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Experimental thrombus formation in mesenteric microvessels: Evaluation of a method
MICROVASCULAR
RESEARCH
6, 108-115 (1973)
TECHNICAL
RE PORT
Experimental Thrombus Formation in Mesenteric Microvessels: Evaluation of a Method1 J. L. GORDON, R. J. EVANS, AND G. A. GRESHAM Department of Pathology, Cambridge University, Cambridge CB2 1 QP, England Received October 25,1972 Platelet thrombi in rats are produced at sites of electrically induced damage in mesenteric venules. The thrombus formation response is dependent on the temperature of the bathing fluid and varies with electrode polarity. Cathodal stimulation causes vasoconstriction and induces platelet aggregation after a slight delay, while anodal stimulation induces the rapid formation of aggregates containing some white cells and erythrocytes, without any vasoconstriction. The effects of cathodal and anodal stimulation can be simulated by topical application of isotonic saline adjusted to high or low pH, respectively. Ultrastructural studies showed that aggregated platelets remaining at the stimulation site lose some of their organelles after 10 min (platelet emboli were found in liver venules of gerbils after thrombi had been produced in the ileal veins). The thrombus formation response cannot be satisfactorily quantified by measuring either rate or duration of embolisation, but recording the number of thrombi produced by a group of “threshold” stimuli is a simple, semi-objective means of assessment, and gives reproducible results in control animals.
INTRODUCTION Experimental studies on thrombogenesis in small blood vesselshave been reported using the microcirculation of the hamster cheekpouch (Berman, 1961;Begentand Born, 1970), the cerebral cortical arteries of rabbits (Honour and Ross Russell, 1962)or rats (Born and Philp, 1965),the regeneratedvesselsof the rabbit ear chamber (Arfors et al., 1968),the ileal vesselsof rats (Didisheim, 1968)or mice (Cowan and Monkhouse, 1966), and the mesentericmicrocirculation of rats (Reber, 1966; Gordon and Gresham, 1970; Wenzel and Richards, 1970). Thesestudies have been performed in animals of different species,in vesselsdiffering widely in size and location, and employing biochemical (Begent and Born, 1970; Wenzel and Richards, 1970) mechanical (Honour and Ross Russell 1962; Born and Philp, 1965),electrical (Reber, 1966; Cowan and Monkhouse, 1966; Didisheim, 1968; Gordon and Gresham, 1970), or thermal (Arfors et al., 1968)stimuli to induce thrombus formation. There is, however, little information available on the effectsof experimental variables on thrombus formation, and the relevance of microcirculation studies to systemic thrombosis
is seldom discussed.
* This study was supported by a grant from Beecham Research Laboratories. Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain.
108
TECHNICAL
REPORT
109
This report summarizes the standardisation and evaluation of one technique for studying thrombus formation in mesenteric microvessels. METHODS Animal Preparation
The technique used was based on that previously described (Gordon and Gresham, 1970),and involved observation of the microcirculation in the exposed mesenterywith an inverted microscope. The mesentery was irrigated by connecting a thin-walled polyethylene catheter (OS-mm bore) to a reservoir of isotonic saline, with the height of the reservoir adjusted to maintain a flow rate of IO-12 drops per min on the preparation. Excesssaline was constantly removed by a cellulose wick at the opposite end of the mesentery. The temperature of this saline drip could be maintained at 37” by a spiral of insulated Nichrome wire around the catheter tip with the ends of the wire connected to a 12-V alternating-current supply. Thrombus formation was studied at sites of vascular damageproduced by a constant voltage, constant-current pulse from a square-wavestimulator. The duration of stimulation was varied to produce the desired effect, and results were recorded on 16-mm colour film, with a tine camera connected to the back of the microscope by an optical tube and beam-splitter. Electrode Corutructiorl
Tungsten wire 0.2 mm in diameter, electropolished in 4 % potassium hydroxide, then cleaned in xylene and ethanol and insulated with resin except for the tip produced excellent microelectrodes; they were more durable than platinum or steel, and less prone to collect tissue at the tip. Electrical Stimulation
Square-wave 150-V pulses provided an easily standardised means of inflicting local vascular damage; the current was maintained at 8 mA by a 1 MSZvariable resistor, and a current-limiting 30 KQ resistor was included to protect the output stageof the stimulator against overloading at high current. Pulse duration was adjusted to produce the desired thrombus formation response, and both current intensity and pulse shape were checked on a cathode ray oscilloscope. RESULTS Animal Species
Rats were the most suitable animals for routine use. No significant relation between thrombus formation and age or sex was found, but animals used routinely were at least 4 mo old, because in younger animals the mesenteric microcirculation was poorly developed. Adult rabbits were unsuitable becauseof their size, and young animals had a sparse mesenteric microcirculation. Peristalsis made accurate alignment of the electrode difficult. This criticism also applied to guinea pigs, in which peristaltic movement was
110
TECHNICAL
REPORT
even stronger. In mice, the mesenteric microcirculation was scanty, and the ileal blood vesselswere too short to allow extensive microscopic observation. The vesselswere usually surrounded by fat, which made it difficult to inflict focal lesions on the walls, and impossible to record thrombus formation in detail. Mongolian gerbils (Meriones unguiculatus), although possessing only a limited mesenteric microcirculation, had ileal vesselsthat were virtually free from perivascular fat, and were sufficiently long to allow detailed microscopic observation. The venous structure allowed more thrombi and emboli to be produced than was poSsible in rat venules, and these animals were used to study the fate of thrombi produced in ileal veins. Anesthetic
In Reber’s (1966) description of thrombus formation in rat mesenteric microvessels, the anesthetic used was sodium allobarbitone, injected intraperitoneally. Our early work, using intraperitoneal anesthetics, established that the microcirculation in many animals was poor, with several vesselsin which the blood had stopped flowing. After an intramuscular injection of pentobarbitone sodium, the incidence of damaged mesenteric microvessels was negligible, and anesthesiawas satisfactory provided that small supplementary injections were given every 40-60 min. Polarity, pH and Thrombus Formation
Quantitative studies of the thrombus formation response were made using both cathodal and anodal stimulation. The mesenteric arterioles were previously used to study thrombus formation in this preparation (Gordon and Gresham, 1970), but it was subsequently found that in venules a more consistent sequenceof thrombus growth and embolization was obtained, probably becauseof the slower and less variable rate of blood flow. The darker colour of venous blood also afforded better conditions for observing and filming thrombi. No difference in thrombus formation response was seen in venules between 20 and 40 pm diameter; all experiments reported here were performed on vesselswithin this size range. Minor changes in the distance between electrode tip and vessel wall altered the response; when the tip was approximately 10 pm from the vessel, the mean pulse duration required to produce a discernible thrombus in 100experiments on 20 rats was 51 + 12 SD msec. If the electrode tip was in direct contact with the vessel and compressedit slightly, the mean pulse duration required to produce a thrombus was only 14.4 & 2.4 SD msec. Stimulation with either cathode or anode was virtually equiactive in producing thrombi, but there were qualitative differences in the responses.Anodal stimulation produced no dhangein vesseldiameter, even in arterioles, and thrombi formed rapidly after stimulation. Cathodal stimulation causedvasoconstriction in arterioles, and there was usually a delay of several secondsbefore thrombi started to form after cathodal stimulation. Thrombi which formed in venules after cathodal stimulation were composed almost entirely of aggregatedplatelets, with occasional leukocytes present, but anodal stimulation produced thrombi in which severalleukocytes and erythrocytes were incorporated.
TECHNICAL REPORT
111
Since the electrical current was passed through the saline bathing the tissue there was some electrolysis, which often manifested in the formation of visible gas bubbles at the microelectrode tip. The gas bubbles dispersed within a few seconds, and less gas evolved after anodal than after cathodal stimulation. After repeated stimulation at one site, changes in the pH of the saline around the electrode tip could be detected by dipping the end of a thin strip of pH multi-indicator paper into the fluid immediately surrounding the electrode tip. Following repeated cathodal stimulation the pH increased to 10 or 11, and after repeated anodal stimulation it fell to 2 or 3. The topical application of isotonic saline, with the pH adjusted to 11 or 2 by sodium hydroxide or hydrochloric acid, produced results similar to those observed after cathodal or anodal stimulation respectively; saline at pH 11 caused intense vasoconstriction in arterioles and formation of platelet aggregates (particularly in venules), while saline at pH 2 caused no vasoconstriction but produced “sludging” in venules, that is, aggregation of all blood cells. Temperature In 40 experiments on 5 animals, the mean duration of stimulation required was 14.2 rt 1.OSD msec with a 37” saline drip, and 19.4 f 1.2 SD msec with a 24” saline drip; the difference was highly significant. No obvious change in flow rate through the microvascular bed was observed during the change in environmental temperature.
FIG. la. Electron micrograph of platelet aggregate in rat mesenteric venule. Stimulus 150 V, 8 mA, 20 msec, with cathode. Fixed in 4% glutaraldehyde, 2 min after stimulation. x 15,600. Endothelium absent, and platelets closely apposed to internal elastic lamina (TEL). M-mitochondria; V-vacuoles; A--n granules; G-glycogen.
112
TECHNICAL REPORT
Structure and Fate of Thrombi The platelets which aggregated at sites of electrically-induced damage in venules were tightly packed but retained most of their internal structures (Fig. la). In thrombi which remained at the site of stimulation, without embolising, the aggregatedplatelets soon lost some of their organelles, and by 10 min after stimulation there were signs of membrane disintegration (Fig. 1b). To investigate the fate of the embolising fragments, thrombi were produced in the ileal veins of four gerbils, and allowed to embolize for 5-10 min. The animals were then killed, and their livers excised, fixed in 10% formalin, and examined histologically. In all animals, small platelet aggregates were seen in several liver venules, apparently adhering to the vesselwalls (Fig. 2). No aggregateswere seenin livers of control animals which were subjected to an identical surgical procedure, but without the production of thrombi. Assessment-of Response The usual pattern after stimulation was an irregular sequenceof thrombus growth and embolization lasting up to 15 min. Thrombi could be produced predictably by applying successiveidentical stimuli at different sites in the sameanimal, but the rate and duration of embolization were variable: in a series of 64 experiments on control animals, the mean number of emboli in the first 5 min after stimulation was 3.95 & 3.96 SD. This variability was not primarily due to inter-animal differences,as illustrated in
FIG. lb. Electron micrograph of platelet aggregate produced as above, but fixed 10 min after stimulation. Platelets with degranulated areas, and “clustering” of remaining organelles. Arrows show points of apparent membrane disintegration.
TECHNICALREPORT
FIG. 2. Light micrograph of platelet embolus (P) in venule of gerbililiver. Fixed 10 min after production of thrombus in ileal vein. Picromallory stain. x 130. 16144
’ 2-
.E lo .E
IOa-
E ,a .-
6-
E
42O, 123456 Stimulations
FIG. 3. Emboli count after production of platelet thrombi in rat mesenteric venules. Six successive thrombi produced in each animal by cathodal pulses, and emboli recorded for 5 min after each stimulus.
114
TECHNICAL
REPORT
Fig. 3, which shows the emboli counts for six successivethrombi produced in each of five animals. Becauseembolization measurementswere variable, we found it preferable to assess the response by determining the pulse duration required to produce a discernible thrombus. Initially, the stimulus which always caused thrombus formation was determined, but later tests showed that a more accurate and objective result was obtained by establishing a “threshold” stimulus (usually 10 to 15 msec in duration) and then recording the number of discernible thrombi produced by ten successivethreshold stimulations. The reproducibility of this system was checked by determining the thrombus formation incidence (TFI) in individual animals, using two groups of ten threshold stimuli, with the second group applied 15 min after the first. In 16 control animals the mean TFI for the first group of stimuli was 8.75 & 1.06 SD, and for the second group, 8.56 & 0.96 SD. This procedure is being usedto test potential anti-thrombotic agentsin z&o, and hence a stimulus was selectedto give a control TFI around 8, so as to facilitate detection of any reduction in responseafter administration of an agent. Where the possibility of an increased thrombus formation response is anticipated, the stimulus may be adjusted to give a control TFI around 5. DISCUSSION The possible use of this type of preparation to study potential anti-thrombotic agents depends on being able to quantify the thrombus formation response.Quantitation by meansof the embolization rate or duration is not satisfactory in this preparation, although these parameters have been used previously to assessthrombus formation in mechanically damaged cerebral vessels (Honour and Ross Russell, 1962) and laserdamaged vesselsin the rabbit ear chamber (Arfors et al., 1968). Visual assessmentof thrombus formation is necessarilysubjective, but we believe that the subjective element is minimised when the responseis assessedon an “all or none” basis (i.e., presenceor absence of a thrombus). Recording the number of thrombi formed by a group of “threshold” stimuli is a simple, semi-objective method of assessmentand we have used this technique successfully for in uivo studies with platelet aggregation inhibitors (Gordon and Evans, in preparation). The relevance of such studies may be questioned on the grounds of the “non-physiological“ nature of the initiating stimulus, but if platelet thrombus formation can be considered as a type of repair process, it should be largely independent of the initial damaging stimulus. Hence, anything which affects thrombogenesis at sites of electrically induced vascular damagemight well have a similar effect on thrombi which form when vessels have been damaged by other factors such as hemodynamic stress or biochemical agents. REFERENCES
ARFORS,K-E., DHALL, D. P., ENGESET,J., HINT, H., MATHESON,N. A., ANDTANGEN,0. (1968). Biolaser endothelial trauma as a means of quantifying platelet aggregation. Nature (London) 218,887-888. BEGENT,N., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature (London) 227,926-930.
TECHNICAL REPORT
115
H. J. (1961). Anticoagulant-induced alterations in haemostasis, platelet thrombosis, and vascular fragility in the peripheral vesselsof the hamster cheek pouch. In “Anticoagulants and Fibrinolysins” (R. L. Macmillan and J. F. Mustard, eds.), pp. 95-107. Lea and Febiger, Philadelphia. BORN, G. V. R., AND PHILP, R. B. (1965). Effect of anesthetics on the duration of embolisation of platelet thrombi in injured blood vessels. Natwe (London) 205, 398400. COWAN, C. R., AND MONKHOUSE, F. C. (1966). Studies on electrically-induced thrombosis and related phenomena. Can. J. Physiol. Pharmacol. 44, 881-886. DIDISHEIM, P. (1968). Inhibition by dipyridamole of arterial thrombosis in rats. Thromb. Diath. Haem-
BERMAN,
orrh.
(Stuttg.)
20, 257-266.
GORDON,J. L., AND GRESHAM,G. A. (1970). The production of thrombosis in rat mesenteric vessels. Zn“Platelets and Vessel Wall-Fibrin Deposition” (G. Schettler, ed), pp. 107-l 14. Thieme, Stuttgart. HONOUR,A. J., AND Ross RUSSELL,R. W. (1962). Experimental platelet embolism. Brit. J. Exp. Pathol. 43,350-362. REBER,K. (1966). A device for the production of well-defined lesions of mesenteric blood vessels, with resulting platelet thrombi. Thromb. Diath. Haemorrh. (Stuttg.) 15,471475. WENZEL, D. G., AND RICHARDS,M. H. (1970). Decreased thrombus formation in rats after chronic nicotine administration. Eur. J. Pharmacol. 10. 143-144.
RESEARCH
6, 108-115 (1973)
TECHNICAL
RE PORT
Experimental Thrombus Formation in Mesenteric Microvessels: Evaluation of a Method1 J. L. GORDON, R. J. EVANS, AND G. A. GRESHAM Department of Pathology, Cambridge University, Cambridge CB2 1 QP, England Received October 25,1972 Platelet thrombi in rats are produced at sites of electrically induced damage in mesenteric venules. The thrombus formation response is dependent on the temperature of the bathing fluid and varies with electrode polarity. Cathodal stimulation causes vasoconstriction and induces platelet aggregation after a slight delay, while anodal stimulation induces the rapid formation of aggregates containing some white cells and erythrocytes, without any vasoconstriction. The effects of cathodal and anodal stimulation can be simulated by topical application of isotonic saline adjusted to high or low pH, respectively. Ultrastructural studies showed that aggregated platelets remaining at the stimulation site lose some of their organelles after 10 min (platelet emboli were found in liver venules of gerbils after thrombi had been produced in the ileal veins). The thrombus formation response cannot be satisfactorily quantified by measuring either rate or duration of embolisation, but recording the number of thrombi produced by a group of “threshold” stimuli is a simple, semi-objective means of assessment, and gives reproducible results in control animals.
INTRODUCTION Experimental studies on thrombogenesis in small blood vesselshave been reported using the microcirculation of the hamster cheekpouch (Berman, 1961;Begentand Born, 1970), the cerebral cortical arteries of rabbits (Honour and Ross Russell, 1962)or rats (Born and Philp, 1965),the regeneratedvesselsof the rabbit ear chamber (Arfors et al., 1968),the ileal vesselsof rats (Didisheim, 1968)or mice (Cowan and Monkhouse, 1966), and the mesentericmicrocirculation of rats (Reber, 1966; Gordon and Gresham, 1970; Wenzel and Richards, 1970). Thesestudies have been performed in animals of different species,in vesselsdiffering widely in size and location, and employing biochemical (Begent and Born, 1970; Wenzel and Richards, 1970) mechanical (Honour and Ross Russell 1962; Born and Philp, 1965),electrical (Reber, 1966; Cowan and Monkhouse, 1966; Didisheim, 1968; Gordon and Gresham, 1970), or thermal (Arfors et al., 1968)stimuli to induce thrombus formation. There is, however, little information available on the effectsof experimental variables on thrombus formation, and the relevance of microcirculation studies to systemic thrombosis
is seldom discussed.
* This study was supported by a grant from Beecham Research Laboratories. Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain.
108
TECHNICAL
REPORT
109
This report summarizes the standardisation and evaluation of one technique for studying thrombus formation in mesenteric microvessels. METHODS Animal Preparation
The technique used was based on that previously described (Gordon and Gresham, 1970),and involved observation of the microcirculation in the exposed mesenterywith an inverted microscope. The mesentery was irrigated by connecting a thin-walled polyethylene catheter (OS-mm bore) to a reservoir of isotonic saline, with the height of the reservoir adjusted to maintain a flow rate of IO-12 drops per min on the preparation. Excesssaline was constantly removed by a cellulose wick at the opposite end of the mesentery. The temperature of this saline drip could be maintained at 37” by a spiral of insulated Nichrome wire around the catheter tip with the ends of the wire connected to a 12-V alternating-current supply. Thrombus formation was studied at sites of vascular damageproduced by a constant voltage, constant-current pulse from a square-wavestimulator. The duration of stimulation was varied to produce the desired effect, and results were recorded on 16-mm colour film, with a tine camera connected to the back of the microscope by an optical tube and beam-splitter. Electrode Corutructiorl
Tungsten wire 0.2 mm in diameter, electropolished in 4 % potassium hydroxide, then cleaned in xylene and ethanol and insulated with resin except for the tip produced excellent microelectrodes; they were more durable than platinum or steel, and less prone to collect tissue at the tip. Electrical Stimulation
Square-wave 150-V pulses provided an easily standardised means of inflicting local vascular damage; the current was maintained at 8 mA by a 1 MSZvariable resistor, and a current-limiting 30 KQ resistor was included to protect the output stageof the stimulator against overloading at high current. Pulse duration was adjusted to produce the desired thrombus formation response, and both current intensity and pulse shape were checked on a cathode ray oscilloscope. RESULTS Animal Species
Rats were the most suitable animals for routine use. No significant relation between thrombus formation and age or sex was found, but animals used routinely were at least 4 mo old, because in younger animals the mesenteric microcirculation was poorly developed. Adult rabbits were unsuitable becauseof their size, and young animals had a sparse mesenteric microcirculation. Peristalsis made accurate alignment of the electrode difficult. This criticism also applied to guinea pigs, in which peristaltic movement was
110
TECHNICAL
REPORT
even stronger. In mice, the mesenteric microcirculation was scanty, and the ileal blood vesselswere too short to allow extensive microscopic observation. The vesselswere usually surrounded by fat, which made it difficult to inflict focal lesions on the walls, and impossible to record thrombus formation in detail. Mongolian gerbils (Meriones unguiculatus), although possessing only a limited mesenteric microcirculation, had ileal vesselsthat were virtually free from perivascular fat, and were sufficiently long to allow detailed microscopic observation. The venous structure allowed more thrombi and emboli to be produced than was poSsible in rat venules, and these animals were used to study the fate of thrombi produced in ileal veins. Anesthetic
In Reber’s (1966) description of thrombus formation in rat mesenteric microvessels, the anesthetic used was sodium allobarbitone, injected intraperitoneally. Our early work, using intraperitoneal anesthetics, established that the microcirculation in many animals was poor, with several vesselsin which the blood had stopped flowing. After an intramuscular injection of pentobarbitone sodium, the incidence of damaged mesenteric microvessels was negligible, and anesthesiawas satisfactory provided that small supplementary injections were given every 40-60 min. Polarity, pH and Thrombus Formation
Quantitative studies of the thrombus formation response were made using both cathodal and anodal stimulation. The mesenteric arterioles were previously used to study thrombus formation in this preparation (Gordon and Gresham, 1970), but it was subsequently found that in venules a more consistent sequenceof thrombus growth and embolization was obtained, probably becauseof the slower and less variable rate of blood flow. The darker colour of venous blood also afforded better conditions for observing and filming thrombi. No difference in thrombus formation response was seen in venules between 20 and 40 pm diameter; all experiments reported here were performed on vesselswithin this size range. Minor changes in the distance between electrode tip and vessel wall altered the response; when the tip was approximately 10 pm from the vessel, the mean pulse duration required to produce a discernible thrombus in 100experiments on 20 rats was 51 + 12 SD msec. If the electrode tip was in direct contact with the vessel and compressedit slightly, the mean pulse duration required to produce a thrombus was only 14.4 & 2.4 SD msec. Stimulation with either cathode or anode was virtually equiactive in producing thrombi, but there were qualitative differences in the responses.Anodal stimulation produced no dhangein vesseldiameter, even in arterioles, and thrombi formed rapidly after stimulation. Cathodal stimulation causedvasoconstriction in arterioles, and there was usually a delay of several secondsbefore thrombi started to form after cathodal stimulation. Thrombi which formed in venules after cathodal stimulation were composed almost entirely of aggregatedplatelets, with occasional leukocytes present, but anodal stimulation produced thrombi in which severalleukocytes and erythrocytes were incorporated.
TECHNICAL REPORT
111
Since the electrical current was passed through the saline bathing the tissue there was some electrolysis, which often manifested in the formation of visible gas bubbles at the microelectrode tip. The gas bubbles dispersed within a few seconds, and less gas evolved after anodal than after cathodal stimulation. After repeated stimulation at one site, changes in the pH of the saline around the electrode tip could be detected by dipping the end of a thin strip of pH multi-indicator paper into the fluid immediately surrounding the electrode tip. Following repeated cathodal stimulation the pH increased to 10 or 11, and after repeated anodal stimulation it fell to 2 or 3. The topical application of isotonic saline, with the pH adjusted to 11 or 2 by sodium hydroxide or hydrochloric acid, produced results similar to those observed after cathodal or anodal stimulation respectively; saline at pH 11 caused intense vasoconstriction in arterioles and formation of platelet aggregates (particularly in venules), while saline at pH 2 caused no vasoconstriction but produced “sludging” in venules, that is, aggregation of all blood cells. Temperature In 40 experiments on 5 animals, the mean duration of stimulation required was 14.2 rt 1.OSD msec with a 37” saline drip, and 19.4 f 1.2 SD msec with a 24” saline drip; the difference was highly significant. No obvious change in flow rate through the microvascular bed was observed during the change in environmental temperature.
FIG. la. Electron micrograph of platelet aggregate in rat mesenteric venule. Stimulus 150 V, 8 mA, 20 msec, with cathode. Fixed in 4% glutaraldehyde, 2 min after stimulation. x 15,600. Endothelium absent, and platelets closely apposed to internal elastic lamina (TEL). M-mitochondria; V-vacuoles; A--n granules; G-glycogen.
112
TECHNICAL REPORT
Structure and Fate of Thrombi The platelets which aggregated at sites of electrically-induced damage in venules were tightly packed but retained most of their internal structures (Fig. la). In thrombi which remained at the site of stimulation, without embolising, the aggregatedplatelets soon lost some of their organelles, and by 10 min after stimulation there were signs of membrane disintegration (Fig. 1b). To investigate the fate of the embolising fragments, thrombi were produced in the ileal veins of four gerbils, and allowed to embolize for 5-10 min. The animals were then killed, and their livers excised, fixed in 10% formalin, and examined histologically. In all animals, small platelet aggregates were seen in several liver venules, apparently adhering to the vesselwalls (Fig. 2). No aggregateswere seenin livers of control animals which were subjected to an identical surgical procedure, but without the production of thrombi. Assessment-of Response The usual pattern after stimulation was an irregular sequenceof thrombus growth and embolization lasting up to 15 min. Thrombi could be produced predictably by applying successiveidentical stimuli at different sites in the sameanimal, but the rate and duration of embolization were variable: in a series of 64 experiments on control animals, the mean number of emboli in the first 5 min after stimulation was 3.95 & 3.96 SD. This variability was not primarily due to inter-animal differences,as illustrated in
FIG. lb. Electron micrograph of platelet aggregate produced as above, but fixed 10 min after stimulation. Platelets with degranulated areas, and “clustering” of remaining organelles. Arrows show points of apparent membrane disintegration.
TECHNICALREPORT
FIG. 2. Light micrograph of platelet embolus (P) in venule of gerbililiver. Fixed 10 min after production of thrombus in ileal vein. Picromallory stain. x 130. 16144
’ 2-
.E lo .E
IOa-
E ,a .-
6-
E
42O, 123456 Stimulations
FIG. 3. Emboli count after production of platelet thrombi in rat mesenteric venules. Six successive thrombi produced in each animal by cathodal pulses, and emboli recorded for 5 min after each stimulus.
114
TECHNICAL
REPORT
Fig. 3, which shows the emboli counts for six successivethrombi produced in each of five animals. Becauseembolization measurementswere variable, we found it preferable to assess the response by determining the pulse duration required to produce a discernible thrombus. Initially, the stimulus which always caused thrombus formation was determined, but later tests showed that a more accurate and objective result was obtained by establishing a “threshold” stimulus (usually 10 to 15 msec in duration) and then recording the number of discernible thrombi produced by ten successivethreshold stimulations. The reproducibility of this system was checked by determining the thrombus formation incidence (TFI) in individual animals, using two groups of ten threshold stimuli, with the second group applied 15 min after the first. In 16 control animals the mean TFI for the first group of stimuli was 8.75 & 1.06 SD, and for the second group, 8.56 & 0.96 SD. This procedure is being usedto test potential anti-thrombotic agentsin z&o, and hence a stimulus was selectedto give a control TFI around 8, so as to facilitate detection of any reduction in responseafter administration of an agent. Where the possibility of an increased thrombus formation response is anticipated, the stimulus may be adjusted to give a control TFI around 5. DISCUSSION The possible use of this type of preparation to study potential anti-thrombotic agents depends on being able to quantify the thrombus formation response.Quantitation by meansof the embolization rate or duration is not satisfactory in this preparation, although these parameters have been used previously to assessthrombus formation in mechanically damaged cerebral vessels (Honour and Ross Russell, 1962) and laserdamaged vesselsin the rabbit ear chamber (Arfors et al., 1968). Visual assessmentof thrombus formation is necessarilysubjective, but we believe that the subjective element is minimised when the responseis assessedon an “all or none” basis (i.e., presenceor absence of a thrombus). Recording the number of thrombi formed by a group of “threshold” stimuli is a simple, semi-objective method of assessmentand we have used this technique successfully for in uivo studies with platelet aggregation inhibitors (Gordon and Evans, in preparation). The relevance of such studies may be questioned on the grounds of the “non-physiological“ nature of the initiating stimulus, but if platelet thrombus formation can be considered as a type of repair process, it should be largely independent of the initial damaging stimulus. Hence, anything which affects thrombogenesis at sites of electrically induced vascular damagemight well have a similar effect on thrombi which form when vessels have been damaged by other factors such as hemodynamic stress or biochemical agents. REFERENCES
ARFORS,K-E., DHALL, D. P., ENGESET,J., HINT, H., MATHESON,N. A., ANDTANGEN,0. (1968). Biolaser endothelial trauma as a means of quantifying platelet aggregation. Nature (London) 218,887-888. BEGENT,N., AND BORN, G. V. R. (1970). Growth rate in vivo of platelet thrombi, produced by iontophoresis of ADP, as a function of mean blood flow velocity. Nature (London) 227,926-930.
TECHNICAL REPORT
115
H. J. (1961). Anticoagulant-induced alterations in haemostasis, platelet thrombosis, and vascular fragility in the peripheral vesselsof the hamster cheek pouch. In “Anticoagulants and Fibrinolysins” (R. L. Macmillan and J. F. Mustard, eds.), pp. 95-107. Lea and Febiger, Philadelphia. BORN, G. V. R., AND PHILP, R. B. (1965). Effect of anesthetics on the duration of embolisation of platelet thrombi in injured blood vessels. Natwe (London) 205, 398400. COWAN, C. R., AND MONKHOUSE, F. C. (1966). Studies on electrically-induced thrombosis and related phenomena. Can. J. Physiol. Pharmacol. 44, 881-886. DIDISHEIM, P. (1968). Inhibition by dipyridamole of arterial thrombosis in rats. Thromb. Diath. Haem-
BERMAN,
orrh.
(Stuttg.)
20, 257-266.
GORDON,J. L., AND GRESHAM,G. A. (1970). The production of thrombosis in rat mesenteric vessels. Zn“Platelets and Vessel Wall-Fibrin Deposition” (G. Schettler, ed), pp. 107-l 14. Thieme, Stuttgart. HONOUR,A. J., AND Ross RUSSELL,R. W. (1962). Experimental platelet embolism. Brit. J. Exp. Pathol. 43,350-362. REBER,K. (1966). A device for the production of well-defined lesions of mesenteric blood vessels, with resulting platelet thrombi. Thromb. Diath. Haemorrh. (Stuttg.) 15,471475. WENZEL, D. G., AND RICHARDS,M. H. (1970). Decreased thrombus formation in rats after chronic nicotine administration. Eur. J. Pharmacol. 10. 143-144.