Morphologic Quantitation of Pseudointima and Effects of

Antiplatelet Drugs on Vascular Prostheses in Goats”

Leslie M. RaInwater, BA, Rochester, Minnesota

Gunnar Plate, MD, Rochester, Minnesota

Peter Glovlczkl, MD, Rochester, Minnesota

Robert C. Bahn, MD, Rochester, Minnesota

Larry H. Holller, MD, Rochester, Minnesota

Michael P. Kaye, MD, Rochester, Minnesota

Since Alexis Carrel first noted an occlusive thrombus propagating from the cut edge of an arterial wall after vascular graft placement [I], numerous investigators have studied the mechanism of both early [Z-4] and late [5-91 occlusion of prosthetic vascular grafts. Because of the inherent thrombogenicity of vascular grafts and the unavoidable mechanical damage that occurs during manipulation of vessels [IO-161, an- tithrombotic agents have been employed to enhance patency in small diameter prostheses [17-211. A sign&ant role in early thrombus formation has been attributed to platelets [2,3,22,23] which are activated by the thrombogenic surface of a graft and by tissue factors generated in the presence of mechanical in- jury to the native vessel [I ,11,12,24,25]. Platelet- inhibiting drugs have been shown to decrease thrombus formation on both prosthetic and autog- enous vein grafts, thus decreasing the rate of early occlusion [18,26-291. In addition to the progression of the underlying disease, myofibroblastic prolifer- ation at the site of the anastomosis is one of the main causes of late graft occlusion [5,7,9,30]. There is now experimental evidence that platelet-derived growth factor plays an important role in this process [31-321. Therefore, it has been postulated that the anti- platelet agent may have a beneficial effect not only on early, but also on late graft patency [17,18,33,34]. Recent data of Bomberger et al [35] indicate that antiplatelet therapy may have a negative effect on endothelial regeneration after intimal injury. There has been no available data thus far on either benefi- cial or detrimental effects of antiplatelet drugs on endothelialization of vascular grafts; therefore, this

From ths Mayo Medical School and the Departments of Surgery and Pa- thology, Mayo Clinic and Foundetlon, Rochester, Minnesota.

* This work has been designated 1984 Prize Essay by a Medical Student by the Society for Cllnical Vasculer Sugery. Leslie M. Rainwater, BA, is the recipient of the prize. l~sqrlnts should be addressed to Michael P. Kaye, MD, Mayo

, MkMesote 56905. Presented at the 12th Annual Meeting of the Society for Clinical Vascular

Surgery, Palm Sprlngs, California, March E-April 1,1984.

volume 148, August 1884

study was designed to investigate endothelialization and development of anastomotic pseudointima on expanded polytetrafluoroethylene (PTFE) grafts and to evaluate the effect of antiplatelet agents on these events.

Material and Methods

Eight female French Alpine goats that weighed 35 to 45 kg were divided into four groups of two goats each for se- quential sacrifice study.* Anesthesia was induced with ether and maintained with halothane. Penicillin G ben- zathine (1.2 million units intramuscularly) and gentamicin (75 mg intravenously) were administered in single doses immediately before surgery. Using aseptic technique, the carotid arteries were exposed bilaterally through a midline incision. Short segments of these arteries were excised, and 10 cm segments of 6 mm diameter expanded PTFE grafts were implanted end to end using running 6-O polypropyl- ene sutures. Two grafts were harvested from each of the two goats killed at 2,4,6, and 8 weeks. Staining the grafts’ surfaces was performed in vivo as described by Caplan et al [36] with injection of 0.5 percent solution of Evans Blue (2 mg/kg intravenously). Three hours later, the goats were anesthetized with pentobarbital(O.5 mg/kg intravenously), the grafts were dissected free, and the carotid arteries proximal to the grafts were cannulated. After flushing with saline solution, the grafts were fixed in situ with buffered glutaraldehyde and formalin (Trump’s solution) at a pressure of 100 mm Hg for 30 minutes to maintain normal artery and graft diameters, and to preserve the endothelial cell morphology in its in vivo state [37-391. Fixation of the grafts was continued at 4°C for 24 hours after removal.

French Alpine goats that weighed 35 to 45 kg were used for the antiplatelet study. The 28 animals surviving the 12 week experimental period were divided into four groups. Group I consisted of eight goats that received no medica- tion; Group II consisted of seven goats that received di- pyridamole, 75 mg orally three times a day beginning 24

l All anirnek received humane care in compliance with the Principles of Laboratory An&a/ Care formulated by the National Society for Medical RessarchendtheGuidefortheCereend~ofLaboretoryAn~~prepared by the National Academy of Sciences and publlshed by National Institutes of Health (NIH Publication 80-23, revised 1978).

195

Rainwater et al

Figure 1. Light photomicrographs of expanded PTFE graft at proximai anastomosis from animals sacrificed 2 weeks (top) and 4 weeks (bottom) postoperatively Arrows indkate the throtius (top) on lumen of the graft and the pseudointima (bottom). a = native artery; p = expanded PTFE graft.

hours before operation and acetylsalicylic acid, 1,300 mg orally one time per day, plus dipyridamole, 75 mg orally three times a day postoperatively; Group III consisted of seven animals that received nifedipine (Procardia,@ sup- plied by Pfizer Laboratories, New York, NY), 30 mg orally three times a day beginning 24 hours preoperatively; and Group IV consisted of six animals that received ibuprofen (Motrin,@ supplied by Upjohn Co., Kalamazoo, MI) 400 mg orally three times a day beginning 24 hours preoperatively. Drug treatment in all groups was continued for 12 weeks. Serum levels of acetylsalicylic acid and ibuprofen were measured 1 and 2 months postoperatively.

Each goat was operated on while under endotracheal anesthesia induced with ether and maintained with halo- thane supplemented with pancuronium. Penicillin G benzathine (1.2 million units intramuscularly) and gen- tamicin (75 mg intravenously) were given preoperatively. The infrarenal segment was dissected free. Five minutes after intravenous administration of 1 mg/kg of heparin, the aorta was clamped, and a 4 cm segment was replaced with 5 cm of 8 mm diameter expanded PTFE vascular graft using running 6-O polypropylene sutures. All surviving goats were sacrificed 12 weeks after surgery, and the grafts were stained in situ using the technique used in the se- quential sacrifice study.

Evaluation: After storage in Trump’s solution over- night, all grafts were opened lengthwise, and photographs were taken. Each photographed graft was enlarged seven times to an 8 by 10 inch color print. After photographic documentation of gross appearance, each graft was divided

into eight longitudinal sections of which three were em- bedded in paraffin and one in plastic to be processed for light microscopy using hematoxylin and eosin, toluidine blue, 0, elastic van Gieson’s and Masson trichrome stains. Selected sections of the remaining four longitudinal sec- tions were examined by transmission and scanning electron microscopy to confirm cell types observed on the light microscopy sections.

For the sequential sacrifice study, all sections processed for light microscopy were reviewed, and histologic de- scriptions of the individual slides were recorded for each of the grafta harvested at 2,4,6, and 8 weeks. Confirmation of histologic cell types was determined by electron mi- croscopy.

For the antiplatelet drug study, all sections for light microscopy were numbered and reviewed without knowl- edge of their group. The thickness of the pseudointima of each longitudinal section was measured and recorded at 272 pm intervals beginning at each anastomosis. Each re- corded thickness was entered into a computer, and the mean thickness was entered into a computer, and the mean thickness of pseudointima was obtained for the total length of the endothelialization from the proximal and distal anastomosis of that section. Further, a mean thickness obtained by averaging the four longitudinal sections was determined for the proximal and distal anastomosis.

The surface area of endothelial cell graft coverage, highlighted with Evans Blue, was outlined with a marking pen on the color print enlargement of the gross specimen. These areas were cross-checked with the measured distance of endothelium from the four longitudinal sections to en- sure their accuracy. The outlined surface areas were traced using a micromeasurement planimetrically programmed computer to calculate the surface area of endothelialization from the proximal and distal anastomoses of each graft. In addition, the mean surface area of endothelialization was calculated for each group. The two-tailed Student’s t test was used to compare and analyze the mean thickness and surface area of ingrowth in all the groups.

Results

Sequential sacrifice study: Figure 1, top is rep- resentative of the four specimens studied at 2 weeks. The native artery adjacent to the expanded PTFE graft was noted to have a disrupted intima, internal elastic lamina, and medial layer at the anastomosis. Endothelial cells covered the disrupted portion of the native vessel but did not extend onto the expanded PTFE graft surface. Increased numbers of myofi- broblasts were seen beneath the endothelial cell coverage adjacent to the expanded PTFE graft. At, the anastomosis, the graft surface contained thrombus composed of red blood cells, mono&es, and polymorphonuclear leukocytes. Little thrombus was noted on the surface of the graft towards its center. At 4 weeks, the specimens continued to have an increased number of myofibroblasts in the medial layer of the native artery adjacent to the anastomosis (Figure 1, bottom). Further, a pseudointima of myofibroblasts covered by endothelial cells extended across the anastomoses onto the luminal surface of the expanded PTFE grafts. The thickness of the pseudointima was greatest at the anastomosis and

196 The American Journal 01 Surgery

Antiplatelet Drugs and Graft Healing

Flgwe 3. Photograph of gross speclmen of graft wtth adjacent anastomotk area from anbnal sacrlfked 12 we&k? postopera- tlvely. New emWh&knn ts m wtttt Eva~ Btue dye. Note Wand of myofltnvbksts cowred with endothellum k, the central potilon. The proximal anastomosls ls on the left,

Flgure 2. Lighf pfwtomhvograph of expmled Pl’FE graft at pmxlmalanestomoslshvman&nalsacrlfked6 weeks(top) and 8 W (bottom) posMperatlve&. Anvw lndkatesPseidWtma. a = natlw artery; p = expanded PTFE grafl.

decreased progressively to a one- to two-cell thick- ness some 2 to 3 mm from the anastomosis. Throm- bus of red blood cells, monocytes, and polymorpho- nuclear leukocytes extended from the distal end of the pseudointima towards the center of the graft. Connective tissue that contained numerous capil- laries and arterioles was seen enveloping the exterior of the graft. The specimens at 6 weeks were similar

F&we 4. Transmbkn electrvn pholo-

= ml cetl; M = rnw. Anvw lndkates en&the- llal cell junctlon.

to those at 4 weeks, except that the thickness of the anastomotic pseudointima, consisting of myofibro- blasts covered with endothelium and the distance of the pseudointima extending from the anastomoses were greater (3 to 5 mm) (Figure 2, top). Increased numbers of monocytes and polymorphonuclear leu- kocytes appeared within the thrombus in the graft lumen. At 8 weeks, the specimens were noted to have an anastomotic pseudointima which continued to increase in thickness and in distance from the anas- tomosis towards the center of the graft (Figure 2, bottom). An occasional island of pseudointima that consisted of myofibroblasts covered by endothelium separate from the pannus ingrowth could be seen grossly (Figure 3) and was confirmed to contain en- dothelial cells and myofibroblasts by transmission electron microscopy (Figure 4). In addition, islands of a single cell layer of endothelium could be seen separate from the pannus ingrowth as shown by scanning electron microscopy (Figure 5) and con-

Vd 148, Augusl1884

Rainwater et al

Figure 5. Scanning electron photomicrograph from animal sacrl- fkxd 12 weeks postoperatively (top). /s/and of a single cell layer of endothelkim (E) Is separate from pannus ingrowth. Scanning electran photomlcrograph of the same Island of endothellum (bottom).

firmed to be endothelial cells by transmission elec- tron microscopy (Figure 6). Connective tissue con- taining numerous capillaries and arterioles was seen incorporating the grafts. There was no capillary in- growth noted within the interstices in any of the control grafts. The luminal third of the grafts con- tained monocytes, myofibroblasts, red blood cells, and occasional polymorphonuclear leukocytes; the middle third was primarily acellular with only an occasional mono&e noted within the interstices; and the outer third of the graft contained red blood cells, monocytes, and an occasional polymorphonuclear leukocyte.

Antiplatelet drug study: Of the 28 goats that survived the 12 week study period, 1 animal in each of the first three groups and 2 animals in Group IV had occluded grafts. These animals were excluded

from the study since our purpose was to document pseudointimal formation in patent grafts. In the control group, the total mean pannus ingrowth at 12 weeks was 11.3 mm. The pannus ingrowth extended a mean of 9.3 mm from the proximal anastomoses and 13.3 mm from the distal anastomoses. The dif- ference, however, was not statistically significant. This group of seven animals had a mean total surface area of endothelialization of 574 mm2 (Figure 7). The mean proximal surface area of endothelialization was 167 mm2 and mean distal surface area was 407 mm2. The mean thickness of neointima at the proximal anastomoses was 110 pm2 and 59 pm2 at the distal anastomoses (Figure 8). Group II animals treated with aspirin and dipyridamole had a mean total surface area of endothelialization of 553 mm2 (Figure 7). The mean proximal surface area of endotheliali- zation was 238 mm2 and the mean distal surface area was 315 mm2. The mean thickness of pseudointima at the proximal anastomoses was 99 pm2 and at the distal anastomoses 67 pm2. None of these data dif- fered significantly from the control group (Figure 8). The Group III animals treated with nifedipine had a mean total surface area of endotheliahzation of 440 mm2 (Figure 7). The mean proximal surface area of endothelialization was 244 mm2 and the mean distal surface area was 196 mm2. The mean thickness of pseudointima at the proximal anastomoses was 106 pm2 and at the distal anastomoses 123 pm2. The distal anastomotic hyperplasia was significantly thicker (p <0.05) than that of either Group I or Group II (Figure 8). The Group IV animals treated with ibuprofen had a mean total surface area of en- dothelialization of 351 mm2 (Figure 7). The mean proximal surface area of endothelialization was 201 mm2 and the distal surface area was 150 mm2. The mean thickness of pseudointima at the proximal anastomoses was 182 pm2. The mean thickness of pseudointima at the distal anastomoses was 184 pm2 and was significantly thicker (p <O.Ol) than that of either Group I or Group II but was not significantly thicker than that of Group III (Figure 8). Mean serum levels of acetylsalicylic acid 1 and 2 months postoperatively were 2.7 and 2.9 mg/dl, respectively. Mean serum levels of ibuprofen varied from a trough level of 7.5 ,clg/ml to a peak level of 30.1 pg/ml in the four animals studied. Nifedipine levels were not de- termined.

Comments

Since early thrombosis and late neointimal hy- perplasia continue to be troublesome for vascular surgeons using small vessel prostheses, a number of pharmacologic interventions have been suggested to alleviate these problems. In an attempt to shed more light on this subject, we designed a study to qualita- tively and quantitatively determine the rate and degree of endothelial coverage and pseudointimal deposition on expanded PTFE grafts placed in the

199 The American Journal ol Surgery

Antiplatelet Drugs and Graft Healing

infrarenal aorta of goats. Furthermore, we adminis- tered platelet-inhibiting drugs to determine whether they have a beneficial or detrimental effect on pseudointimal formation. Sauvage et al [40] reported the rate of pannus ingrowth in vascular prostheses in human subjects, dogs, pigs, and calves. No such studies have been conducted in the goat model, and accordingly, our initial studies were directed at the documentation of the degree and type of graft cov- erage. As delineated by our data, there was progres- sive coverage on the luminal surface of the graft over a 12 week period, at which time the pannus formation had extended a mean of 9.3 mm from the proximal anastomoses and 13.3 mm from the distal anasto- moses. These results from the goat model indicate that this model appears to be comparable to the calf model in terms of the rate of pannus formation. In our control group studies, we were also able to doc- ument that the mean thickness of the proximal pannus was 110.2 pm, whereas the mean thickness of the distal pannus averaged only 58.5 pm. Our re- sults were also similar to those reported by Brook et

q Qroup I, n=7

0 GrouO II. n=f3 I

*Co q Group IV, n=4 Surface

area (mm*)

200

Proximal Oiatal Total

Fm 7. llksn surfsor, area covered by emWheKum 12 wwks waW-a_.

al [41] who undertook a sequential study of expanded PTFE grafts in dogs in which they found neoen- dothelium beginning to develop on the 12th post- operative day with the new intimal lining becoming progressively thicker over the course of an 8 month period. As did Brook et al [41], we noted that only fibroblasts and collagen penetrated the graft and that there was no capillary ingrowth into the graft al- though capillaries and arterioles were present in the external connective tissue. The histologic changes noted in our study were also similar to those noted by Hanel et al [42], who compared two different types of commercially available standard PTFE grafts in dogs. As in their study, at 12 weeks we noted a zone in the midwall of the graft that was relatively acel- lular compared with the inner and outer thirds. We found evidence which suggest that the lack of cellu- larity within the midwall of the expanded PTFE graft is dependent on the internodal distance, which

Thickness cpJ*, loo

Mean proximal

F&m8.MI?8n f.f&hW~plVX~anddlrwgM~

12wnkrpmtoperat/wly. 77h9sk7#eastuhkIndkafesp<O.O5 whencalqm3dwwldllmorarrp!or(4oy,~.Th6~~ indkate~ p < 0.0 1 when compared with either Group I or Group Il.

vdtmw Mu, he#u# 1984 199

Rainwater et al

at 20 to 30 pm, appeared to slow down cellular in- growth. Of great interest in our study was the finding of islands of endothelial growth separate from the anastomotic pannus ingrowth. Although these findings have been noted by others in both the dog and the pig models [43-461, the source of these cells has been a subject of great debate. Stump et al [43] and O’Neal et al [44] have suggested that endothelial cells or their primordial precursors may be borne in blood and randomly stick to the prosthetic surface. We propose the theory that the pannus ingrowth of endothelial cells loosely attaches to the graft, and subsequently, islands slide further downstream, similar in nature to a glacier. The finding in our study of little cellularity in the middle third of the graft and no capillary ingrowth is compatible with either the bloodborne theory of endothelialization or our glacial theory. Of further interest was the fact that in our studies, areas of myofibroblastic pseudointima completely separate from the pannus ingrowth could be seen on the luminal surface in the midportion of the expanded PTFE grafts. Such findings on ex- panded PTFE grafts have not been previously re- ported, although Stump et al [43] and O’Neal et al [44] have noted fibroblasts and smooth muscle cells beneath an island of endothelium in their prepara- tion of Dacron* hubs suspended within a vascular prosthesis. The reports of these investigators suggest that myofibroblasts may also be borne by the blood in nature. Whether the precursor cells of myofibro- blasts or endothelial cells circulating within the blood adhere to the graft surface and subsequently differ- entiate or, as we suggest, migrate in a glacial fashion was not specifically addressed in this study. None- theless, the process of coverage of the graft surface in the goat model was demonstrated to be similar to that in other animal models and hence acts as a firm base for the studies of pharmacologic intervention. Aspirin and dipyridamole have been demonstrated by a number of investigators to prevent early thrombosis and subsequent late graft failure [17,47-501. Oblath et al [50] found a significant de- crease in the development of pseudointimal fibrous hyperplasia in treated animals in a 4 month study employing 4 mm expanded PTFE grafts. Further, Green et al [17], in a double-blind clinical trial using expanded PTFE for above-knee grafts, found a sig- nificant increase in late patency when dipyridarnole and aspirin were used compared with patency in the control group. The data from our study reported herein indicate that aspirin and dipyridamole have no detrimental effect on the healing process of ex- panded PTFE grafts when compared with that of the control group. With the work of Fuster et al [51], Dewanjee et al [26], and Pumphrey et al [27] dem- onstrating that the calcium antagonists, verapamil and nifedipine, were effective in reducing platelet deposition, we chose to employ nifedipine to deter- mine whether or not its effect on platelets would

cause alterations in the rate of healing or deposition of thickness of pseudointima in the grafts studied. The data from our studies indicated a statistically significant increase in anastomotic pseudointimal hyperplasia and a trend toward reduction of endo- thelial coverage in the expanded PTFE grafts in the group given nifedipine when compared with that in our untreated control group and the aspirin and di- pyridamole treated group. It seems, therefore, that caution must be used in recommending nifedipine as an agent to enhance long-term patency of vascular prostheses. In a similar fashion, ibuprofen has been shown to effectively decrease platelet deposition in PTFE grafts in short-term studies [26,28] and has enhanced the 30 day patency rate in both rats and dogs I.21 ,521. Although these studies did demonstrate enhanced patency rates, the degree of graft endo- thelial coverage and the thickness of graft pseu- dointimal hyperplasia were not analyzed in detail. The results of our study suggest a trend toward an increase in anastomotic pseudointimal hyperplasia and a decrease in the rate of endothelial coverage. Because of the small number of animals involved in this study, we believe that before reaching definitive conclusions, further investigational work is needed to properly assess the effects of ibuprofen on long- term patency.

Previous work from our laboratory and others has well documented the beneficial effect of antiplatelet treatment in enhancing early patency and reducing thrombus formation in vascular grafts. The long- term effects of these drugs remain controversial. Our data herein did not demonstrate a decrease in late pseudointimal hyperplasia consequent to antiplatelet drug administration.

Summary

The natural process of endothelialization, pseu- dointimal formation, and connective tissue incor- poration of the expanded PTFE grafts in the goat was documented through histologic examination of specimens harvested at 2,4,6, and 8 weeks. The goats demonstrated a progressive increase in pseudointi- ma1 pannus ingrowth from the anastomoses at a rate of 11.3 mm over a 12 week period. Histologic changes according to time of vascular graft incorporation in the goat model were found to be comparable to those of the dog, pig, and calf models reported in the lit- erature. Platelet-inhibiting drugs, aspirin, dipy- ridamole, nifedipine, and ibuprofen were adminis- tered to goats after replacement of their infrarenal aorta with 5 cm of 8 mm diameter expanded PTFE grafts. The effects of the drugs on graft endothelial- ization and anastomotic pseudointimal formation was compared with those in the untreated control group after 12 weeks. Aspirin and dipyridamole had no detrimental effect on the healing process com- pared with the untreated control group. Studies with nifedipine and ibuprofen did not demonstrate a de-

200 The American Journal of Surgery

Antiplatelet Drugs and Graft Healing

crease in pseudointimal hyperplasia. Antiplatelet treatment resulted in no significant change in the rate of endothelialization of expanded PTFE grafts.

bosis in patients on hemodialysis by lowdose aspirin. N Engl J Med 1979;301:577-9.

21. Claus PL, Gloviczki P, Hollier LH, Kaye MP. Patency of polyte- trafluoroethylene microarterial prostheses improved by ibuprofen. Am J Sum 1982:144:180-5.

Acknowledgmenti We are grateful for the technical assistance of 0. Arlan Hildestad, Michael L. Kapinos, Gerald E. McGrath, Robert Mieras, and Kathy J. To- pham.

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