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Comparison of myointimal hyperplasia in laser-assisted and suture anastomosed arteries
Comparison of myointimal hyperplasia in laser-assisted and suture anastomosed arteries A preliminary report M a t t h e w R. Quigley, M . D . , Julian E. Bailes, M . D . , H a u C. K w a a n , M . D . , L e o n a r d J. Cerullo, M.D., and Spencer Block, B.S., Chicago, Ill. Use o f the milfiwatt CO2 laser to perform microvascular anastomoses is associated with characteristic histologic changes, including intimal hyperplasia and medial necrosis. The extent o f myoinfimal proliferation after both suture and laser-assisted vascular anastomosis was assessed in the rat femoral artery model. At 2 weeks the average intimal height o f the laser-anastomosed vessels was 11.7 -+ 2.2 gtm (mean _+ standard error o f the mean) vs. 21.3 -+ 3.2 ~ m for sutured arteries (p < 0.05). By 6 weeks the groups were equivalent (laser, 25.6 -+ 4.6 ~m; suture, 17.3 -+ 1.2 Ixm; p, not significant). The medial changes associated with the laser-assisted method appear to inhibit the proliferative response at 2 weeks but are reversed by 6 weeks. (J VAse SURG 1986; 4:217-9.)
Myointimal hyperplasia (MH) is a proliferative response of vascular smooth muscle subsequent to intimal injury. This process has been implicated in the progressive stenosis of vessels occasionally observed after coronary artery bypass grafting ~ and carotid endarterectomy. 2'3Most evidence points to M H as an etiologic factor in atherosclerotic disease4 and possibly in vasospasm after subarachnoid hemorrhage, s Reports from our laboratory have delineated the vessel histology observed with laser-assisted vascular anastomosis (LAVA), in which significant M H was observed. 6'7 Therefore, we initiated this study to compare the proliferative response occurring after conventional microsurgical anastomosis and arteries joined by LAVA. MATERIAL AND METHODS
Twenty Sprague-Dawley rats (weighing 250 to 350 gm) were operated on according to standards of care outlined by the Northwestern University Medical School Animal Research Committee. A LAVA was performed on the right femoral artery as previously described. 6"8 Briefly, three stay sutures of 10-0 nylon were placed 120 degrees apart for coaptation. Laser energy was then applied along the From the Division of Neurosurgery, Department of Surgery, Department of Medicine, Northwestern University Medical School and Hematology/OncologySection, Veterans Administration LakesideMedicalCenter. Reprint requests: Hau C. Kwaan,M.D., Chief, Hematology,Department of Surgery, Northwestern University,303 East Chicago Ave., Chicago, IL 60611.
everted vessel edges held taut between two of the stays. The application was in 0.1-second bursts at 65 to 75 mW with a 150 ~m spot delivered by a milfiwatt COz laser (model 7600, Bioquantum, Houston, Tex.) through an operating microscope at × 40 magnification. Copious irrigation was applied between laser applications and the end point was a subjective tissue change to a fight brown color. This process was repeated along the other two sectors formed by the stays. The left underwent a standard microsurgical anastomosis with the use of 8 to 10 sutures of 10-0 nylon with 70 Cm needles. 9 Animals were killed at 2 or 6 weeks by antegrade aortic perfusion with Zenker's solution. The femoral vessels were dissected free from the inguinal ligament to the superficial epigastric artery, embedded in paraffin, stained by Movat technique, 1° and cut in 5 ~m sections perpendicular to the long axis of the vessel. Quantitation o f myointimal proliferation. Sections of each vessel were visually scanned to determine the site of the anastomosis. From this reference point, four additional sections were selected on each side of the anastomosis at 50 p,m increments both proximal and distal. This procedure yielded nine sections altogether, which were taken as representative of a given tissue sample (Fig. 1). The area of proliferation was quantitated by means of an Ibas image analysis system (Zeiss, New York, N.Y.) interfaced with a real-time video display from a × 40 microscopic image. After computer image enhancement, an operator driving a cursor outlined the site of proliferation, that 217
Journal of VASCULAR SURGERY
218 Qu~qley et al.
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Fig. 1. Schematic of method for selecting representative sections to quantitate myointimal hyperplasia.
Fig. 2. A, Unenhanced image of tissue section. B, Specimen subject to computer enhancement by expanding gray scale. C, Operator-driven cursor line placed along internal elastic lamina (arrows) to allow calculation of intimal area. Table I. Mean values of intimal height for laser-assisted and suture anastomosed groups Intinud he~ht (t~rn) Time 2 wk 6 wk
Laser 11.7 (n 25.6 (n p <
+ 2.2 = 7) -+ 4.6 = 7) 0.02
Suture 2 1 . 3 + 3.2 (n = 6) 17.3 -+ 1.2 (n = 7) p = NS
p < 0.05 p = NS
N S = n o t signiticant, p > 0.05. NOTE: D a t a are expressed as m e a n values -+ s t a n d a r d error o f the mean.
being the area inside the internal elastic lamina (IEL) (Fig. 2). The area of proliferation was divided by the perimeter of the IEL to derive the intimal height for each section. The nine values were averaged to obtain the height for each sample. Groups were compared by the comparison of independent means test with
the Student t distribution. Analysis was not performed by paired samples because of an insufficient number of paired samples. RESULTS Three deaths occurred before sacrifice (two at 2 weeks and one at 6 weeks), leaving 34 vessels available for study. Vessel occlusion or improper preparation invalidated another seven specimens, leaving 27 arteries for quantitation. Group means at 2 and 6 weeks are shown in Table I. Intimal height was significantly less among the laser group than the suture cohort at 2 weeks (p < 0.05). However, this trend was reversed at 6 weeks with laser proliferation exceeding suture, the difference not being significant. Not significant also was the change in average myointimal height from 2 to 6 weeks among the suture group, although the LAVA cohort showed a significant increase in myointimal thickening during this same period (p < 0.02).
Volume 4 Number 3 September 1986
DISCUSSION Recent reports point to the importance o f m y o i n timal proliferation regarding late vascular stenosis ~-3 and atherosclerosis. 4 Studies f r o m o u r laboratory addressing the usefulness o f L A V A have indicated the presence o f p r o f o u n d medial injury a b o u t the anast o m o t i c site, in addition to significant M H . 6'7 H o w ever medial d a m a g e was not seen a m o n g suture controls. T h e results o f the current investigation w o u l d indicate that the medial loss associated with L A V A inhibits M H w h e n c o m p a r e d with suture controls at 2 weeks, a time p o i n t w h e n proliferation is maximal, following air drying or balloon catheter injury m o d els. ",~2 H o w e v e r , at 6 weeks the L A V A vessels have evidently o v e r c o m e this inhibition. Research p e r f o r m e d in a n u m b e r o f institutions has highlighted a n u m b e r o f p r o b l e m s associated with L A V A , the p r e d o m i n a n t one being aneurysm formation. 13,~4 T h e enhanced M H at 6 weeks observed in this study raises the question as to w h e t h e r over the long term, L A V A vessels will display M H regression as in other models or progress to early atherosclerotic lesions. This question indicates that further studies are necessary before the technique can find its way into clinical use. We gratefully acknowledge the technical assistance of Clara Lastre and the aid of Bonnie Osborn in manuscript preparation. REFERENCES
1. Cashin WL, Sanmarco ME, Nessim SA, Blankenhom DH. Accelerated progression of atherosclerosis in coronary vessels with minimal lesions that are bypassed. N Engl J Med 1984; 311:824-8.
Laser proliferation 219
2. Cossman D, Cadow AD, Stein A, Matsumoto G. Early restenosis after carotid endarterectomy. Arch Surg 1978; 113:275-8. 3. Palmaz JC, Hunter G, Carson SN, French SW. Postoperative carotid restenosis due to neointimal fibrovascular hyperplasia. Radiology 1983; 148:699-702. 4. Ross R, Glomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976; 295:420-5. 5. Takemae T, Bronson J, Alksae JF. Intimal proliferation of cerebral arteries after subarachnoid blood injection in pigs. J Neurosurg 1984; 61:494-500. 6. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ, Brown JT, Lastre CL, Monma D. Microvascular anastomosis using the milfiwatt CO2 laser. Lasers Surg Med 1985; 5:357-65. 7. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ. Histological comparison of suture versus laser-assisted vascular anastomosis. Surg Forum 1985; 36:508-10. 8. Bailes JE, Quigley MR, Kwaan HC, CeruUo LJ, Brown JT. Fibrinolytic activity following laser-assisted vascular anastomosis. Microsurgery 1985; 6:163-8. 9. Acland RD, Tractenberg L. Histopathology of small arteries following experimental microvascular anastomosis. Plast Reconstr Surg 1977; 59:868-75. 10. Movat HZ. Demonstration of all connective tissue elements in a single reaction. Arch Pathol 1955; 60:289-92. 11. Clowes AW, Ryan GB, Breslow JL, Karnovsky MJ. Absence of enhanced intimal thickening in the response of the carotid artery to endothelial injury in hypercholesterolemic rats. Lab Invest 1976; 35:6-17. 12. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab Invest 1983; 49:32733. 13. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ, Brown JT. Aneurysm formation following low power COs laser-assisted vascular anastomosis. Neurosurgery 1986; 18:292-9. 14. Pribil S, Powers SK. Carotid artery end-to-end anastomosis in the rat using the argon laser. J Neurosurg 1985; 63:771-5.
Myointimal hyperplasia (MH) is a proliferative response of vascular smooth muscle subsequent to intimal injury. This process has been implicated in the progressive stenosis of vessels occasionally observed after coronary artery bypass grafting ~ and carotid endarterectomy. 2'3Most evidence points to M H as an etiologic factor in atherosclerotic disease4 and possibly in vasospasm after subarachnoid hemorrhage, s Reports from our laboratory have delineated the vessel histology observed with laser-assisted vascular anastomosis (LAVA), in which significant M H was observed. 6'7 Therefore, we initiated this study to compare the proliferative response occurring after conventional microsurgical anastomosis and arteries joined by LAVA. MATERIAL AND METHODS
Twenty Sprague-Dawley rats (weighing 250 to 350 gm) were operated on according to standards of care outlined by the Northwestern University Medical School Animal Research Committee. A LAVA was performed on the right femoral artery as previously described. 6"8 Briefly, three stay sutures of 10-0 nylon were placed 120 degrees apart for coaptation. Laser energy was then applied along the From the Division of Neurosurgery, Department of Surgery, Department of Medicine, Northwestern University Medical School and Hematology/OncologySection, Veterans Administration LakesideMedicalCenter. Reprint requests: Hau C. Kwaan,M.D., Chief, Hematology,Department of Surgery, Northwestern University,303 East Chicago Ave., Chicago, IL 60611.
everted vessel edges held taut between two of the stays. The application was in 0.1-second bursts at 65 to 75 mW with a 150 ~m spot delivered by a milfiwatt COz laser (model 7600, Bioquantum, Houston, Tex.) through an operating microscope at × 40 magnification. Copious irrigation was applied between laser applications and the end point was a subjective tissue change to a fight brown color. This process was repeated along the other two sectors formed by the stays. The left underwent a standard microsurgical anastomosis with the use of 8 to 10 sutures of 10-0 nylon with 70 Cm needles. 9 Animals were killed at 2 or 6 weeks by antegrade aortic perfusion with Zenker's solution. The femoral vessels were dissected free from the inguinal ligament to the superficial epigastric artery, embedded in paraffin, stained by Movat technique, 1° and cut in 5 ~m sections perpendicular to the long axis of the vessel. Quantitation o f myointimal proliferation. Sections of each vessel were visually scanned to determine the site of the anastomosis. From this reference point, four additional sections were selected on each side of the anastomosis at 50 p,m increments both proximal and distal. This procedure yielded nine sections altogether, which were taken as representative of a given tissue sample (Fig. 1). The area of proliferation was quantitated by means of an Ibas image analysis system (Zeiss, New York, N.Y.) interfaced with a real-time video display from a × 40 microscopic image. After computer image enhancement, an operator driving a cursor outlined the site of proliferation, that 217
Journal of VASCULAR SURGERY
218 Qu~qley et al.
50p 5p I
I
i ~ ~ , ,
,
k
,i
L
1
,
,
,\t
I
I
,
,
lilllllJ~
lllllllllll
I
1
!iilillill
lJlllr]lrlF i
illlllllll
illlllllll
I
I
i
i
'
,
I I
IIIIIlllll
,
IIIIIIIIII
q
i i
! --
I ~
-
,
g'Lj/_2 Jy ,
Fig. 1. Schematic of method for selecting representative sections to quantitate myointimal hyperplasia.
Fig. 2. A, Unenhanced image of tissue section. B, Specimen subject to computer enhancement by expanding gray scale. C, Operator-driven cursor line placed along internal elastic lamina (arrows) to allow calculation of intimal area. Table I. Mean values of intimal height for laser-assisted and suture anastomosed groups Intinud he~ht (t~rn) Time 2 wk 6 wk
Laser 11.7 (n 25.6 (n p <
+ 2.2 = 7) -+ 4.6 = 7) 0.02
Suture 2 1 . 3 + 3.2 (n = 6) 17.3 -+ 1.2 (n = 7) p = NS
p < 0.05 p = NS
N S = n o t signiticant, p > 0.05. NOTE: D a t a are expressed as m e a n values -+ s t a n d a r d error o f the mean.
being the area inside the internal elastic lamina (IEL) (Fig. 2). The area of proliferation was divided by the perimeter of the IEL to derive the intimal height for each section. The nine values were averaged to obtain the height for each sample. Groups were compared by the comparison of independent means test with
the Student t distribution. Analysis was not performed by paired samples because of an insufficient number of paired samples. RESULTS Three deaths occurred before sacrifice (two at 2 weeks and one at 6 weeks), leaving 34 vessels available for study. Vessel occlusion or improper preparation invalidated another seven specimens, leaving 27 arteries for quantitation. Group means at 2 and 6 weeks are shown in Table I. Intimal height was significantly less among the laser group than the suture cohort at 2 weeks (p < 0.05). However, this trend was reversed at 6 weeks with laser proliferation exceeding suture, the difference not being significant. Not significant also was the change in average myointimal height from 2 to 6 weeks among the suture group, although the LAVA cohort showed a significant increase in myointimal thickening during this same period (p < 0.02).
Volume 4 Number 3 September 1986
DISCUSSION Recent reports point to the importance o f m y o i n timal proliferation regarding late vascular stenosis ~-3 and atherosclerosis. 4 Studies f r o m o u r laboratory addressing the usefulness o f L A V A have indicated the presence o f p r o f o u n d medial injury a b o u t the anast o m o t i c site, in addition to significant M H . 6'7 H o w ever medial d a m a g e was not seen a m o n g suture controls. T h e results o f the current investigation w o u l d indicate that the medial loss associated with L A V A inhibits M H w h e n c o m p a r e d with suture controls at 2 weeks, a time p o i n t w h e n proliferation is maximal, following air drying or balloon catheter injury m o d els. ",~2 H o w e v e r , at 6 weeks the L A V A vessels have evidently o v e r c o m e this inhibition. Research p e r f o r m e d in a n u m b e r o f institutions has highlighted a n u m b e r o f p r o b l e m s associated with L A V A , the p r e d o m i n a n t one being aneurysm formation. 13,~4 T h e enhanced M H at 6 weeks observed in this study raises the question as to w h e t h e r over the long term, L A V A vessels will display M H regression as in other models or progress to early atherosclerotic lesions. This question indicates that further studies are necessary before the technique can find its way into clinical use. We gratefully acknowledge the technical assistance of Clara Lastre and the aid of Bonnie Osborn in manuscript preparation. REFERENCES
1. Cashin WL, Sanmarco ME, Nessim SA, Blankenhom DH. Accelerated progression of atherosclerosis in coronary vessels with minimal lesions that are bypassed. N Engl J Med 1984; 311:824-8.
Laser proliferation 219
2. Cossman D, Cadow AD, Stein A, Matsumoto G. Early restenosis after carotid endarterectomy. Arch Surg 1978; 113:275-8. 3. Palmaz JC, Hunter G, Carson SN, French SW. Postoperative carotid restenosis due to neointimal fibrovascular hyperplasia. Radiology 1983; 148:699-702. 4. Ross R, Glomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976; 295:420-5. 5. Takemae T, Bronson J, Alksae JF. Intimal proliferation of cerebral arteries after subarachnoid blood injection in pigs. J Neurosurg 1984; 61:494-500. 6. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ, Brown JT, Lastre CL, Monma D. Microvascular anastomosis using the milfiwatt CO2 laser. Lasers Surg Med 1985; 5:357-65. 7. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ. Histological comparison of suture versus laser-assisted vascular anastomosis. Surg Forum 1985; 36:508-10. 8. Bailes JE, Quigley MR, Kwaan HC, CeruUo LJ, Brown JT. Fibrinolytic activity following laser-assisted vascular anastomosis. Microsurgery 1985; 6:163-8. 9. Acland RD, Tractenberg L. Histopathology of small arteries following experimental microvascular anastomosis. Plast Reconstr Surg 1977; 59:868-75. 10. Movat HZ. Demonstration of all connective tissue elements in a single reaction. Arch Pathol 1955; 60:289-92. 11. Clowes AW, Ryan GB, Breslow JL, Karnovsky MJ. Absence of enhanced intimal thickening in the response of the carotid artery to endothelial injury in hypercholesterolemic rats. Lab Invest 1976; 35:6-17. 12. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab Invest 1983; 49:32733. 13. Quigley MR, Bailes JE, Kwaan HC, Cerullo LJ, Brown JT. Aneurysm formation following low power COs laser-assisted vascular anastomosis. Neurosurgery 1986; 18:292-9. 14. Pribil S, Powers SK. Carotid artery end-to-end anastomosis in the rat using the argon laser. J Neurosurg 1985; 63:771-5.