- Email: [email protected]
Vascular reactions to laser in vivo
MICROVASCULAR
RESEARCH
8,132-138 (1974)
Vascular
Reactions MARY
to Laser
In Viva’
P. WIEDEMAN
Temple University School of Medicine, Department of Physiology, 3420 North Broad Street, Philadelphia, Pennsylvania 19140 Received January 17,1974 Platelet aggregates form in arterial vessels following exposure of the vessels to a single pulse ruby-red laser beam. Usually, a small cluster of red blood cells occupies the center of the aggregate. It appears that endothelial damage is not required for the platelet aggregation, but that platelet activity is initiated by rupture of red blood cells, possibly through the release of ADP from the injured red cells. Also, no shape change is noted in platelets that adhere to the vessel wall or to each other.
INTRODUCTION Factual information for an understanding of the cause of spontaneous thrombus formation in human blood vessels is being gathered at a rapid rate. Investigators continue to introduce new methods to examine the relationship between the blood vessel wall and the cellular components of the blood in an effort to determine what physical and chemical changes in the endothelial lining of the blood vessel causes it to attract platelets and evoke their aggregation. In vitro studies have revealed that adenosine diphosphate (ADP) activates platelet aggregation, and that platelets release ADP themselves to prolong the aggregation. In vitro studies have also uncovered endogenous materials, such as collagen, which cause a change in the shape of platelets and subsequent aggregation. It has been postulated that injury to vascular endothelium, resulting in exposure of underlying collagen, is the primary cause of platelet aggregation which precedes thrombus formation. Several methods of damaging the endothelial lining of blood vesselsduring in viuo microscopic observation have been used. Mechanical, electrical, and thermal injuries are included. Thermal injury can be controlled and discretely localized by the use of a laser beam. Investigators utilizing this method include Kochen and Baez (1965), Arfors et al. (1968), Kovacs et al. (1973), and Wiedeman and Margulies (1972). Several findings reported from this laboratory indicated that there were significant differences between in vivo and in vitro platelet aggregation, and also it was demonstrated that endothelial damage was not the sole prerequisite for platelet aggregation (Wiedeman and Margulies, 1972; Wiedeman, 1973). Also, McKenzie, Arfors, and Matheson, and Hovig, McKenzie, and Arfors have suggested that platelet aggregate formation following laser-induced microvascular injury was primarily an adenosine phosphate mediated reaction, the source possibly red blood cells, and they have shown, by ultrastructural studies, that very little endothelial cell damage is incurred when producing aggregates,by laser injury. The material presented here includes further studies in pursuit of the mechanism ’ Supported by the Specialized Center for Research-Thrombosis 132
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
Grant Hl 14216-04.
VASCULAR REACTIONS TO LASER
133
underlying platelet aggregation and adherence to a blood vessel wall following the delivery of a laser beam as the vehicle of injury. MATERIALS
AND METHODS
The site selected for microscopic observation of blood cells and blood vessels subjected to a laser beam was the wing of the little brown bat (Myotis lucifugus). The unanesthetized bat is placed in a holder and one wing is spreadacrossa large microscope slide bounded by a plastic frame which has fittings to secure spring clips which, in
FIG. 1. Diagrammatic representation of a bat wing artery cannulated for perfusion of materials in arterial vessels. The perfused solution pushes arterial blood upstream and replaces it in the arterial vessels of the side branch.
turn, hold the wing in place. The major artery entering the wing is cannulated at its most distal portion with a tapered glass can&a. Perfusion of solutions, when desired, is made retrograde to arterial inflow until reaching an upstream branch of the cannulated artery at which point the perfused material enters the branch and travels in the direction of normal arterial inflow (Fig. 1). This vessel, classified as a first-order vesselor artery, with a diameter of approximately 52.6 pm, or a branch of it, classified as a second-order vessel, or small artery, with a diameter of approximately 19.0 Ltrn were used in the study. The epithelium covering the area to be observed is removed to permit better visualization at high magnification in that the epithelial cells are heavily pigmented and partially obscure the vesselsand their contents. No further preparation of the animal is required and microscopic observation of blood vesselsand flow can be made at magnifications of 400-1200 diam with good resolution and fine detail. A ruby-red biolaser that delivers a single pulse is mounted on the trinocular head of the microscope. The laser beam produces a discrete, local thermal injury. By trial and error it was determined that an input of 150-170 J was sufficient to produce an immediate platelet aggregate that adhered to the vessel wall. The aggregate was considered, subjectively, to be active aslong asit continued to build up by the additional adherence of platelets and by producing emboli. After an average time of 4 min and 19 set, platelets from the flowing blood were no longer attracted to the initial clump, indicating a cessation of the liberation of aggregating material. The stability of the platelet aggregatewas indicated by the cessation of emboli production. Solutions perfused included buffered saline, platelet rich plasma, washed red blood cells, I % solution of Congo Red in saline, plasma, hemolysate, and red cell ghosts.
134
MARY P. WIEDEMAN
The PRP, washed RBCs, plasma, hemolysate, and red cell ghosts were prepared from canine blood samples. There was no indication of any lack of compatibility between bat and canine blood. Whole blood was never injected, but no vascular activity or untoward effects on cellular components was obvious when PRP, washed RBCs, plasma, or hemolysate preparations of canine blood were introduced into the bat. The hemolysate was prepared by freezing washed red blood cells and later centrifuging the thawed material and withdrawing the hemolysate. All infusions were made within 1-2 hr of preparation. RESULTS 1. Delivery of a Single Pulse Ruby-Red Laser Beam to an Intact Arterial Normal Blood Flow
Vessel with
Immediately after the laser beam had struck the vessel,aggregation of platelets was visible. It was noted that the initial aggregatecontinued to attract platelets from the blood flowing past the exposed site, occasionally to an extent that caused occlusion of the vessel. In several seconds after occlusion flow was usually reinstituted because of a diminution of the aggregate through embolization. A striking feature was that 50-80 pm upstream from the aggregate,platelets becamevisible, rolling along the wall of the vessel.Some adhered to the aggregatewhile others were swept by in the flowing blood. This suggestedthat some area around the site may have had subliminal damage that altered the endothelium sufficiently to cause adherenceof the platelets. However, on occasions where laser injury was produced in an arterial branch, platelets were seenfalling out of the blood stream of the parent vesselto collect at the site of injury. These two observations would suggest some chemotaxic activity originated from the major site of injury. The fact that platelets began to adhere to the vessel wall upstream, rather than downstream, from the injured site, and the fact that platelets were attracted from rapidly flowing blood in a parent vessel toward an injury some distance away in a branch cannot be accounted for simply by blood flow characteristics. A discrepancy with reports of in vivo studies was noted regarding a change in shape of platelets thought to be concurrent with adhesiveness.Platelets in a test tube become spherical preceding their adherence to one another when mixed with collagen (Hovig et al., 1968). However, in flowing blood in a vesselin a living animal, no change from disk shape to sphere shape could be seen in platelets rolling along the wall toward the aggregate nor in platelets that were adhering to one another. It is suggestedthat the change in shape from disk to sphere seenin vitro is a test tube artifact. It has previously been reported by Poliwoda (1969) that no change in platelet shape occurs in aggregates forming after electrical injury. Stehbens and Biscoe (1967) report that ultrastructure studies show no spherical platelets in excisedcarotid bodies with platelet aggregates. French, MacFarlane, and Sanders (1964) found no change in platelets forming clumps after arterial transection. One other characteristic of the platelet aggregate produced by the laser beam was observed. In most of the white clusters of adhering platelets, a small clump of red blood cells could be seen. The entire shape of each cell was not always definable, and they had the appearance of having partially “melted” together. Logically, they could be coagulated by heat of the laser.
VASCULAR
REACTIONS
TO LASER
135
Attention was directed toward the clump of amorphous red blood cells to ascertain how they participate, if at all, in the formation of the platelet aggregate.One observation was that platelets from the flowing blood were attracted to the red blood cells, and at the cessation of platelet activity, the massof red cells remained fixed to the wall of the vessel. In other instances, especially in arterial vessels,the inner core of red blood cells while covered with platelets, would wash from the wall and be carried downstream. When this occurred in the first secondsafter laser injury, platelet activity would continue around the site, evidenced by rebuilding of a platelet aggregatewhich usually broke off either entirely or in part to reform again. This continued for several minutes after disappearance of the red blood cell clump. The very noticeable line of platelets rolling slowly along the vessel wall toward the site of the original aggregate continued, in some experiments, as long as 2 hr. It appeared that the red blood cell clump did actively participate in formation of the platelet aggregate, and perfusion studies were introduced to learn to what degree the red blood cells were responsible and if there was truly endothelial damage. 2. Perfkion
of an Arterial Vessel at the Time of Laser Injq
a. B@k-ed saline solution. It was reasoned that if the platelet aggregateformed as a result of thermal injury to the endothelial lining of the blood vesselwithout involvement of blood, a laser beam delivered when the vesselwas filled with buffered saline would injure the wall and when blood flow returned, platelets would adhere to the injured site. The perfusion of buffered saline was accomplished as described, the laser beam was delivered while the arterial vesselwas filled with buffered saline, and when blood flow returned, no platelet aggregateformed at the site. b. Platelet rich plasma (PRP). To be certain that platelets would be available for immediate reaction to the damaged endothelium, PRP was perfused through an arterial vesselat the time of delivery of a laser beam. No aggregateof platelets occurred at the time of laser delivery or subsequently when blood flow returned. c. Congo Red. To overcome the criticism that colorless fiuids such as buffered saline and PRP might not absorb as much energy from the ruby-red laser beam as whole blood, a solution of Congo Red (dye dissolved in buffered saline) that appeared to have the same rednessas blood was perfused through an arterial vesselat the time of exposure to a laser beam. When blood flow returned to the arterial vessel at the cessation of perfusion, no platelet aggregateformed. d. Washed red blood cells. The foregoing results gave strong indication that some component of whole blood other than plasma or platelets was needed to occupy the arterial vessel subjected to the laser beam if a platelet aggregate was to appear as a sequel to the thermal insult. Red blood cells had been noted as “melted” after laser, and so washed red cells were perfused during delivery of a laser beam and it was seen that a clump of them formed to act as a core for platelet adherencewhen normal blood how was restored. e. Hemolysate. Having identified the red blood cell as the primary factor, two components remained to be tested which were the hemolysate and the red cell ghosts. Perfusion of hemolyzed blood separatedfrom cell bodies by centrifugation resulted in the formation of a solid mass following exposure to laser to which platelets adhered
136
MARY P. WIEDEMAN
when blood flowed over it. Red cell ghosts caused no such massto be formed. It was concluded, therefore, that the contents of erythrocytes were necessaryfor the formation of a platelet aggregateas a result of exposure of flowing blood in a vesselto a ruby-red laser beam. $ Hemolysate in vitro. To observe the massproduced by laser when the hemolysate was exposed, hemolysate was placed on a glass microscope slide and hit with 150 J input as had been done in the experiments in the living animal. The result, as seen microscopically, was a heat coagulated mass,firmly adhering to the glass slide. g. Flowing blood in a glass cannula. To further demonstrate that the blood vessel wall was not necessaryfor the production of a platelet aggregate in flowing blood, blood was drawn through a glass capillary tube and a laser beam was delivered. The result was a clump of red blood cells surrounded by platelets, occasionally sticking to the glass wall of the capillary tube. h. Acetylsalicylic acid. Attempts to retard laser-induced platelet aggregation by pretreatment of animals with acetylsalicylic acid failed. Intravenous infusion of 166 mg/kg body wt of ASA had no effect on the duration of platelet activity. It was established in control animals that platelet activity persisted for 4 min, 19 set after laser injury. During the first 3 hr after exposure to laser, platelet activity continued in pretreated animals for 4 min, 15 set in the first hour, 4 min, 7 set between the first and second hour, and 3 min, 53 set between the second and third hour (Wiedeman, 1973). If it is a fact that ASA inhibits only collagen-induced platelet aggregation as shown in vitro, the lack of inhibition demonstrated here would further support the contention that laser-induced platelet aggregation does not originate from collagen exposure. Kovacs et al. (1973) inhibited laser-induced aggregates,but produced a microburn on the vesselwall by perfusing carbon particles or dye to assurea site for heat generation on the endothelium. Arfors et al. (1972) were not able to demonstrate inhibition using the same method as reported here. In summary, platelet aggregatesformed in arterial vesselsfollowing the delivery of a single pulse ruby-red laser beam when whole blood, washed red blood cells, or hemolysate was flowing through the blood vesselat the time the laser beam struck. No platelet aggregateformed if the blood vesselwas perfused with saline, PRP, or Congo Red at the time of exposure to the laser beam. DISCUSSION It is well established that damageto endothelial cells lining the walls of blood vessels promotes the adherence of platelets to the wall and to each other, thereby forming a platelet aggregate(Spaet and Erichson, 1966).It is believed that collagen or collagenous material underlying the endothelium releases ADP on exposure and ADP is the primary factor in promoting platelet aggregation. Furthermore, platelets exposed to collagen in vitro are seento assumea spherical shapeand this shapechange is equated with the beginning of “stickiness’ of the platelet. Aggregation of platelets exposed to collagen in the test tube follows (Zucker and Borrelli, 1962).A vascular lesion is thought to be the primary requisite for platelet aggregation which precedesthe formation of a thrombus. The findings presentedhere require a different explanation for the sequenceof events
VASCULAR REACTIONS TO LASER
137
following the exposure of flowing blood to a laser beam. McKenzie et al. (1972), using the same technique for injury to blood vesselsin the rabbit ear chamber, were also impressed with the heat-coagulated red blood cells forming the core of the platelet aggregateand postulated that no endothelial damage had transpired. Electron micrographs of the laser exposed blood vesselssupported their belief in that they showed little, if any, endothelial damage. They also postulated that the mechanism, other than collagen exposure which could explain the microscopically observedplatelet aggregates, was an ADP mediated reaction. Begent and Born have demonstrated that ADP, in the absenceof wall injury, can causeadherenceof platelets by the application of ADP by iontophoresis on the wall of a blood vessel. The explanation that is most attractive is that ADP is liberated from the red blood cells which have been damaged by localized heat from the laser. The heat coagulated red cells, releasing ADP, could initiate platelet adhesivenessand then aggregation. The subsequent rebuilding of the aggregatesfollowing embolization could be attributed to the release reaction in which platelets themselves supply the ADP necessary for continued adhesivenessand clumping. A major unresolved question centers around two observations. First, if the endothelium is undamaged by the laser beam, what causesthe heat coagulated red blood cells to adhere to the vesselwall in the area which has been subjectedto the laser beam? Second, if the endothelium is undamaged by the laser beam, what causes platelets upstream from the site of the aggregateto roll slowly along the vesselwalls as if they had become sticky or adhesive? A suggested explanation, unsupported by experimental evidence, may be that some reaction between the vessel wall and the first cluster of damaged cells activates a protective system which induces platelet adhesiveness and aggregation, thus preventing further deterioration of vascular endothelium. This possibility should be explored. ACKNOWLEDGMENT The author acknowledges the splendid technical assistance of Miss Claire Bernardin. REFERENCES ARFORS,K. E., DHALL, D. P., ENGESET,J., HINT, H., MATHESON,K. A., AND TAGEN,0 (1968). Biolaser endothelial trauma as a means of quantifying platelet activity in cko. Nature(London) 218,887-888. ARFORS,K. E., BERGQUIST,D., BYGDEMAN,S., MCKENZIE, F. N., AND SVENSJO, E. (1972). The effect of the platelet release reaction on platelet behavior in oitro and in oklo. &a&J. Haematol. 9,322-332. BEGENT, N., AND BORN, G. V. R. (1970). Growth rate in uico of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature (London) 227, 926-930. FRENCH, J. E., MACFARLANE, R. G., AND SANDERS, A. G. (1964). The structure of haemostatic plugs and experimental thrombi in small arteries. British J. Exp. Pathol. 45,467-474. How;, T., JORGENSEN,L., PACKHAM, M. A., AND MUSTARD,J. F. (1968). Platelet adherence to fibrin and collagen. J. Lab. C/in. Med. 71,29-40. HOVIG, T., MCKENZIE, F. N., AND ARFORS, K. E. (1974). Measurement of platelet response to laserinduced microvascular injury. Ultrastr. Studies, to be published. KOCHEN, J. A., AND BAEZ, S. (1965). Vascular and intravascular effects of pulsed laser micro-beam. Bibl. Anat. 7, 46-49. KOVACS, I. B., COALAY, L., AND GOROG, D. (1973). Laser-induced thrombosis in the microcirculation of the hamster cheek pouch and its inhibition by acetylsalicylic acid. Microvasc. Res. 6, 194-201. MCKENZIE, F. N., ARFORS, K. E., HOVIG, T., AND MATHESON, N. A. (1972). The mechanism of platelet aggregation at sites of laser-induced microvascular injury-pharmacological and ultrastructural
138
MARY P. WIEDEMAN
studies. In “7th Conf. Europ. Sot. for Microcirculation.” Aberdeen. p. 142. Bib/. Anat. (1973) 12, 18G192, Karger, Basel. MCKENZIE, F. N., ARFORS, K. E., AND MATHESON,N. A. Measurement of platelet response to laserinduced microvascular injury. To be published. POUWODA, H., HAGEMANN, G., AND ZURWEHME,D. (1969). Quantitative analysis concerning the early phase of white thrombi. Bibl. Anat. 10,489-493. SPAET, T. H., AND ERICHSON, R. B. (1966). The vascular wall in the pathogenesis of thrombosis. Thromb. Diath. Haemorrh.
Suppl. 21,67-86.
STEHBENS,W. E., AND BISCOE,T. J. (1967). The ultrastructure of early platelet aggregation in Go. Amer. J. Pathol. 50,212-224.
WIEDEMAN,M. P., AND MARGULIES,E. H. (1973). Factors affecting production of platelet aggregates. In “7th European Conference on Microcirculation.” Aberdeen, Scotland, Part II, Karger-Basel. Bibl. Anat. 12,193-197. WIEDEMAN,M. P. (1973). Platelet aggregates induced by red blood cell injury. In “Oxygen Transport to Tissues” (D. F. Bruley and H. I. Bicher, eds.). Aduan. Exp. Med. Biol. 37B, 681-686. ZUCKER,M. B., AND BORRELLI,J. (1962). Platelet clumping produced by connective tissue suspensions and by collagen. Proc. Sot. Exp. Biol. Med. 109, 779-787.
RESEARCH
8,132-138 (1974)
Vascular
Reactions MARY
to Laser
In Viva’
P. WIEDEMAN
Temple University School of Medicine, Department of Physiology, 3420 North Broad Street, Philadelphia, Pennsylvania 19140 Received January 17,1974 Platelet aggregates form in arterial vessels following exposure of the vessels to a single pulse ruby-red laser beam. Usually, a small cluster of red blood cells occupies the center of the aggregate. It appears that endothelial damage is not required for the platelet aggregation, but that platelet activity is initiated by rupture of red blood cells, possibly through the release of ADP from the injured red cells. Also, no shape change is noted in platelets that adhere to the vessel wall or to each other.
INTRODUCTION Factual information for an understanding of the cause of spontaneous thrombus formation in human blood vessels is being gathered at a rapid rate. Investigators continue to introduce new methods to examine the relationship between the blood vessel wall and the cellular components of the blood in an effort to determine what physical and chemical changes in the endothelial lining of the blood vessel causes it to attract platelets and evoke their aggregation. In vitro studies have revealed that adenosine diphosphate (ADP) activates platelet aggregation, and that platelets release ADP themselves to prolong the aggregation. In vitro studies have also uncovered endogenous materials, such as collagen, which cause a change in the shape of platelets and subsequent aggregation. It has been postulated that injury to vascular endothelium, resulting in exposure of underlying collagen, is the primary cause of platelet aggregation which precedes thrombus formation. Several methods of damaging the endothelial lining of blood vesselsduring in viuo microscopic observation have been used. Mechanical, electrical, and thermal injuries are included. Thermal injury can be controlled and discretely localized by the use of a laser beam. Investigators utilizing this method include Kochen and Baez (1965), Arfors et al. (1968), Kovacs et al. (1973), and Wiedeman and Margulies (1972). Several findings reported from this laboratory indicated that there were significant differences between in vivo and in vitro platelet aggregation, and also it was demonstrated that endothelial damage was not the sole prerequisite for platelet aggregation (Wiedeman and Margulies, 1972; Wiedeman, 1973). Also, McKenzie, Arfors, and Matheson, and Hovig, McKenzie, and Arfors have suggested that platelet aggregate formation following laser-induced microvascular injury was primarily an adenosine phosphate mediated reaction, the source possibly red blood cells, and they have shown, by ultrastructural studies, that very little endothelial cell damage is incurred when producing aggregates,by laser injury. The material presented here includes further studies in pursuit of the mechanism ’ Supported by the Specialized Center for Research-Thrombosis 132
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
Grant Hl 14216-04.
VASCULAR REACTIONS TO LASER
133
underlying platelet aggregation and adherence to a blood vessel wall following the delivery of a laser beam as the vehicle of injury. MATERIALS
AND METHODS
The site selected for microscopic observation of blood cells and blood vessels subjected to a laser beam was the wing of the little brown bat (Myotis lucifugus). The unanesthetized bat is placed in a holder and one wing is spreadacrossa large microscope slide bounded by a plastic frame which has fittings to secure spring clips which, in
FIG. 1. Diagrammatic representation of a bat wing artery cannulated for perfusion of materials in arterial vessels. The perfused solution pushes arterial blood upstream and replaces it in the arterial vessels of the side branch.
turn, hold the wing in place. The major artery entering the wing is cannulated at its most distal portion with a tapered glass can&a. Perfusion of solutions, when desired, is made retrograde to arterial inflow until reaching an upstream branch of the cannulated artery at which point the perfused material enters the branch and travels in the direction of normal arterial inflow (Fig. 1). This vessel, classified as a first-order vesselor artery, with a diameter of approximately 52.6 pm, or a branch of it, classified as a second-order vessel, or small artery, with a diameter of approximately 19.0 Ltrn were used in the study. The epithelium covering the area to be observed is removed to permit better visualization at high magnification in that the epithelial cells are heavily pigmented and partially obscure the vesselsand their contents. No further preparation of the animal is required and microscopic observation of blood vesselsand flow can be made at magnifications of 400-1200 diam with good resolution and fine detail. A ruby-red biolaser that delivers a single pulse is mounted on the trinocular head of the microscope. The laser beam produces a discrete, local thermal injury. By trial and error it was determined that an input of 150-170 J was sufficient to produce an immediate platelet aggregate that adhered to the vessel wall. The aggregate was considered, subjectively, to be active aslong asit continued to build up by the additional adherence of platelets and by producing emboli. After an average time of 4 min and 19 set, platelets from the flowing blood were no longer attracted to the initial clump, indicating a cessation of the liberation of aggregating material. The stability of the platelet aggregatewas indicated by the cessation of emboli production. Solutions perfused included buffered saline, platelet rich plasma, washed red blood cells, I % solution of Congo Red in saline, plasma, hemolysate, and red cell ghosts.
134
MARY P. WIEDEMAN
The PRP, washed RBCs, plasma, hemolysate, and red cell ghosts were prepared from canine blood samples. There was no indication of any lack of compatibility between bat and canine blood. Whole blood was never injected, but no vascular activity or untoward effects on cellular components was obvious when PRP, washed RBCs, plasma, or hemolysate preparations of canine blood were introduced into the bat. The hemolysate was prepared by freezing washed red blood cells and later centrifuging the thawed material and withdrawing the hemolysate. All infusions were made within 1-2 hr of preparation. RESULTS 1. Delivery of a Single Pulse Ruby-Red Laser Beam to an Intact Arterial Normal Blood Flow
Vessel with
Immediately after the laser beam had struck the vessel,aggregation of platelets was visible. It was noted that the initial aggregatecontinued to attract platelets from the blood flowing past the exposed site, occasionally to an extent that caused occlusion of the vessel. In several seconds after occlusion flow was usually reinstituted because of a diminution of the aggregate through embolization. A striking feature was that 50-80 pm upstream from the aggregate,platelets becamevisible, rolling along the wall of the vessel.Some adhered to the aggregatewhile others were swept by in the flowing blood. This suggestedthat some area around the site may have had subliminal damage that altered the endothelium sufficiently to cause adherenceof the platelets. However, on occasions where laser injury was produced in an arterial branch, platelets were seenfalling out of the blood stream of the parent vesselto collect at the site of injury. These two observations would suggest some chemotaxic activity originated from the major site of injury. The fact that platelets began to adhere to the vessel wall upstream, rather than downstream, from the injured site, and the fact that platelets were attracted from rapidly flowing blood in a parent vessel toward an injury some distance away in a branch cannot be accounted for simply by blood flow characteristics. A discrepancy with reports of in vivo studies was noted regarding a change in shape of platelets thought to be concurrent with adhesiveness.Platelets in a test tube become spherical preceding their adherence to one another when mixed with collagen (Hovig et al., 1968). However, in flowing blood in a vesselin a living animal, no change from disk shape to sphere shape could be seen in platelets rolling along the wall toward the aggregate nor in platelets that were adhering to one another. It is suggestedthat the change in shape from disk to sphere seenin vitro is a test tube artifact. It has previously been reported by Poliwoda (1969) that no change in platelet shape occurs in aggregates forming after electrical injury. Stehbens and Biscoe (1967) report that ultrastructure studies show no spherical platelets in excisedcarotid bodies with platelet aggregates. French, MacFarlane, and Sanders (1964) found no change in platelets forming clumps after arterial transection. One other characteristic of the platelet aggregate produced by the laser beam was observed. In most of the white clusters of adhering platelets, a small clump of red blood cells could be seen. The entire shape of each cell was not always definable, and they had the appearance of having partially “melted” together. Logically, they could be coagulated by heat of the laser.
VASCULAR
REACTIONS
TO LASER
135
Attention was directed toward the clump of amorphous red blood cells to ascertain how they participate, if at all, in the formation of the platelet aggregate.One observation was that platelets from the flowing blood were attracted to the red blood cells, and at the cessation of platelet activity, the massof red cells remained fixed to the wall of the vessel. In other instances, especially in arterial vessels,the inner core of red blood cells while covered with platelets, would wash from the wall and be carried downstream. When this occurred in the first secondsafter laser injury, platelet activity would continue around the site, evidenced by rebuilding of a platelet aggregatewhich usually broke off either entirely or in part to reform again. This continued for several minutes after disappearance of the red blood cell clump. The very noticeable line of platelets rolling slowly along the vessel wall toward the site of the original aggregate continued, in some experiments, as long as 2 hr. It appeared that the red blood cell clump did actively participate in formation of the platelet aggregate, and perfusion studies were introduced to learn to what degree the red blood cells were responsible and if there was truly endothelial damage. 2. Perfkion
of an Arterial Vessel at the Time of Laser Injq
a. B@k-ed saline solution. It was reasoned that if the platelet aggregateformed as a result of thermal injury to the endothelial lining of the blood vesselwithout involvement of blood, a laser beam delivered when the vesselwas filled with buffered saline would injure the wall and when blood flow returned, platelets would adhere to the injured site. The perfusion of buffered saline was accomplished as described, the laser beam was delivered while the arterial vesselwas filled with buffered saline, and when blood flow returned, no platelet aggregateformed at the site. b. Platelet rich plasma (PRP). To be certain that platelets would be available for immediate reaction to the damaged endothelium, PRP was perfused through an arterial vesselat the time of delivery of a laser beam. No aggregateof platelets occurred at the time of laser delivery or subsequently when blood flow returned. c. Congo Red. To overcome the criticism that colorless fiuids such as buffered saline and PRP might not absorb as much energy from the ruby-red laser beam as whole blood, a solution of Congo Red (dye dissolved in buffered saline) that appeared to have the same rednessas blood was perfused through an arterial vesselat the time of exposure to a laser beam. When blood flow returned to the arterial vessel at the cessation of perfusion, no platelet aggregateformed. d. Washed red blood cells. The foregoing results gave strong indication that some component of whole blood other than plasma or platelets was needed to occupy the arterial vessel subjected to the laser beam if a platelet aggregate was to appear as a sequel to the thermal insult. Red blood cells had been noted as “melted” after laser, and so washed red cells were perfused during delivery of a laser beam and it was seen that a clump of them formed to act as a core for platelet adherencewhen normal blood how was restored. e. Hemolysate. Having identified the red blood cell as the primary factor, two components remained to be tested which were the hemolysate and the red cell ghosts. Perfusion of hemolyzed blood separatedfrom cell bodies by centrifugation resulted in the formation of a solid mass following exposure to laser to which platelets adhered
136
MARY P. WIEDEMAN
when blood flowed over it. Red cell ghosts caused no such massto be formed. It was concluded, therefore, that the contents of erythrocytes were necessaryfor the formation of a platelet aggregateas a result of exposure of flowing blood in a vesselto a ruby-red laser beam. $ Hemolysate in vitro. To observe the massproduced by laser when the hemolysate was exposed, hemolysate was placed on a glass microscope slide and hit with 150 J input as had been done in the experiments in the living animal. The result, as seen microscopically, was a heat coagulated mass,firmly adhering to the glass slide. g. Flowing blood in a glass cannula. To further demonstrate that the blood vessel wall was not necessaryfor the production of a platelet aggregate in flowing blood, blood was drawn through a glass capillary tube and a laser beam was delivered. The result was a clump of red blood cells surrounded by platelets, occasionally sticking to the glass wall of the capillary tube. h. Acetylsalicylic acid. Attempts to retard laser-induced platelet aggregation by pretreatment of animals with acetylsalicylic acid failed. Intravenous infusion of 166 mg/kg body wt of ASA had no effect on the duration of platelet activity. It was established in control animals that platelet activity persisted for 4 min, 19 set after laser injury. During the first 3 hr after exposure to laser, platelet activity continued in pretreated animals for 4 min, 15 set in the first hour, 4 min, 7 set between the first and second hour, and 3 min, 53 set between the second and third hour (Wiedeman, 1973). If it is a fact that ASA inhibits only collagen-induced platelet aggregation as shown in vitro, the lack of inhibition demonstrated here would further support the contention that laser-induced platelet aggregation does not originate from collagen exposure. Kovacs et al. (1973) inhibited laser-induced aggregates,but produced a microburn on the vesselwall by perfusing carbon particles or dye to assurea site for heat generation on the endothelium. Arfors et al. (1972) were not able to demonstrate inhibition using the same method as reported here. In summary, platelet aggregatesformed in arterial vesselsfollowing the delivery of a single pulse ruby-red laser beam when whole blood, washed red blood cells, or hemolysate was flowing through the blood vesselat the time the laser beam struck. No platelet aggregateformed if the blood vesselwas perfused with saline, PRP, or Congo Red at the time of exposure to the laser beam. DISCUSSION It is well established that damageto endothelial cells lining the walls of blood vessels promotes the adherence of platelets to the wall and to each other, thereby forming a platelet aggregate(Spaet and Erichson, 1966).It is believed that collagen or collagenous material underlying the endothelium releases ADP on exposure and ADP is the primary factor in promoting platelet aggregation. Furthermore, platelets exposed to collagen in vitro are seento assumea spherical shapeand this shapechange is equated with the beginning of “stickiness’ of the platelet. Aggregation of platelets exposed to collagen in the test tube follows (Zucker and Borrelli, 1962).A vascular lesion is thought to be the primary requisite for platelet aggregation which precedesthe formation of a thrombus. The findings presentedhere require a different explanation for the sequenceof events
VASCULAR REACTIONS TO LASER
137
following the exposure of flowing blood to a laser beam. McKenzie et al. (1972), using the same technique for injury to blood vesselsin the rabbit ear chamber, were also impressed with the heat-coagulated red blood cells forming the core of the platelet aggregateand postulated that no endothelial damage had transpired. Electron micrographs of the laser exposed blood vesselssupported their belief in that they showed little, if any, endothelial damage. They also postulated that the mechanism, other than collagen exposure which could explain the microscopically observedplatelet aggregates, was an ADP mediated reaction. Begent and Born have demonstrated that ADP, in the absenceof wall injury, can causeadherenceof platelets by the application of ADP by iontophoresis on the wall of a blood vessel. The explanation that is most attractive is that ADP is liberated from the red blood cells which have been damaged by localized heat from the laser. The heat coagulated red cells, releasing ADP, could initiate platelet adhesivenessand then aggregation. The subsequent rebuilding of the aggregatesfollowing embolization could be attributed to the release reaction in which platelets themselves supply the ADP necessary for continued adhesivenessand clumping. A major unresolved question centers around two observations. First, if the endothelium is undamaged by the laser beam, what causesthe heat coagulated red blood cells to adhere to the vesselwall in the area which has been subjectedto the laser beam? Second, if the endothelium is undamaged by the laser beam, what causes platelets upstream from the site of the aggregateto roll slowly along the vesselwalls as if they had become sticky or adhesive? A suggested explanation, unsupported by experimental evidence, may be that some reaction between the vessel wall and the first cluster of damaged cells activates a protective system which induces platelet adhesiveness and aggregation, thus preventing further deterioration of vascular endothelium. This possibility should be explored. ACKNOWLEDGMENT The author acknowledges the splendid technical assistance of Miss Claire Bernardin. REFERENCES ARFORS,K. E., DHALL, D. P., ENGESET,J., HINT, H., MATHESON,K. A., AND TAGEN,0 (1968). Biolaser endothelial trauma as a means of quantifying platelet activity in cko. Nature(London) 218,887-888. ARFORS,K. E., BERGQUIST,D., BYGDEMAN,S., MCKENZIE, F. N., AND SVENSJO, E. (1972). The effect of the platelet release reaction on platelet behavior in oitro and in oklo. &a&J. Haematol. 9,322-332. BEGENT, N., AND BORN, G. V. R. (1970). Growth rate in uico of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature (London) 227, 926-930. FRENCH, J. E., MACFARLANE, R. G., AND SANDERS, A. G. (1964). The structure of haemostatic plugs and experimental thrombi in small arteries. British J. Exp. Pathol. 45,467-474. How;, T., JORGENSEN,L., PACKHAM, M. A., AND MUSTARD,J. F. (1968). Platelet adherence to fibrin and collagen. J. Lab. C/in. Med. 71,29-40. HOVIG, T., MCKENZIE, F. N., AND ARFORS, K. E. (1974). Measurement of platelet response to laserinduced microvascular injury. Ultrastr. Studies, to be published. KOCHEN, J. A., AND BAEZ, S. (1965). Vascular and intravascular effects of pulsed laser micro-beam. Bibl. Anat. 7, 46-49. KOVACS, I. B., COALAY, L., AND GOROG, D. (1973). Laser-induced thrombosis in the microcirculation of the hamster cheek pouch and its inhibition by acetylsalicylic acid. Microvasc. Res. 6, 194-201. MCKENZIE, F. N., ARFORS, K. E., HOVIG, T., AND MATHESON, N. A. (1972). The mechanism of platelet aggregation at sites of laser-induced microvascular injury-pharmacological and ultrastructural
138
MARY P. WIEDEMAN
studies. In “7th Conf. Europ. Sot. for Microcirculation.” Aberdeen. p. 142. Bib/. Anat. (1973) 12, 18G192, Karger, Basel. MCKENZIE, F. N., ARFORS, K. E., AND MATHESON,N. A. Measurement of platelet response to laserinduced microvascular injury. To be published. POUWODA, H., HAGEMANN, G., AND ZURWEHME,D. (1969). Quantitative analysis concerning the early phase of white thrombi. Bibl. Anat. 10,489-493. SPAET, T. H., AND ERICHSON, R. B. (1966). The vascular wall in the pathogenesis of thrombosis. Thromb. Diath. Haemorrh.
Suppl. 21,67-86.
STEHBENS,W. E., AND BISCOE,T. J. (1967). The ultrastructure of early platelet aggregation in Go. Amer. J. Pathol. 50,212-224.
WIEDEMAN,M. P., AND MARGULIES,E. H. (1973). Factors affecting production of platelet aggregates. In “7th European Conference on Microcirculation.” Aberdeen, Scotland, Part II, Karger-Basel. Bibl. Anat. 12,193-197. WIEDEMAN,M. P. (1973). Platelet aggregates induced by red blood cell injury. In “Oxygen Transport to Tissues” (D. F. Bruley and H. I. Bicher, eds.). Aduan. Exp. Med. Biol. 37B, 681-686. ZUCKER,M. B., AND BORRELLI,J. (1962). Platelet clumping produced by connective tissue suspensions and by collagen. Proc. Sot. Exp. Biol. Med. 109, 779-787.