JOURNAL
OF SURGICAL
RESEARCH
60,
179-187
(1991)
Significance of the Endothelial Lining in Prevention of lntimal Thickening of Autogenous Vein Grafts in Dogs HIROSHI EGUCHI, M.D., TAKASHI YUKIZANE, Second
Department
KENICHIRO OKADOME, M.D., SHINSUKE MII, M.D., M.D., AND KEIZO SUGIMACHI, M.D., F.A.C.S.
of Surgery,
Submitted
Faculty
of Medicine,
for publication
Kyushu
University,
November
9, 1989
Fukuoka
812, Japan
Over the years we found that late graft failure due to intimal thickening tended to occur under conditions of abnormal flow and it was frequently observed in poor run-off cases and in grafts with an abnormal flow and a low value of variation in wall shear stress in a cardiac cycle (r-variation) [g-12]. We describe herein processes related to repair and to the permeation of fibrinogen, in an early period and under abnormal flow conditions following implantation of autogenous vein grafts in dogs.
To better comprehend the role of the endothelial lining in occurrence and development of intimal thickening in arterially implanted autogenous vein grafts, two models of canine femoral arteries were prepared. In the Group I model, the implanted autogenous vein graft was kept under a normal flow condition for 2 to 4 weeks after implantation, then was exposed to an abnormal flow (poor run-off). In case of a 3- to 4-week normal flow, intimal thickening was practically nil. Scanning electron microscopic studies showed that this 3- to 4-week period corresponded to that of re-endothelialization of the autogenous vein grafts. Immunohistochemical studies of fibrinogen distribution in the autogenous vein graft wall in the Group II model revealed that the permeation of fibrinogen was enhanced in case of an abnormal flow condition for about 2 weeks after the implantation. We interpret these observations to mean that intimal thickening was induced by an accelerated permeability in the presence of abnormal flow conditions until full re-endothelialization after the implantation. 0 1991 Academic Press, Inc.
MATERIALS
AND
METHODS
Forty adult mongrel dogs of either sex weighing between 15 and 26 kg were divided into two groups. In the 24 dogs in Group I, in which an autogenous vein graft had been implanted under normal flow condition, the condition of flow was changed to an abnormal one at 2 to 4 weeks after the implantation. In 16 dogs in Group II, autogenous vein grafts were implanted under both normal and abnormal flow conditions to observe re-endothelialization and permeation of plasma fibrinogen into the graft wall. All procedures were performed following the intravenous administration of pentobarbital(30 mg/ kg) and sterile conditions were maintained throughout. During all procedures and in both groups, cephalosporin (1 g) was given intravenously before the surgery, and aminoglucosid (50 mg) was given intramuscularly 1 day after the surgery.
INTRODUCTION
Autogenous vein graft is presently the most suitable bypass graft available for reconstruction of peripheral arteries; however, late graft failure is not uncommon. Intimal thickening is one of the major factors leading to late graft failure [l, 21. Several factors, including intimal damage, delayed reendothelialization, and enhanced permeability to macromolecules, have been implicated in the development of intimal thickening [ 3-51. Recently, hemodynamic factors have been given increasing attention. Shin et al. [6] and Berguer et al. [7] reported that a low flow rate or reduction of blood flow was closely related to intimal thickening. Schwartz et al. [8] described that low wall shear resulted in decreased endothelial cell stimulation, producing a state of disrepair and prolonged platelet adhesion to the exposed subendothelium, and stimulation of fibroblasts and smooth muscle cells.
Experimental
Model in Group I (Fig. 1)
At the first operation, an autogenous vein was grafted to the unilateral femoral artery, under conditions of normal blood flow. Following a skin incision on the upper thigh, the femoral artery and vein were exposed. A 4-cm segment of the femoral vein was dissected for the graft, with meticulous care to avoid unnecessary injury to the vein wall. The femoral artery, 3 cm in length, was removed and replaced with a reversed vein graft in an endto-end fashion with 7-O polypropylene monofilament sutures. 179
0022-4804/91$1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
180
JOURNAL
OF
SURGICAL
RESEARCH:
VOL.
50,
NO.
1991
done in the femoral artery, proximal to the previous occlusion (abnormal flow group). At this time, as controls, graft implantation was performed in the contralateral femoral artery, under conditions of a normal blood flow (normal flow group). The dogs were killed at 1,2,3, and 4 weeks. Four specimens were taken at each period, from each group. Two were examined using a scanning electron microscope and the other two were stained immunohistochemically to observe the permeation of fibrinogen into the wall of the graft.
GROUPI
Hemodynamic
mange to abmrmd, flow Condltlon
FIG.
1.
Experimental
models
A”togenO”I ve>n grafting under abnormalflow
of Group
I.
After implantation, the dogs were kept under conditions of normal flow for 2 to 4 weeks, then, according to the peripheral arterial segmental occlusion described by Morinaga et al. [lo], a second operation was done. A skin incision was made on the lower thigh, the popliteal artery and all branches of femoral and popliteal arteries, distal to previous implanted vein graft, except for the first branch of the distal caudal femoral artery, were ligated and dissected. With these procedures, blood flow in the graft became abnormal and experimental groups were established. We named these three experimental groups 2-weeks group (N = 8), 3-weeks group (N = S), and 4-weeks group (N = 8), respectively, according to the time under normal blood flow conditions. As controls, two models were set up and the contralatera1 femoral artery was used. In the abnormal flow group (N = 12), an autogenous vein graft was placed on the femoral artery which had been subjected to segmental occlusion. Namely, the graft was exposed to an abnormal blood flow from the time of implantation. In the normal flow group (N = 12), an autogenous vein graft was placed on the femoral artery, under conditions of a normal blood flow and the graft was never exposed to an abnormal blood flow. Four dogs in each group (2-, 3-, and 4-weeks group) were killed at 1 month and 3 months, respectively, and histologic examinations were made of the excised grafts. Grafts of the corresponding contralateral control groups were concomitantly removed. Experimental
2, FEBRUARY
Measurements
In the dogs of Groups I and II, flow measurement was performed when the dogs were prepared and at the time when the dogs were killed. The flow probe was applied on the distal artery of the vein graft, and the mean flow rate and flow wave form were measured with an electromagnetic flowmeter (Nihon Koden, MF-27, Tokyo, Japan). Variation of wall shear stress in a cardiac cycle (T-variation) was calculated using a computational flow wave form analyzer, as described [ 111. Histological Examination Thickening
and Measurement
of Intimal
The grafts from Group I were stretched on the board and were fixed with 10% buffered formalin solution. Three cross sections were obtained 5 mm distant from both proximal and distal anastomotic sites and the midportion of the graft and processed for light microscopy. Microscopic sections were stained with hematoxylin-eosin, elastica van Gieson’s, and Azan-Mallory. The thickness of the intima was measured with an ocular cytometer at six points around each cross-sectional circumference and the average value of the entire graft was calculated. Statistical comparison of the intimal thickness was made using Student’s t test. Scanning Electron Microscopic
Observations
In Group II, following washing out the blood with 0.1 mole/liter cacodylate buffer (pH 7.4) through a catheter inserted from the proximal iliac artery, the graft was fixed by perfusion of a mixture of 1% paraformaldehyde
GROWII
Model in Group II (Fig. 2)
Using the same approach to attain an abnormal blood flow group in Group I, first a peripheral segmental occlusion was made on the unilateral femoral artery. Two weeks later with the development of an adequate number of collateral vessels, autogenous vein grafting was
Under “Dl,lldl flow Under dbnonndl flow A”tOgeno”I “L-1” grafting
DlSt.3, poor run-off model
FIG.
2.
Experimental
models
of Group
II.
EGUCHI
ET AL.: INTIMAL
THICKENING
and 1.25% glutaraldehyde in 0.1 mole/liter cacodylate buffer pH 7.4 (Karnovsky’s fixative, diluted 1:3) [13] for 15 min with 100 cmH,O pressure, then removed. The specimen was immersed in 1:l diluted Karnovsky’s fixative overnight and three segments were removed from the adjacent proximal and distal anastomotic sites and midportion of the graft, and processed for scanning electron microscopic observation. To confirm that measurements related to intimal thickening were not influenced by fixation processes, intimal thickening of the perfusion-fixed sections at 4 weeks after implantation in Group II was measured using the same method as for Group I and the data were compared with findings in the control group at 1 month after implantation in Group I. Immunohistochemical
Thickness (Pm) 400 **
RESULTS Group
I
Data (Table 1)
Mean flow rate and r-variation in the abnormal flow condition after segmental occlusion in the experimental
TABLE Hemodynamic
Data
1 on Group Flow rate (ml/min)
* P < 0.05. 0.01.
** P
OF SURGICAL
RESEARCH
60,
179-187
(1991)
Significance of the Endothelial Lining in Prevention of lntimal Thickening of Autogenous Vein Grafts in Dogs HIROSHI EGUCHI, M.D., TAKASHI YUKIZANE, Second
Department
KENICHIRO OKADOME, M.D., SHINSUKE MII, M.D., M.D., AND KEIZO SUGIMACHI, M.D., F.A.C.S.
of Surgery,
Submitted
Faculty
of Medicine,
for publication
Kyushu
University,
November
9, 1989
Fukuoka
812, Japan
Over the years we found that late graft failure due to intimal thickening tended to occur under conditions of abnormal flow and it was frequently observed in poor run-off cases and in grafts with an abnormal flow and a low value of variation in wall shear stress in a cardiac cycle (r-variation) [g-12]. We describe herein processes related to repair and to the permeation of fibrinogen, in an early period and under abnormal flow conditions following implantation of autogenous vein grafts in dogs.
To better comprehend the role of the endothelial lining in occurrence and development of intimal thickening in arterially implanted autogenous vein grafts, two models of canine femoral arteries were prepared. In the Group I model, the implanted autogenous vein graft was kept under a normal flow condition for 2 to 4 weeks after implantation, then was exposed to an abnormal flow (poor run-off). In case of a 3- to 4-week normal flow, intimal thickening was practically nil. Scanning electron microscopic studies showed that this 3- to 4-week period corresponded to that of re-endothelialization of the autogenous vein grafts. Immunohistochemical studies of fibrinogen distribution in the autogenous vein graft wall in the Group II model revealed that the permeation of fibrinogen was enhanced in case of an abnormal flow condition for about 2 weeks after the implantation. We interpret these observations to mean that intimal thickening was induced by an accelerated permeability in the presence of abnormal flow conditions until full re-endothelialization after the implantation. 0 1991 Academic Press, Inc.
MATERIALS
AND
METHODS
Forty adult mongrel dogs of either sex weighing between 15 and 26 kg were divided into two groups. In the 24 dogs in Group I, in which an autogenous vein graft had been implanted under normal flow condition, the condition of flow was changed to an abnormal one at 2 to 4 weeks after the implantation. In 16 dogs in Group II, autogenous vein grafts were implanted under both normal and abnormal flow conditions to observe re-endothelialization and permeation of plasma fibrinogen into the graft wall. All procedures were performed following the intravenous administration of pentobarbital(30 mg/ kg) and sterile conditions were maintained throughout. During all procedures and in both groups, cephalosporin (1 g) was given intravenously before the surgery, and aminoglucosid (50 mg) was given intramuscularly 1 day after the surgery.
INTRODUCTION
Autogenous vein graft is presently the most suitable bypass graft available for reconstruction of peripheral arteries; however, late graft failure is not uncommon. Intimal thickening is one of the major factors leading to late graft failure [l, 21. Several factors, including intimal damage, delayed reendothelialization, and enhanced permeability to macromolecules, have been implicated in the development of intimal thickening [ 3-51. Recently, hemodynamic factors have been given increasing attention. Shin et al. [6] and Berguer et al. [7] reported that a low flow rate or reduction of blood flow was closely related to intimal thickening. Schwartz et al. [8] described that low wall shear resulted in decreased endothelial cell stimulation, producing a state of disrepair and prolonged platelet adhesion to the exposed subendothelium, and stimulation of fibroblasts and smooth muscle cells.
Experimental
Model in Group I (Fig. 1)
At the first operation, an autogenous vein was grafted to the unilateral femoral artery, under conditions of normal blood flow. Following a skin incision on the upper thigh, the femoral artery and vein were exposed. A 4-cm segment of the femoral vein was dissected for the graft, with meticulous care to avoid unnecessary injury to the vein wall. The femoral artery, 3 cm in length, was removed and replaced with a reversed vein graft in an endto-end fashion with 7-O polypropylene monofilament sutures. 179
0022-4804/91$1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
180
JOURNAL
OF
SURGICAL
RESEARCH:
VOL.
50,
NO.
1991
done in the femoral artery, proximal to the previous occlusion (abnormal flow group). At this time, as controls, graft implantation was performed in the contralateral femoral artery, under conditions of a normal blood flow (normal flow group). The dogs were killed at 1,2,3, and 4 weeks. Four specimens were taken at each period, from each group. Two were examined using a scanning electron microscope and the other two were stained immunohistochemically to observe the permeation of fibrinogen into the wall of the graft.
GROUPI
Hemodynamic
mange to abmrmd, flow Condltlon
FIG.
1.
Experimental
models
A”togenO”I ve>n grafting under abnormalflow
of Group
I.
After implantation, the dogs were kept under conditions of normal flow for 2 to 4 weeks, then, according to the peripheral arterial segmental occlusion described by Morinaga et al. [lo], a second operation was done. A skin incision was made on the lower thigh, the popliteal artery and all branches of femoral and popliteal arteries, distal to previous implanted vein graft, except for the first branch of the distal caudal femoral artery, were ligated and dissected. With these procedures, blood flow in the graft became abnormal and experimental groups were established. We named these three experimental groups 2-weeks group (N = 8), 3-weeks group (N = S), and 4-weeks group (N = 8), respectively, according to the time under normal blood flow conditions. As controls, two models were set up and the contralatera1 femoral artery was used. In the abnormal flow group (N = 12), an autogenous vein graft was placed on the femoral artery which had been subjected to segmental occlusion. Namely, the graft was exposed to an abnormal blood flow from the time of implantation. In the normal flow group (N = 12), an autogenous vein graft was placed on the femoral artery, under conditions of a normal blood flow and the graft was never exposed to an abnormal blood flow. Four dogs in each group (2-, 3-, and 4-weeks group) were killed at 1 month and 3 months, respectively, and histologic examinations were made of the excised grafts. Grafts of the corresponding contralateral control groups were concomitantly removed. Experimental
2, FEBRUARY
Measurements
In the dogs of Groups I and II, flow measurement was performed when the dogs were prepared and at the time when the dogs were killed. The flow probe was applied on the distal artery of the vein graft, and the mean flow rate and flow wave form were measured with an electromagnetic flowmeter (Nihon Koden, MF-27, Tokyo, Japan). Variation of wall shear stress in a cardiac cycle (T-variation) was calculated using a computational flow wave form analyzer, as described [ 111. Histological Examination Thickening
and Measurement
of Intimal
The grafts from Group I were stretched on the board and were fixed with 10% buffered formalin solution. Three cross sections were obtained 5 mm distant from both proximal and distal anastomotic sites and the midportion of the graft and processed for light microscopy. Microscopic sections were stained with hematoxylin-eosin, elastica van Gieson’s, and Azan-Mallory. The thickness of the intima was measured with an ocular cytometer at six points around each cross-sectional circumference and the average value of the entire graft was calculated. Statistical comparison of the intimal thickness was made using Student’s t test. Scanning Electron Microscopic
Observations
In Group II, following washing out the blood with 0.1 mole/liter cacodylate buffer (pH 7.4) through a catheter inserted from the proximal iliac artery, the graft was fixed by perfusion of a mixture of 1% paraformaldehyde
GROWII
Model in Group II (Fig. 2)
Using the same approach to attain an abnormal blood flow group in Group I, first a peripheral segmental occlusion was made on the unilateral femoral artery. Two weeks later with the development of an adequate number of collateral vessels, autogenous vein grafting was
Under “Dl,lldl flow Under dbnonndl flow A”tOgeno”I “L-1” grafting
DlSt.3, poor run-off model
FIG.
2.
Experimental
models
of Group
II.
EGUCHI
ET AL.: INTIMAL
THICKENING
and 1.25% glutaraldehyde in 0.1 mole/liter cacodylate buffer pH 7.4 (Karnovsky’s fixative, diluted 1:3) [13] for 15 min with 100 cmH,O pressure, then removed. The specimen was immersed in 1:l diluted Karnovsky’s fixative overnight and three segments were removed from the adjacent proximal and distal anastomotic sites and midportion of the graft, and processed for scanning electron microscopic observation. To confirm that measurements related to intimal thickening were not influenced by fixation processes, intimal thickening of the perfusion-fixed sections at 4 weeks after implantation in Group II was measured using the same method as for Group I and the data were compared with findings in the control group at 1 month after implantation in Group I. Immunohistochemical
Thickness (Pm) 400 **
RESULTS Group
I
Data (Table 1)
Mean flow rate and r-variation in the abnormal flow condition after segmental occlusion in the experimental
TABLE Hemodynamic
Data
1 on Group Flow rate (ml/min)
* P < 0.05. 0.01.
** P
