The Care of Vascular Endothelium in Pediatric Surgery P. B. MANSFIELD, M.D.,* D. G. HALL, M.D.,t G. Di BENEDETTO, M.D.,4 L. R. SAUVAGE,§ A. R. WECHEZAK"
The influence of manipulating blood vessels by clamping, drying, stretching and surgical anastomotic technique on the subsequent integrity of blood vessel endothelium has been studied in the dog. Thrombogenesis at the vessel surface was found in all specimnens where loss of endothelium had exposed subendothelial tissues. Whether thrombotic occlusion of the vessel followed depended upon several local factors including the amount of surface area damaged and volume of blood flow in the vessel. All modes of vessel manipulation studied led to significant endothelial damage. To prevent this damage in the clinical setting all possible methods to avoid the exposure of subendothelial tissues should be used. Endothelial integrity is the cornerstone to success in handling blood vessels and avoiding thrombogenesis. This study suggests that clamping of blood vessels should always be done with a minimum of force, only preventing blood flow, and not crushing the vessel wall. Endothelial surfaces should never be allowed to become dry. Stretching should be avoided as it can lead to thrombosis within intact blood vessels when the endothelium is disrupted. Suture anastomotic techniques should be used which minimize endothelial trauma and thus avoid subendothelial tissue reactions which in turn may jeopardize long-term patency and growth at anastomotic sites. In pediatric cases where growth is anticipated interrupted suture technique should be used. This study suggests that many techniques currently used in vascular surgery may be compromising to short and long-term blood vessel patency.
S PECIAL CARE IS NEEDED in the
handling, clamping
and suturing of blood vessels in the infant and small child. The repair of direct vascular trauma, decompressing shunts for portal hypertension, aortopulmonary shunts and the preservation of distal circulation during extensive tumor surgery are common examples. In many cases, long-term function is dependent on growth at * Chief of Cardiovascular Surgery, Children's Orthopedic Hospital and Medical Center; Director, RCRC; Chief of Cardiovascular Surgery, Providence Medical Center; Clinical Assistant Professor, University of Washington School of Medicine Seattle, Washington. t Attending Surgeon, Children's Orthopedic Hospital and Medic A Center. t Physiology Director, RCRC. § Associate Head of Cardiac Surgery, Children's Orthopedic Hospital and Medical Center; Director, RCRC; Clinical Associate Professor, University of Washington School of Medicine. "Tissue Culture Director, RCRC. Submitted for publication: August 23, 1977.
From the Department of Surgery, Children's Orthopedic Hospital and Medical Center, and the Reconstructive Cardiovascular Research Center (RCRC), Providence Medical Center, Seattle, Washington
anastomotic sites and avoidance of thrombus generated within the vessels. Our previous studies of endothelium and healing of blood vessels'12'1920 suggested that: 1) endothelium is very easily damaged; 2) subendothelial tissue reaction at anastomotic sites is minimized when intact endothelium is present at the margins; and 3) endothelial integrity is necessary to prevent platelet activation and potential intravascular thrombogenesis." This study evaluates the influence of commonly used vascular surgical techniques on endothelial integrity, subendothelial tissue reaction and thrombogenesis in the dog. Materials and Methods Twenty-one mongrel dogs weighing 13-40 kg were used for these studies. Surital was used for induction (17 mg/kg), a cuffed endotracheal tube was inserted and anesthesia maintained with Halothane (0.5-1.0%). Ventilation was provided by a Harvard Respirator (Special Model #624) at a rate of 20/min and a tidal volume of 15 cc/kg. Dissection of the carotid arteries (CA), external jugular veins (EJV), femoral arteries (FA) and femoral veins (FV) was carried out on both sides of the animals, ligating and dividing any branches 4-5 mm away from the vessel. The vessels were kept moist at all times and damage other than required by the protocol was care-
fully avoided. Vascular Clamp Study Potts vascular clamps were used for this study. After dissecting the vessels, marking sutures were placed on the adventitia where the damage would be done. The clamp was left in place for 10 minutes, then released, re-establishing the blood flow through the damaged vessel (Fig. 1). No anticoagulants were used. Two different clamping pressures were used: 1) using
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minimum occlusive force (the minimum force necessary to stop blood flow through the vessel) and 2) full occlusive force of the clamp. Twenty-seven arteries and 27 veins were used to evaluate the damage done by the full occlusive and 30 arteries and 30 veins for the minimum occlusive. Specimens were removed after ten minutes, one hour, three hours, six hours, 12 hours, one day, two days, one week, two weeks and four weeks.
Endothelial Drying Study For this study, 12 vessels were used (six arteries and six veins). After dissecting the vessel, two Potts clamps, using minimum occlusive force were applied to the vessel. Two 2 cm incisions were made and the vessel was irrigated with Hanks Balanced Salt Solution (HBSS) without Ca++ and Hg++ to remove blood elements. Retracting stitches (6-0 Prolene) were placed in the adventitia at each side of the incisions to keep the vessels open (Fig. 1) and the proximal incision was left to dry at room temperature while the distal one was irrigated at all times with HBSS and a moist lap sponge was kept over the exposed section. To prevent the moisture from spreading to the dry section, a third clamp was placed between the two sections. After up to one hour, the incisions were closed with a continuous 6-0 Prolene suture, the clamps released, and the blood flow re-established for two days to one week.
Stretching Study Seven arteries and 7 veins used for this study were carefully dissected and a 2 cm section of each was stretched between fingers to one and a half to two times its original length. Specimens were retrieved at two days and one week. Fogarty Stripping A Fogarty embolectomy catheter (8-10 Fr.) with a 5 cc balloon was used to strip the intimal surface of six arteries and six veins. After dissecting the vessels, a Potts vascular clamp was placed on the proximal site to stop blood flow and a 0.5-1.0 cm incision made longitudinally on the vessel. The catheter was advanced into the vessel for 5 cm, the balloon inflated and the catheter pulled out. The vessel was then irrigated with HBSS to remove debris. The incision was closed with a 6.0 Prolene suture. Specimens were obtained at two days and one week. Anastomotic Techniques Study End-to-end anastomosis of a segment of femoral vein to the carotid arteries was performed in four paired
FIG. 1. Drawing of manipulations of blood vessels used in this study. At the left, a vessel is cross-clamped between marking sutures which are used for later site identification. On the right are the techniques used to compare the effects of air drying on endothelium. One opening is kept continually moist, the other is allowed to dry. The central clamp prevents fluid spillover into the drying area.
studies. End-to-side anastomosis of a segment of femoral vein around a ligated carotid artery was performed in three paired studies. Each study consisted of two shunts, one in each carotid artery. Two different anastomotic techniques were used (Fig. 2). In one carotid artery a retracting stitch was placed in the adventita at the tip of the vein. Further handling of the vessel during the two anastomoses was limited to very careful use of forceps on only the adventitia. Care was taken to avoid touching the intimal surface of the vessel with any instrument ("no touch" technique). In the other carotid artery, the vein was handled with forceps only while the anastomoses were being performed. The full wall thickness was grasped with the forceps including the intimal surface. However, handling of the vessel was kept to the minimum necessary for the performance of a good anastomosis. Throughout these procedures, care was taken to avoid drying or undue tension on the vessel. Both the femoral vein and the carotid artery section were frequently irrigated with Mg++- and Ca++-free HBSS. All anastomoses were performed using a 6-0 Prolene running suture as growth was not studied during these shortterm experiments.
SacrificelSpecimen Preparation At the time of sacrifice, the animals again underwent Surital (17 mg/kg) induction, intubation and maintenance with Halothane anesthesia. The incision sites were reopened and the vessels carefully exposed. Three min-
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48-72 hours at 40 and then to 0. IM sodium cacodylate buffer at 4°. The specimens were held in 0. IM sodium cacodylate buffer at 40 for a minimum of one hour until critical point drying was done in a Bowmar SP 900 unit. The specimens were then coated with a 300 A layer of carbon and gold palladium. The specimens were studied in a Cambridge Stereoscan Mark II-A electron microscope. Photographs were taken with Polaroid 55 P/N film. We found that great care was necessary during the preparation of SEM specimens to minimize "processing artifacts." Handling of specimens was kept at a minimum and surface drying was carefully avoided. Results
FIG. 2. Drawing of the two different anastomotic techniques evaluated in this study. Insert: "No touch" technique where an adventitial suture is placed for movement control of the open end of the vessel. During suturing, only the adventitia is handled with forceps, the intima is never touched (middle right). At the lower left is a commonly used technique where the entire vessel wall, both adventitial and intimal surfaces, are handled with forceps during suturing.
utes prior to removal of the specimens, 2 mg/kg of
sodium heparin was administered intravenously. Immediately after excision, the specimens were placed in Ca++- and Mg++-free HBSS and thoroughly irrigated to remove extraneous blood. The specimens were then placed in fresh Hanks, rinsed again, opened and photographed. Each specimen was then processed for both histologic and scanning electron microscopic (SEM) evaluation. Half of each specimen was fixed in 10% buffered formalin for histologic processing, sectioned and stained with hematoxylin and eosin. The other half was processed for scanning electron microscopy. The SEM sections were fixed in 6.25% glutaraldehyde in 0. 1M sodium cacodylate buffer for six to 24 hours at 4°. After fixation, the specimens were transferred to 7.5% sucrose in 0. 1M sodium cacodylate buffer for
Vascular Clamp Study The Potts vascular clamp used in this study caused significant endothelial damage within all vessels to which it was applied. In every vessel the endothelial layer was disrupted and the subendothelial tissues exposed. It was not possible to determine if the intima was cut and retracted or whether the clamp crushed the cells to expose subendothelial tissues. Actual crush injury to the remainder of the vessel wall varied directly with wall thickness. Hence, the carotid artery showed the greatest amount of crush injury, the femoral artery less, and the veins the least at any given clamping force. Minimum Occlusive Force A typical example of the damage done when minimal occlusive force was used is shown in Figures 3-5. The endothelial loss was almost circumferentially complete in most specimens. Areas of minimal damage were occasionally found, especially in venous specimens where in two specimens a short portion of intact endothelial surface was found (Fig. 3, c). The ridging seen on this arterial endothelium is related to the underlying internal elastic lamina, and is found when specimens have not been fixed at normal vascular pressures.5 The regenerating endothelium however does not form ridges and this suggests that the inner elastic lamina has also been damaged. H&E sections confirmed that damage to the inner elastic lamina was invariably present with the clamp used. The response of platelets, white blood cells and fibrin production (P-W-F response) to the exposed subendothelial tissues was similar to reports of others."12'4'8'9'14'18 Initially, platelets, then WBC attachment followed by fibrin formation occurred within the first hour after clamp release. A dynamic balance of thrombus generation and removal reached equilibrium usually by
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FIG. 3. (left) Scanning electron micrograph (SEM) of endothelial surface of canine femoral artery two days after a ten minute application of a Potts clamp (two locations) at minimum occlusive force. There has been discrete loss of endothelium in the distribution of the clamp blade. (right) Line drawing of SEM at left, showing the two areas where the clamp was applied A and B. Dotted square area is enlarged in Figures 4 and 5. C is an area of nondisrupted endothelium frequently seen in minimum occlusive specimens.
24 hours. The depth and degree of this thrombogenic response varied and appeared to be influenced by both rheologic factors as well as the surface area of exposed subendothelial tissue. Thrombus depth was less in high
flow, high velocity vessels (arteries) and more prominent in lower velocity vessels (veins). Since the clamp did less damage to the veins than to the arteries, quantitative evaluation of this relationship was not possible.
FIG. 4. (left) SEM of endothelial damage from minimum force clamping after two days (from Fig. 4B). There has been a clean separation of endothelial margins which tapers toward the tip of the clamp where an undamaged area is present at the bottom (x 108). (right) Line drawing of SEM at left. Dotted square shows area seen at higher magnification in Figure 5. DM: damage margin. ER: endothelial ridges seen in non-pressurized arterial specimens.5
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FIG. 5. (left) SEM of area damaged as in Figures 3 and 4. At only two days, there is almost complete covering of the previously denuded area by regenerating endothelial cells. Only occasional WBC and platelets are seen where covering is not quite complete. Surviving endothelium at the damage margins shows no evidence of platelet adhesion or aggregation (x542). (right) Line drawing of SEM at left to identify structures. DM: damage margins (surviving endothelial margins). RE: regenerating endothelial cells. WBC: white blood cells.
Regeneration of endothelium occurred from the margins of the surviving endothelium. After minimum occlusive clamping, cells began to cross the exposed subendothelial tissue as early as 12 hours after injury. It was often complete by 48 hours with minimal, if any, evidence of residual P-W-F response found in the area. The new endothelial covering consisted of a flattened monolayer of endothelial cells which did not form ridging on fixation without pressurization (Fig. 5). Surviving endothelial cells at the damage margin had also regained full antithrombogenic capability and platelets and WBC were no longer seen on their surface. Maximum Occlusive Force When the maximum occlusive clamp force was applied to a vessel, the endothelial damage was severe (Fig. 6). The depth of damage varied with vessel wall thickness, being greatest in the thicker walled arteries. In addition to the damage due to crush in the cleft area, endothelial disruption back from the edge of the clamp site was also found (Fig. 6, ED). Subsequent experiments suggest this was caused by local stretching of endothelial surface. Within the cleft, active thrombus generation was always found, until complete endothelial covering had occurred. The response of platelets, WBC and fibrin generation (P-W-F) was similar in time course to the minimum occlusive studies. However, the degree of response
was much greater, with more thrombus generated and the reaction extended well back from the clamp damage site. Endothelium 1-3 mm away had attached platelets and WBC's with increasing incidence ofthrombus present as one scanned toward the cleft. An equilibrium state of thrombus generation and removal was reached later than in the minimum occlusion studies. Equilibrium was rarely found before 48 hours after injury. Endothelial regeneration started from the surviving endothelial cells at the damage margins, but was not well defined at 48 hours (Fig. 7). Surviving cells at the damage margin showed evidence of platelet and WBC attachment. Endothelial cells were found bridging between adjacent ridges. At seven days (Fig. 8) most of the specimens showed a complete covering of the previously exposed subendothelial tissues and no surface reaction with platelets or WBC. Surviving endothelial cells at the damage margin had regained full antithrombogenic capability by the seventh day in all specimens.
Endothelial Drying Study In this study, the endothelial surface of a vein or artery was exposed to the air until dry, the vessel repaired and blood flow restarted. A continuing reaction led to severe endothelial cell damage. Two days following drying, from 50 to 100% of the endothelium had become necrotic or detached and an intense WBC and
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FIG. 6. (left) SEM of endothelial surface of canine carotid artery one week after a ten minute application of a Potts clamp at full occlusive power. There remains a deep cleft in the vessel wall with some regenerating endothelial cells at the damage margins. The majority of the cleft remains denuded of endothelium, with platelet, WBC and fibrin complexes on the surface (see Fig. 11 and compare damage to minimum occlusive force damage in Fig. 3) (x45) (right) Line drawing to identify structures in SEM at left. DC: damage cleft. ED: endothelial damage, now regenerated, back from the cleft. RE: regenerated endothelium.
mild platelet reaction covered the exposed subendothelial tissues. Figure 9a shows the response as seen under the light microscope. In the SEM photo of the same specimen there are dead and dying endothelial
cells covered with WBC's (lower right) and some individual platelets. Just above and to the left of center is a group of endothelial cells with less damage and fewer WBC's on the surface. Endothelial regeneration
FIG. 7. (left) SEM of endothelial surface of canine femoral artery two days after a ten minute application of a Potts clamp at maximum occlusive force. Endothelial regeneration at the damage margin has begun slowly (compare to Fig. 5) and WBC and platelet adhesion to the surviving endothelial margins suggests cellular damage there. Platelets, WBC and fibrin cover the exposed subendothelial tissues (x522). (right) Line drawing to identify structures in SEM at left. RE: regenerating endothelium. P-W-F-: platelets, WBC and fibrin complexes on surface of subendothelial tissues. PL: Platelet attachment on cells at surviving endothelial margin DM.
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FIG. 8. (left) SEM of endothelial surface of canine femoral artery, seven days after a ten minute application of a Potts clamp at full occlusive force. Endothelial regeneration over the subendothelial tissues is complete and the surface no longer attracts platelets, WBC and fibrin. The surviving endothelial cells at the damage margins are also once again nonthrombogenic at their surface (compare to Figures 4-6) (x 108). (right) Line drawing to identify structures in SEM at left. RE: regenerated endothelium. DM: surviving endothelial cells at damage margin.
FIG. 9a. Light photomicrograph of canine jugular vein two days after endothelial surface was exposed to drying in air. There is a marked infiltrate of white blood cells at the vessel surface (lumen above). No intact endothelial cells can be recognized (x200).
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FIG. 9b. SEM of surface of same specimen Figure 9a. Massive WBC adhesion and single platelet adhesion without aggregation is seen. Central area of less severe cell damage has fewer WBC
(x505).
had begun in one-week specimens but due to the large surface area damaged, covering was not complete within the seven day experimental time period. In contrast were the findings when the exposed surface had been kept moist with Hanks balanced salt solution. In Figure 9c the endothelium is seen to be intact without surface reaction. The moist surface results were identical to control studies in which the vessels had not been handled before specimen removal and had only been in contact with blood. Stretching Study These studies demonstrated that longitudinal stretching of intact arteries or veins to one and one-half to two times their own length (even in very short segments) led to endothelial damage (Fig. 10). Splitting of endothelium with damage margin retraction (greater in arteries than in veins) occurred at right angles to the longitudinally applied force. Large areas of subendothelial tissues were exposed and led to a marked platelet, WBC and fibrin reaction on the surface. Despite
this exposed surface area, all veins and arteries remained patent and the surface reactions did not encroach significantly on the lumen.
Fogarty Catheter Study These studies demonstrated, as have others,3 that passing an inflated balloon catheter down either a vein or an artery resulted in removal and/or destruction of all the endothelial cells in that segment. The following surface reaction was intense and led to complete thrombotic occlusion in two of six veins and one of six arteries. In the vessels which remained patent, active thrombogenesis was seen on the exposed subendothelial tissues at two days and seven days (Fig. 1 1). Endothelial regeneration which began at the damage margins was not complete at 1 week following the injury. Anastomotic Techniques Study Both a common vascular anastomotic technique and the "no touch" technique (Fig. 2) worked satisfactorily
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FIG. 9c. Light photomicrograph of canine jugular vein two days after endothelial surface was exposed but kept moist in Hanks balanced salt solution. Same animal and vein as in (Figures 9a and b) (lumen above). The endothelium is intact, without evidence of degeneration or surface reaction. Result is identical to histology of control veins never opened and always exposed to blood (x200).
and all anastomoses were patent. Once the "no touch" technique had become familiar to the surgeon, there was no time difference for completing the anastomosis with either technique. Initial end-to-side studies were changed to end-to-end studies when thrombus was found in the blind ends of the carotid artery which had been ligated to divert blood through the vein graft. Thrombus was frequently seen at the anastomosis using the common vascular technique. Scanning electron microscopy revealed significant differences in the anastomotic surfaces depending on the technique used. Handling the vein with forceps which touch both sides of the wall (common technique) (Fig. 12) led to disruption of the venous endothelium, exposure of subendothelial tissues and local thrombus formation at seven days. The "no touch" technique which avoids touching the endothelial surface did not disrupt the venous endothelial surface and smooth even healing of venous and arterial endothelium at seven days was the rule (Fig. 13).
Discussion
Work which led to the tissue culture techniques needed to maintain and serially passage endothelial cells in tissue culture demonstrated the extremely delicate nature of these cells.20 These cultured cells were grown as a monolayer on the surface of a nonporous prosthetic graft which was inserted into the descending aorta of the autologous calf. No significant reaction occurred in the subendothelial tissues at the aorta to graft anastomosis, and no thrombus was found on the intact surface. Without the endothelial cell covering, a marked subendothelial reaction occurred in the aorta and a heavy layer of thrombus was generated on the surface of the graft. It appeared that the endothelial lining of the artificial graft, which is a monolayer and does not have subendothelial tissues, had, by contact inhibition, stopped the usual tissue reaction in the aortic wall.12 Techniques to culture human endothelial cells were subsequently developed,19 but as in the animal cultures,
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FIG. 10. (left) SEM of endothelial surface of canine femoral artery two days after stretching of the segment to 1.5-2 times its original length. There has been severe splitting and retraction of the endothelium with exposure of large areas of subendothelial tissues. Vessel was not opened at the time of stretching. (right) Line drawing to identify structures in SEM at left. DM: retracted damage margins. SE: exposed subendothelial tissues. $ shows direction of stretching force originally applied.
the cells were found to be easily damaged and finicky about their environment. It was the combination of no subendothelial tissue reaction, when the endothelium was intact, and the ease with which these specialized cells could be damaged that led us to study the effects
of commonly used surgical techniques on endothelial integrity and subendothelial tissue reaction. Postoperative growth, patency and function of anastomoses appear to depend upon the prevention of subendothelial tissue exposure and subsequent thrombogenesis.
FIG. I1. (left) SEM of endothelial surface of canine femoral artery seven days after Fogarty catheter was passed through the area. The vessel patent. This high magnification photo is an excellent example of the platelet, WBC and fibrin reaction which occurs at the surface of subendothelial tissues when exposed by loss of their endothelial covering (x2035). (right) Line drawing to show structures in SEM at left. PL: platelets. F: fibrin. WBC: white blood cells. was
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FIG. 12. (left) SEM of an end-to-side anastomosis between canine femoral vein (above) and carotid artery (below) seven days after surgery. Usual vascular techniques of holding both sides of the vein wall with forceps during suture placement were used (see Fig. 2, lower left). The surface of the vein (above the suture line) is disrupted and folded back endothelium with surface thrombus is present. Large areas of exposed subendothelial tissues remain (x22). (right) Line drawing to identify structures in SEM at left. '-SL--*: suture line. SE: exposed subendothelial tissues. DE: disrupted endothelium. AE: arterial endothelium. VE: venous endothelium.
One of the most basic functions of endothelium is to prevent exposure of the subendothelial tissues to
circulating blood elements." Once the tissues are exposed, thrombogenesis is initiated and the stage is set for variable degrees of subsequent thrombus generation.'5 The loss of endothelium also appears to precede intimal hyperplasia and subendothelial scarring.8"6 This subendothelial reaction may account for the lack of growth at some vascular anastomoses in infants and children. This study demonstrated that physical trauma, identical to that occurring during vascular surgical procedures, can result in complete loss of endothelium in the damaged area. '5"17 The clamps used in these studies imparted both cutting and crushing injuries to blood vessel walls. The endothelium was always damaged. The remaining wall damage was proportional to wall thickness and clamp force (Figs. 3, 5-7). The greater the clamping force, the longer it took for endothelial regeneration and therefore the longer the time active thrombogenesis was present. Cells adjacent to the damage margins were found to be the source of regenerating endothelium. When the injury was severe, the start of endothelial regeneration was delayed. The surface area of damage was also greater with increasing clamp force, so the time to complete endothelial covering was further prolonged in these animals.
In addition to clamp damage, drying of endothelial surfaces led to subsequent endothelial desquamation and a particularly intense leukocyte response. Platelets were present on the vessel surface but usually singly and without evidence of aggregation and the release reaction.4 Since the arterial clamp studies did not show ridging in the area of injury, (Fig. 4) (nonpressurized fixation) we assumed, and histologic sections confirmed, the loss of the internal elastic lamina. In contrast, the injury caused by drying did not involve loss of the elastic lamina as shown by histologic sections (Fig. 9). Ridging was seen in surfacedried (unpressurized fixation) arterial specimens suggesting the internal elastic lamina was functionally intact.6'8 Platelets adhere to minimal damage areas on endothelial surfaces but have not been found to undergo aggregation and the release reaction at these sites. Aggregation and release has not been seen on the elastic lamina" either. Thus, the damage from drying may not lead to complete exposure of subendothelial tissues. Long-term studies need to be done to evaluate whether chronic damage can occur with an intact inner elastic lamina. Reversible intimal thickening is known to occur between the inner elastic lamina and the vessel lumen in rat carotid arteries.6 Stretching caused endothelial disruption which included the internal elastic lamina (see absence of
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VE SL
AE FIG. 13. (left) SEM of an end-to-side anastomosis between canine femoral vein (above) and carotid artery (below) seven days after surgery. Same animal was as in Figure 12. The "no touch" technique of anastomosis was used. The venous endothelium is intact, there is no exposed subendothelial tissue. Note the difference in surface texture between fixed arterial and fixed venous endothelium. The arterial surface is corrugated with ridges while the venous surface appears as a smooth cobblestone surface. Arterial surfaces become smooth if fixed at above normal diastolic pressures (x22).5 (right) Line drawing to identify structures in the SEM at the left. SL: suture line. VE: venous endothelium. S: suture. AE: arterial endothelium.
ridging in Fig. 10). The usual reaction to exposed subendothelium occurred with platelet, WBC and fibrin formation on the surface. Fogarty balloon endothelial stripping removed both endothelium and the internal elastic lamina in some arteries but only the endothelium in others. When both were removed a marked platelet, WBC and fibrin reaction was seen (Fig. 11). When only the endothelium was removed, single adherent but nonaggregated platelets were seen without significant numbers of WBC's or fibrin. Several other modes of endothelial damage not evaluated in this study are known.7"3"5"7 Whether thrombus progresses to occlusion of the lumen is dependent on several factors16 among which are: blood flow and turbulence,10 fibrinolytic activity,'2 rate of endothelial regeneration, presence of anticoagulants, and the surface area of exposed subendothelial tissues. Embolic phenomena have not been evaluated during this study. The clinical implications of these findings may have significance especially for the infant and child. It takes less thrombus to occlude a small vessel than a large vessel. Damage which leads to subendothelial proliferation may influence later development of a vessel or growth at the site of anastomoses. To avoid damage and create the best environment for later growth one should use all means possible to prevent endothelial damage. Some methods suggested
by our findings include: 1) minimal clamping pressure to stop flow in the vessel; 2) avoid drying of endothelial surfaces; 3) avoid stretching of vessels including tension at suture lines; 4) use the minimum inflation pressure needed to effect balloon embolectomies; 5) use or devise interrupted suture anastomotic techniques which minimize endothelial trauma. Summary The influence of clamping, drying, stretching and surgical anastomotic technique on the subsequent integrity of blood vessel endothelium has been studied in the dog. Thrombogenesis at the vessel surface was found in all specimens where loss of endothelium had exposed subendothelial tissues. Whether thrombotic occlusion of the vessel followed depended upon several local factors including surface area damaged and blood flow in the vessel. All methods of influence studied led to significant endothelial damage and suggested the following clinical implications: 1) Endothelial integrity is the cornerstone to success in handling blood vessels and avoiding thrombogenesis-prevention of exposure of subendothelial tissues is the goal. 2) Clamping of blood vessels should always be done with a minimum of force.
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3) Endothelial surfaces should never be allowed to become dry. 4) Stretching can lead to thrombosis within intact blood vessels when the endothelium is disrupted. 5) Interrupted suture anastomotic techniques should be used which minimize endothelial trauma and thus avoid subendothelial tissue reactions which in turn may jeopardize long-term patency and growth at anastomotic sites.
8.
9.
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11.
Acknowledgments The authors are particularly grateful to Paulette Uto and Wendy Miller for their expert technical assistance during these experiments, Arnold Schmidt at the Materials Analysis Center of the University of Washington for his operation of the electron microscope, Marena Slatt for help in preparing the manuscript and Trese Rand for her illustrative interpretation of techniques and results.
References 1. Ashford, T. P. and Freiman, D. G.: Platelet Aggregation at Sites of Minimal Endothelial Injury: An Electron Microscopic Study. Am. Jour. Pathol. 53:599, 1968. 2. Baumgertner, H. R.: The Subendothelial Surface and Thrombosis. Throm. Diath. Halmorth. Suppl., 59:91, 1974. 3. Baumgartner, H. R.: Sterierman, M. B. and Spaet, T. H.: Adhesion of Platelets to Subendothelial Surface: Distinct from Adhesion to Collagen. Experientia, 27:283, 1971. 4. Born, G. V. R.: Current Ideas on the Mechanism of Platelet Aggregation. Ann. N.Y. Acad. Sci, 201:4, 1972. 5. Clark, J. M. and Glagov, S.: Luminal Surface of Distended Arteries by Scanning Electron Microscopy: Eliminating Configurational and Technical Artifacts. Br. J. Exp. Pathol., 57:129, 1975. 6. Clowes, A. W., Ryan, G. B., Breslow, J. L. and Karnovsky, M. J.: Absence of Enhanced Intimal Thickening in the Response of the Carotid Arterial Wall to Endothelial Injury in Hypercholesterolemic Rats. Lab. Invest., 35:6, 1975. 7. Elemer, G., Kereny, T. and Jekkinek, H.: Scanning (SEM) and Transmission (TEM) Electronmicroscopic Studies on Post-
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ischemic Endothelial Lesions Following Recirculation. Atherosclerosis, 24:219, 1976. Fishman, J. A., Ryan, J. B. and Karnosvsky, M. J.: Endothelial Regeneration in the Rat. Carotid Artery and the Significance of Endothelial Denudation in the Pathogenesis of Myintimal Thickening. Lab Invest., 32:339, 1975. Gertz, S. D., Rennels, M. L., Forbes, M. S. et al.: Endothelial Cell Damage by Temporary Arterial Occlusion with Surgical Clips. J. Neurosurg., 45:514, 1976. Leonard, E. F.: The Role of Flow in Thrombogenesis. Bull. N.Y. Acad. Med., 48:273, 1972. Mansfield, P. B., Wechezak, A. R., Di Benedetto, G. and Sauvage, L. R.: Antithrombogenic Functions of the Endothelium. NIH, NHLBI Symposium. In Vascular Grafts: Current Status and Future Trends. New York, AppletonCentury Crofts, Sawyer, P. M. and Kaplitt, M. J. (ed.) pg 130-146, 1978. Mansfield, P. B., Sauvage, L. R., and Smith, J. C.: Factors Influencing Thrombus Generation in Artificial Hearts. In Coronary Artery Medicine and Surgery: Concepts & Controversies. New York, Appleton-Century-Crofts, 1975, p. 968. Ramos, J. R., Berger, K., Mansfield, P. B. and Sauvage, L. R.: Histologic Fate and Endothelial Changes of Distended and Nondistended Vein Grafts. Ann Surg., 183:205, 1976. Sheppard, B. L. and French, J. E.: Platelet Adhesion in the Rabbit Abdominal Aorta Following the Removal of the Endothelium: A Scanning and Transmission Electron Microscopical Study. Proc. R. Sec. Lond. B., 176:427, 1971. Slayback, J. B., Bowen, W. W. and Hinshaw, D. B.: Intimal Injury from Arterial Clamps. Am. J. Surg., 132: 183, 1976. Spaet, T. H., Gaynor, E. and Stemerman, M. B.: Thrombosis, Atherosclerosis and Endothelium. Am. Heart J., 87:661, 1974. Stoner, G. E., Chisolm, G. M., Srinivasan, S., et al.: Vascular Injury and Thrombosis; A Scanning Electron Microscopic Study. Thrombosis Res., 4:699, 1974. Ts'ao, C.: Graded Endothelial Injury of the Rabbit Aorta with Special Reference to Platelet Desposition. Arch. Pathol., 90:222, 1970. Wechezak, A. R., Mansfield, P. B. and Way, S. A.: Platelet Interaction with Cultured Endothelial Cells Following In Vitro Injury. Artery, 1:508, 1975. Wechexak, A. R. and Mansfield, P. B. Isolation and Growth Characteristics of Cell Lines from Bovine Venous Endothelium. In Vitro, 9:39, 1973.
The influence of manipulating blood vessels by clamping, drying, stretching and surgical anastomotic technique on the subsequent integrity of blood vessel endothelium has been studied in the dog. Thrombogenesis at the vessel surface was found in all specimnens where loss of endothelium had exposed subendothelial tissues. Whether thrombotic occlusion of the vessel followed depended upon several local factors including the amount of surface area damaged and volume of blood flow in the vessel. All modes of vessel manipulation studied led to significant endothelial damage. To prevent this damage in the clinical setting all possible methods to avoid the exposure of subendothelial tissues should be used. Endothelial integrity is the cornerstone to success in handling blood vessels and avoiding thrombogenesis. This study suggests that clamping of blood vessels should always be done with a minimum of force, only preventing blood flow, and not crushing the vessel wall. Endothelial surfaces should never be allowed to become dry. Stretching should be avoided as it can lead to thrombosis within intact blood vessels when the endothelium is disrupted. Suture anastomotic techniques should be used which minimize endothelial trauma and thus avoid subendothelial tissue reactions which in turn may jeopardize long-term patency and growth at anastomotic sites. In pediatric cases where growth is anticipated interrupted suture technique should be used. This study suggests that many techniques currently used in vascular surgery may be compromising to short and long-term blood vessel patency.
S PECIAL CARE IS NEEDED in the
handling, clamping
and suturing of blood vessels in the infant and small child. The repair of direct vascular trauma, decompressing shunts for portal hypertension, aortopulmonary shunts and the preservation of distal circulation during extensive tumor surgery are common examples. In many cases, long-term function is dependent on growth at * Chief of Cardiovascular Surgery, Children's Orthopedic Hospital and Medical Center; Director, RCRC; Chief of Cardiovascular Surgery, Providence Medical Center; Clinical Assistant Professor, University of Washington School of Medicine Seattle, Washington. t Attending Surgeon, Children's Orthopedic Hospital and Medic A Center. t Physiology Director, RCRC. § Associate Head of Cardiac Surgery, Children's Orthopedic Hospital and Medical Center; Director, RCRC; Clinical Associate Professor, University of Washington School of Medicine. "Tissue Culture Director, RCRC. Submitted for publication: August 23, 1977.
From the Department of Surgery, Children's Orthopedic Hospital and Medical Center, and the Reconstructive Cardiovascular Research Center (RCRC), Providence Medical Center, Seattle, Washington
anastomotic sites and avoidance of thrombus generated within the vessels. Our previous studies of endothelium and healing of blood vessels'12'1920 suggested that: 1) endothelium is very easily damaged; 2) subendothelial tissue reaction at anastomotic sites is minimized when intact endothelium is present at the margins; and 3) endothelial integrity is necessary to prevent platelet activation and potential intravascular thrombogenesis." This study evaluates the influence of commonly used vascular surgical techniques on endothelial integrity, subendothelial tissue reaction and thrombogenesis in the dog. Materials and Methods Twenty-one mongrel dogs weighing 13-40 kg were used for these studies. Surital was used for induction (17 mg/kg), a cuffed endotracheal tube was inserted and anesthesia maintained with Halothane (0.5-1.0%). Ventilation was provided by a Harvard Respirator (Special Model #624) at a rate of 20/min and a tidal volume of 15 cc/kg. Dissection of the carotid arteries (CA), external jugular veins (EJV), femoral arteries (FA) and femoral veins (FV) was carried out on both sides of the animals, ligating and dividing any branches 4-5 mm away from the vessel. The vessels were kept moist at all times and damage other than required by the protocol was care-
fully avoided. Vascular Clamp Study Potts vascular clamps were used for this study. After dissecting the vessels, marking sutures were placed on the adventitia where the damage would be done. The clamp was left in place for 10 minutes, then released, re-establishing the blood flow through the damaged vessel (Fig. 1). No anticoagulants were used. Two different clamping pressures were used: 1) using
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minimum occlusive force (the minimum force necessary to stop blood flow through the vessel) and 2) full occlusive force of the clamp. Twenty-seven arteries and 27 veins were used to evaluate the damage done by the full occlusive and 30 arteries and 30 veins for the minimum occlusive. Specimens were removed after ten minutes, one hour, three hours, six hours, 12 hours, one day, two days, one week, two weeks and four weeks.
Endothelial Drying Study For this study, 12 vessels were used (six arteries and six veins). After dissecting the vessel, two Potts clamps, using minimum occlusive force were applied to the vessel. Two 2 cm incisions were made and the vessel was irrigated with Hanks Balanced Salt Solution (HBSS) without Ca++ and Hg++ to remove blood elements. Retracting stitches (6-0 Prolene) were placed in the adventitia at each side of the incisions to keep the vessels open (Fig. 1) and the proximal incision was left to dry at room temperature while the distal one was irrigated at all times with HBSS and a moist lap sponge was kept over the exposed section. To prevent the moisture from spreading to the dry section, a third clamp was placed between the two sections. After up to one hour, the incisions were closed with a continuous 6-0 Prolene suture, the clamps released, and the blood flow re-established for two days to one week.
Stretching Study Seven arteries and 7 veins used for this study were carefully dissected and a 2 cm section of each was stretched between fingers to one and a half to two times its original length. Specimens were retrieved at two days and one week. Fogarty Stripping A Fogarty embolectomy catheter (8-10 Fr.) with a 5 cc balloon was used to strip the intimal surface of six arteries and six veins. After dissecting the vessels, a Potts vascular clamp was placed on the proximal site to stop blood flow and a 0.5-1.0 cm incision made longitudinally on the vessel. The catheter was advanced into the vessel for 5 cm, the balloon inflated and the catheter pulled out. The vessel was then irrigated with HBSS to remove debris. The incision was closed with a 6.0 Prolene suture. Specimens were obtained at two days and one week. Anastomotic Techniques Study End-to-end anastomosis of a segment of femoral vein to the carotid arteries was performed in four paired
FIG. 1. Drawing of manipulations of blood vessels used in this study. At the left, a vessel is cross-clamped between marking sutures which are used for later site identification. On the right are the techniques used to compare the effects of air drying on endothelium. One opening is kept continually moist, the other is allowed to dry. The central clamp prevents fluid spillover into the drying area.
studies. End-to-side anastomosis of a segment of femoral vein around a ligated carotid artery was performed in three paired studies. Each study consisted of two shunts, one in each carotid artery. Two different anastomotic techniques were used (Fig. 2). In one carotid artery a retracting stitch was placed in the adventita at the tip of the vein. Further handling of the vessel during the two anastomoses was limited to very careful use of forceps on only the adventitia. Care was taken to avoid touching the intimal surface of the vessel with any instrument ("no touch" technique). In the other carotid artery, the vein was handled with forceps only while the anastomoses were being performed. The full wall thickness was grasped with the forceps including the intimal surface. However, handling of the vessel was kept to the minimum necessary for the performance of a good anastomosis. Throughout these procedures, care was taken to avoid drying or undue tension on the vessel. Both the femoral vein and the carotid artery section were frequently irrigated with Mg++- and Ca++-free HBSS. All anastomoses were performed using a 6-0 Prolene running suture as growth was not studied during these shortterm experiments.
SacrificelSpecimen Preparation At the time of sacrifice, the animals again underwent Surital (17 mg/kg) induction, intubation and maintenance with Halothane anesthesia. The incision sites were reopened and the vessels carefully exposed. Three min-
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48-72 hours at 40 and then to 0. IM sodium cacodylate buffer at 4°. The specimens were held in 0. IM sodium cacodylate buffer at 40 for a minimum of one hour until critical point drying was done in a Bowmar SP 900 unit. The specimens were then coated with a 300 A layer of carbon and gold palladium. The specimens were studied in a Cambridge Stereoscan Mark II-A electron microscope. Photographs were taken with Polaroid 55 P/N film. We found that great care was necessary during the preparation of SEM specimens to minimize "processing artifacts." Handling of specimens was kept at a minimum and surface drying was carefully avoided. Results
FIG. 2. Drawing of the two different anastomotic techniques evaluated in this study. Insert: "No touch" technique where an adventitial suture is placed for movement control of the open end of the vessel. During suturing, only the adventitia is handled with forceps, the intima is never touched (middle right). At the lower left is a commonly used technique where the entire vessel wall, both adventitial and intimal surfaces, are handled with forceps during suturing.
utes prior to removal of the specimens, 2 mg/kg of
sodium heparin was administered intravenously. Immediately after excision, the specimens were placed in Ca++- and Mg++-free HBSS and thoroughly irrigated to remove extraneous blood. The specimens were then placed in fresh Hanks, rinsed again, opened and photographed. Each specimen was then processed for both histologic and scanning electron microscopic (SEM) evaluation. Half of each specimen was fixed in 10% buffered formalin for histologic processing, sectioned and stained with hematoxylin and eosin. The other half was processed for scanning electron microscopy. The SEM sections were fixed in 6.25% glutaraldehyde in 0. 1M sodium cacodylate buffer for six to 24 hours at 4°. After fixation, the specimens were transferred to 7.5% sucrose in 0. 1M sodium cacodylate buffer for
Vascular Clamp Study The Potts vascular clamp used in this study caused significant endothelial damage within all vessels to which it was applied. In every vessel the endothelial layer was disrupted and the subendothelial tissues exposed. It was not possible to determine if the intima was cut and retracted or whether the clamp crushed the cells to expose subendothelial tissues. Actual crush injury to the remainder of the vessel wall varied directly with wall thickness. Hence, the carotid artery showed the greatest amount of crush injury, the femoral artery less, and the veins the least at any given clamping force. Minimum Occlusive Force A typical example of the damage done when minimal occlusive force was used is shown in Figures 3-5. The endothelial loss was almost circumferentially complete in most specimens. Areas of minimal damage were occasionally found, especially in venous specimens where in two specimens a short portion of intact endothelial surface was found (Fig. 3, c). The ridging seen on this arterial endothelium is related to the underlying internal elastic lamina, and is found when specimens have not been fixed at normal vascular pressures.5 The regenerating endothelium however does not form ridges and this suggests that the inner elastic lamina has also been damaged. H&E sections confirmed that damage to the inner elastic lamina was invariably present with the clamp used. The response of platelets, white blood cells and fibrin production (P-W-F response) to the exposed subendothelial tissues was similar to reports of others."12'4'8'9'14'18 Initially, platelets, then WBC attachment followed by fibrin formation occurred within the first hour after clamp release. A dynamic balance of thrombus generation and removal reached equilibrium usually by
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FIG. 3. (left) Scanning electron micrograph (SEM) of endothelial surface of canine femoral artery two days after a ten minute application of a Potts clamp (two locations) at minimum occlusive force. There has been discrete loss of endothelium in the distribution of the clamp blade. (right) Line drawing of SEM at left, showing the two areas where the clamp was applied A and B. Dotted square area is enlarged in Figures 4 and 5. C is an area of nondisrupted endothelium frequently seen in minimum occlusive specimens.
24 hours. The depth and degree of this thrombogenic response varied and appeared to be influenced by both rheologic factors as well as the surface area of exposed subendothelial tissue. Thrombus depth was less in high
flow, high velocity vessels (arteries) and more prominent in lower velocity vessels (veins). Since the clamp did less damage to the veins than to the arteries, quantitative evaluation of this relationship was not possible.
FIG. 4. (left) SEM of endothelial damage from minimum force clamping after two days (from Fig. 4B). There has been a clean separation of endothelial margins which tapers toward the tip of the clamp where an undamaged area is present at the bottom (x 108). (right) Line drawing of SEM at left. Dotted square shows area seen at higher magnification in Figure 5. DM: damage margin. ER: endothelial ridges seen in non-pressurized arterial specimens.5
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FIG. 5. (left) SEM of area damaged as in Figures 3 and 4. At only two days, there is almost complete covering of the previously denuded area by regenerating endothelial cells. Only occasional WBC and platelets are seen where covering is not quite complete. Surviving endothelium at the damage margins shows no evidence of platelet adhesion or aggregation (x542). (right) Line drawing of SEM at left to identify structures. DM: damage margins (surviving endothelial margins). RE: regenerating endothelial cells. WBC: white blood cells.
Regeneration of endothelium occurred from the margins of the surviving endothelium. After minimum occlusive clamping, cells began to cross the exposed subendothelial tissue as early as 12 hours after injury. It was often complete by 48 hours with minimal, if any, evidence of residual P-W-F response found in the area. The new endothelial covering consisted of a flattened monolayer of endothelial cells which did not form ridging on fixation without pressurization (Fig. 5). Surviving endothelial cells at the damage margin had also regained full antithrombogenic capability and platelets and WBC were no longer seen on their surface. Maximum Occlusive Force When the maximum occlusive clamp force was applied to a vessel, the endothelial damage was severe (Fig. 6). The depth of damage varied with vessel wall thickness, being greatest in the thicker walled arteries. In addition to the damage due to crush in the cleft area, endothelial disruption back from the edge of the clamp site was also found (Fig. 6, ED). Subsequent experiments suggest this was caused by local stretching of endothelial surface. Within the cleft, active thrombus generation was always found, until complete endothelial covering had occurred. The response of platelets, WBC and fibrin generation (P-W-F) was similar in time course to the minimum occlusive studies. However, the degree of response
was much greater, with more thrombus generated and the reaction extended well back from the clamp damage site. Endothelium 1-3 mm away had attached platelets and WBC's with increasing incidence ofthrombus present as one scanned toward the cleft. An equilibrium state of thrombus generation and removal was reached later than in the minimum occlusion studies. Equilibrium was rarely found before 48 hours after injury. Endothelial regeneration started from the surviving endothelial cells at the damage margins, but was not well defined at 48 hours (Fig. 7). Surviving cells at the damage margin showed evidence of platelet and WBC attachment. Endothelial cells were found bridging between adjacent ridges. At seven days (Fig. 8) most of the specimens showed a complete covering of the previously exposed subendothelial tissues and no surface reaction with platelets or WBC. Surviving endothelial cells at the damage margin had regained full antithrombogenic capability by the seventh day in all specimens.
Endothelial Drying Study In this study, the endothelial surface of a vein or artery was exposed to the air until dry, the vessel repaired and blood flow restarted. A continuing reaction led to severe endothelial cell damage. Two days following drying, from 50 to 100% of the endothelium had become necrotic or detached and an intense WBC and
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FIG. 6. (left) SEM of endothelial surface of canine carotid artery one week after a ten minute application of a Potts clamp at full occlusive power. There remains a deep cleft in the vessel wall with some regenerating endothelial cells at the damage margins. The majority of the cleft remains denuded of endothelium, with platelet, WBC and fibrin complexes on the surface (see Fig. 11 and compare damage to minimum occlusive force damage in Fig. 3) (x45) (right) Line drawing to identify structures in SEM at left. DC: damage cleft. ED: endothelial damage, now regenerated, back from the cleft. RE: regenerated endothelium.
mild platelet reaction covered the exposed subendothelial tissues. Figure 9a shows the response as seen under the light microscope. In the SEM photo of the same specimen there are dead and dying endothelial
cells covered with WBC's (lower right) and some individual platelets. Just above and to the left of center is a group of endothelial cells with less damage and fewer WBC's on the surface. Endothelial regeneration
FIG. 7. (left) SEM of endothelial surface of canine femoral artery two days after a ten minute application of a Potts clamp at maximum occlusive force. Endothelial regeneration at the damage margin has begun slowly (compare to Fig. 5) and WBC and platelet adhesion to the surviving endothelial margins suggests cellular damage there. Platelets, WBC and fibrin cover the exposed subendothelial tissues (x522). (right) Line drawing to identify structures in SEM at left. RE: regenerating endothelium. P-W-F-: platelets, WBC and fibrin complexes on surface of subendothelial tissues. PL: Platelet attachment on cells at surviving endothelial margin DM.
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FIG. 8. (left) SEM of endothelial surface of canine femoral artery, seven days after a ten minute application of a Potts clamp at full occlusive force. Endothelial regeneration over the subendothelial tissues is complete and the surface no longer attracts platelets, WBC and fibrin. The surviving endothelial cells at the damage margins are also once again nonthrombogenic at their surface (compare to Figures 4-6) (x 108). (right) Line drawing to identify structures in SEM at left. RE: regenerated endothelium. DM: surviving endothelial cells at damage margin.
FIG. 9a. Light photomicrograph of canine jugular vein two days after endothelial surface was exposed to drying in air. There is a marked infiltrate of white blood cells at the vessel surface (lumen above). No intact endothelial cells can be recognized (x200).
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FIG. 9b. SEM of surface of same specimen Figure 9a. Massive WBC adhesion and single platelet adhesion without aggregation is seen. Central area of less severe cell damage has fewer WBC
(x505).
had begun in one-week specimens but due to the large surface area damaged, covering was not complete within the seven day experimental time period. In contrast were the findings when the exposed surface had been kept moist with Hanks balanced salt solution. In Figure 9c the endothelium is seen to be intact without surface reaction. The moist surface results were identical to control studies in which the vessels had not been handled before specimen removal and had only been in contact with blood. Stretching Study These studies demonstrated that longitudinal stretching of intact arteries or veins to one and one-half to two times their own length (even in very short segments) led to endothelial damage (Fig. 10). Splitting of endothelium with damage margin retraction (greater in arteries than in veins) occurred at right angles to the longitudinally applied force. Large areas of subendothelial tissues were exposed and led to a marked platelet, WBC and fibrin reaction on the surface. Despite
this exposed surface area, all veins and arteries remained patent and the surface reactions did not encroach significantly on the lumen.
Fogarty Catheter Study These studies demonstrated, as have others,3 that passing an inflated balloon catheter down either a vein or an artery resulted in removal and/or destruction of all the endothelial cells in that segment. The following surface reaction was intense and led to complete thrombotic occlusion in two of six veins and one of six arteries. In the vessels which remained patent, active thrombogenesis was seen on the exposed subendothelial tissues at two days and seven days (Fig. 1 1). Endothelial regeneration which began at the damage margins was not complete at 1 week following the injury. Anastomotic Techniques Study Both a common vascular anastomotic technique and the "no touch" technique (Fig. 2) worked satisfactorily
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FIG. 9c. Light photomicrograph of canine jugular vein two days after endothelial surface was exposed but kept moist in Hanks balanced salt solution. Same animal and vein as in (Figures 9a and b) (lumen above). The endothelium is intact, without evidence of degeneration or surface reaction. Result is identical to histology of control veins never opened and always exposed to blood (x200).
and all anastomoses were patent. Once the "no touch" technique had become familiar to the surgeon, there was no time difference for completing the anastomosis with either technique. Initial end-to-side studies were changed to end-to-end studies when thrombus was found in the blind ends of the carotid artery which had been ligated to divert blood through the vein graft. Thrombus was frequently seen at the anastomosis using the common vascular technique. Scanning electron microscopy revealed significant differences in the anastomotic surfaces depending on the technique used. Handling the vein with forceps which touch both sides of the wall (common technique) (Fig. 12) led to disruption of the venous endothelium, exposure of subendothelial tissues and local thrombus formation at seven days. The "no touch" technique which avoids touching the endothelial surface did not disrupt the venous endothelial surface and smooth even healing of venous and arterial endothelium at seven days was the rule (Fig. 13).
Discussion
Work which led to the tissue culture techniques needed to maintain and serially passage endothelial cells in tissue culture demonstrated the extremely delicate nature of these cells.20 These cultured cells were grown as a monolayer on the surface of a nonporous prosthetic graft which was inserted into the descending aorta of the autologous calf. No significant reaction occurred in the subendothelial tissues at the aorta to graft anastomosis, and no thrombus was found on the intact surface. Without the endothelial cell covering, a marked subendothelial reaction occurred in the aorta and a heavy layer of thrombus was generated on the surface of the graft. It appeared that the endothelial lining of the artificial graft, which is a monolayer and does not have subendothelial tissues, had, by contact inhibition, stopped the usual tissue reaction in the aortic wall.12 Techniques to culture human endothelial cells were subsequently developed,19 but as in the animal cultures,
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FIG. 10. (left) SEM of endothelial surface of canine femoral artery two days after stretching of the segment to 1.5-2 times its original length. There has been severe splitting and retraction of the endothelium with exposure of large areas of subendothelial tissues. Vessel was not opened at the time of stretching. (right) Line drawing to identify structures in SEM at left. DM: retracted damage margins. SE: exposed subendothelial tissues. $ shows direction of stretching force originally applied.
the cells were found to be easily damaged and finicky about their environment. It was the combination of no subendothelial tissue reaction, when the endothelium was intact, and the ease with which these specialized cells could be damaged that led us to study the effects
of commonly used surgical techniques on endothelial integrity and subendothelial tissue reaction. Postoperative growth, patency and function of anastomoses appear to depend upon the prevention of subendothelial tissue exposure and subsequent thrombogenesis.
FIG. I1. (left) SEM of endothelial surface of canine femoral artery seven days after Fogarty catheter was passed through the area. The vessel patent. This high magnification photo is an excellent example of the platelet, WBC and fibrin reaction which occurs at the surface of subendothelial tissues when exposed by loss of their endothelial covering (x2035). (right) Line drawing to show structures in SEM at left. PL: platelets. F: fibrin. WBC: white blood cells. was
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FIG. 12. (left) SEM of an end-to-side anastomosis between canine femoral vein (above) and carotid artery (below) seven days after surgery. Usual vascular techniques of holding both sides of the vein wall with forceps during suture placement were used (see Fig. 2, lower left). The surface of the vein (above the suture line) is disrupted and folded back endothelium with surface thrombus is present. Large areas of exposed subendothelial tissues remain (x22). (right) Line drawing to identify structures in SEM at left. '-SL--*: suture line. SE: exposed subendothelial tissues. DE: disrupted endothelium. AE: arterial endothelium. VE: venous endothelium.
One of the most basic functions of endothelium is to prevent exposure of the subendothelial tissues to
circulating blood elements." Once the tissues are exposed, thrombogenesis is initiated and the stage is set for variable degrees of subsequent thrombus generation.'5 The loss of endothelium also appears to precede intimal hyperplasia and subendothelial scarring.8"6 This subendothelial reaction may account for the lack of growth at some vascular anastomoses in infants and children. This study demonstrated that physical trauma, identical to that occurring during vascular surgical procedures, can result in complete loss of endothelium in the damaged area. '5"17 The clamps used in these studies imparted both cutting and crushing injuries to blood vessel walls. The endothelium was always damaged. The remaining wall damage was proportional to wall thickness and clamp force (Figs. 3, 5-7). The greater the clamping force, the longer it took for endothelial regeneration and therefore the longer the time active thrombogenesis was present. Cells adjacent to the damage margins were found to be the source of regenerating endothelium. When the injury was severe, the start of endothelial regeneration was delayed. The surface area of damage was also greater with increasing clamp force, so the time to complete endothelial covering was further prolonged in these animals.
In addition to clamp damage, drying of endothelial surfaces led to subsequent endothelial desquamation and a particularly intense leukocyte response. Platelets were present on the vessel surface but usually singly and without evidence of aggregation and the release reaction.4 Since the arterial clamp studies did not show ridging in the area of injury, (Fig. 4) (nonpressurized fixation) we assumed, and histologic sections confirmed, the loss of the internal elastic lamina. In contrast, the injury caused by drying did not involve loss of the elastic lamina as shown by histologic sections (Fig. 9). Ridging was seen in surfacedried (unpressurized fixation) arterial specimens suggesting the internal elastic lamina was functionally intact.6'8 Platelets adhere to minimal damage areas on endothelial surfaces but have not been found to undergo aggregation and the release reaction at these sites. Aggregation and release has not been seen on the elastic lamina" either. Thus, the damage from drying may not lead to complete exposure of subendothelial tissues. Long-term studies need to be done to evaluate whether chronic damage can occur with an intact inner elastic lamina. Reversible intimal thickening is known to occur between the inner elastic lamina and the vessel lumen in rat carotid arteries.6 Stretching caused endothelial disruption which included the internal elastic lamina (see absence of
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VE SL
AE FIG. 13. (left) SEM of an end-to-side anastomosis between canine femoral vein (above) and carotid artery (below) seven days after surgery. Same animal was as in Figure 12. The "no touch" technique of anastomosis was used. The venous endothelium is intact, there is no exposed subendothelial tissue. Note the difference in surface texture between fixed arterial and fixed venous endothelium. The arterial surface is corrugated with ridges while the venous surface appears as a smooth cobblestone surface. Arterial surfaces become smooth if fixed at above normal diastolic pressures (x22).5 (right) Line drawing to identify structures in the SEM at the left. SL: suture line. VE: venous endothelium. S: suture. AE: arterial endothelium.
ridging in Fig. 10). The usual reaction to exposed subendothelium occurred with platelet, WBC and fibrin formation on the surface. Fogarty balloon endothelial stripping removed both endothelium and the internal elastic lamina in some arteries but only the endothelium in others. When both were removed a marked platelet, WBC and fibrin reaction was seen (Fig. 11). When only the endothelium was removed, single adherent but nonaggregated platelets were seen without significant numbers of WBC's or fibrin. Several other modes of endothelial damage not evaluated in this study are known.7"3"5"7 Whether thrombus progresses to occlusion of the lumen is dependent on several factors16 among which are: blood flow and turbulence,10 fibrinolytic activity,'2 rate of endothelial regeneration, presence of anticoagulants, and the surface area of exposed subendothelial tissues. Embolic phenomena have not been evaluated during this study. The clinical implications of these findings may have significance especially for the infant and child. It takes less thrombus to occlude a small vessel than a large vessel. Damage which leads to subendothelial proliferation may influence later development of a vessel or growth at the site of anastomoses. To avoid damage and create the best environment for later growth one should use all means possible to prevent endothelial damage. Some methods suggested
by our findings include: 1) minimal clamping pressure to stop flow in the vessel; 2) avoid drying of endothelial surfaces; 3) avoid stretching of vessels including tension at suture lines; 4) use the minimum inflation pressure needed to effect balloon embolectomies; 5) use or devise interrupted suture anastomotic techniques which minimize endothelial trauma. Summary The influence of clamping, drying, stretching and surgical anastomotic technique on the subsequent integrity of blood vessel endothelium has been studied in the dog. Thrombogenesis at the vessel surface was found in all specimens where loss of endothelium had exposed subendothelial tissues. Whether thrombotic occlusion of the vessel followed depended upon several local factors including surface area damaged and blood flow in the vessel. All methods of influence studied led to significant endothelial damage and suggested the following clinical implications: 1) Endothelial integrity is the cornerstone to success in handling blood vessels and avoiding thrombogenesis-prevention of exposure of subendothelial tissues is the goal. 2) Clamping of blood vessels should always be done with a minimum of force.
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3) Endothelial surfaces should never be allowed to become dry. 4) Stretching can lead to thrombosis within intact blood vessels when the endothelium is disrupted. 5) Interrupted suture anastomotic techniques should be used which minimize endothelial trauma and thus avoid subendothelial tissue reactions which in turn may jeopardize long-term patency and growth at anastomotic sites.
8.
9.
10.
11.
Acknowledgments The authors are particularly grateful to Paulette Uto and Wendy Miller for their expert technical assistance during these experiments, Arnold Schmidt at the Materials Analysis Center of the University of Washington for his operation of the electron microscope, Marena Slatt for help in preparing the manuscript and Trese Rand for her illustrative interpretation of techniques and results.
References 1. Ashford, T. P. and Freiman, D. G.: Platelet Aggregation at Sites of Minimal Endothelial Injury: An Electron Microscopic Study. Am. Jour. Pathol. 53:599, 1968. 2. Baumgertner, H. R.: The Subendothelial Surface and Thrombosis. Throm. Diath. Halmorth. Suppl., 59:91, 1974. 3. Baumgartner, H. R.: Sterierman, M. B. and Spaet, T. H.: Adhesion of Platelets to Subendothelial Surface: Distinct from Adhesion to Collagen. Experientia, 27:283, 1971. 4. Born, G. V. R.: Current Ideas on the Mechanism of Platelet Aggregation. Ann. N.Y. Acad. Sci, 201:4, 1972. 5. Clark, J. M. and Glagov, S.: Luminal Surface of Distended Arteries by Scanning Electron Microscopy: Eliminating Configurational and Technical Artifacts. Br. J. Exp. Pathol., 57:129, 1975. 6. Clowes, A. W., Ryan, G. B., Breslow, J. L. and Karnovsky, M. J.: Absence of Enhanced Intimal Thickening in the Response of the Carotid Arterial Wall to Endothelial Injury in Hypercholesterolemic Rats. Lab. Invest., 35:6, 1975. 7. Elemer, G., Kereny, T. and Jekkinek, H.: Scanning (SEM) and Transmission (TEM) Electronmicroscopic Studies on Post-
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