered the denuded area showed irregular nuclei, fewer cytoplasmic filaments with relatively developed pinocytotic vesicles, and the endothelial basement membrane was not thick (Fig. 4).

Vein graft. The inner surface, including the site of the anastomoses, was completely covered by regenerated

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T. Isomura et al.: Free arterial grafts

uniform endothelial cells with moderately devetoped microvilli and pores. The regenerated cells up to this period were small and the density of the cells covering the inner surface of the graft varied among the harvested grafts. No evidence of smooth muscle cell migration and hyperplasia was noted in the vein graft removed in this period. In each phase, the morphological changes were uniform among the different arterial grafts, while the regeneration of endothelial cells, which was observed after the 7th day of implantation in the vein graft, showed slight variations in the regenerated cell number among the different vessels. Prostacyclin production (Fig. 5) The production of 6-keto-PGFl« by the vein grafts was 2.49 + 0.74pg/mg (n = 10) immediately after harvesting, and 2.20 + 0.89pg/mg (n = 5) at lh, 2.56 + 1.24pg/mg (n = 5) at 7 days, 7.13 + 2.60pg/mg (n = 5) at 14 days, and 13.56 + 5.36pg/mg (n = 5) at 21 days after implantation. The level had thus increased significantly after the implantation of the free vein graft. The production of 6-keto-PGFl~ by the arterial grafts was 35.94 + 4.28pg/mg immediately after harvest (n = 10), and 33.32 + 3.79pg/mg (n = 5) at l h , 35.77 + 3.56pg/mg (n = 5) at 7 days, 36.43 + 3.56pg/mg (n = 5) at 14 days, and 34.73 + 5.02pg/mg (n = 5) at 21 days after implantation. There were no significant differences among the phases. However, by comparing the levels, the production per tissue weight after implantation showed significant differences between the two types of grafts (P < 0.01).

The production of 6-keto-PGFl~ per graft area unit was 3.72 + 1.27pg/mm 2 for vein grafts and 7.43pg/ mm a for arterial grafts at 21 days after implantation, a singificant difference (P < 0.01).

Discussion In reports on implanted grafts, the long-term patency of the venous graft has been described as poor. In 1961, Ejrup et al. [2] reported evidence of atherosclerotic changes in the venous graft. These sclerotic changes were found angiographically in 42% of the SVGs 5 years after CABG. Svensen et al. [3] considered that the changes were caused by atherosclerotic alterations in the venous graft after endothelial cell injury. Ross [4] considered that atherosclerotic changes in the vein graft might be caused by response to the injury of the endothelial cells. In these studies, the late changes in the venous grafts tend to be mainly related to hyperplasia and atherosclerosis, and seemed to influence the late patency. Although we observed the morphological and functional changes of implanted arterial and vein grafts for 3 weeks in this model, the difference between the two grafts was seen from the early phase of implantation. The endothelial cells in the venous graft detached after preservation of the graft in saline at 20°C. Thus, the venous graft seemed to be easily affected by the preserved solution. The endothelial cells, however, started to regenerate 7 days after the graft implantation and the internal surface of the graft was completely covered by regenerated endothelial cells with abundant microvilli

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Fig. 5. Prostacyclin production of the arterial and vein grafts after implantation. The level of 6-ketoPGFI~ was significantly higher (P

Morphological and functional study of free arterial grafts.

Morphological and functional changes of free arterial grafts in dogs were studied for 3 weeks after implantation and the changes were compared to thos...
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