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1 6I2. II5.3:6I2.62L THE EFFECT OF CHICAGO BLUE AND CHLORAZOLI BLUE ON THE CLOTTING TIME OF THE BLOOD AND ON OVULATION IN THE RABBIT. BY F. W. ROGERS BRAMBELL AND A. S. PARKEES (Foulerton Student of the Royal Society). (From the Department of Zoology, University College of North Wales, Bangor.) I. INTRODUCTION. THE late Dr A. R. Fee had in mind the possibility of performing cross, circulation experiments on the rabbit as a means of investigating certain problems connected with ovulation. Such experiments would involve the use of an anti-coagulant to prevent clotting of the blood for at least 12 hours. The high price of heparine rendered its use on the extensive scale contemplated almost impossible. In considering this problem it was suggested to us by Dr H. P. Gilding that the anti-coagulant properties he and Dr Peyton Rous had observed in Chicago blue 6 B might be utilized. As a preliminary to any cross-circulation experiments it was necessary to investigate the duration of the action of the dye and to ascertain whether it had any effect on ovulation. The present paper is concerned with a brief description of the results of these initial experiments. It was not intended to investigate these properties in detail, but merely to determine whether the dye prolonged the coagulation time sufficiently for our purpose, and to determine the necessary dose. To increase the general usefulness of the work, some additional experiments were carried out on the prevention of coagulation in vitro. II. TECHNIQUE. Source of dye. The dye employed belongs to the diazo group of the organic dyes. Two samples were used, the first (Chicago blue 6 B) was manufactured by the General Dyestuffs Corporation and purified by Dr Gilding by precipitation and dialysis under toluol [193]. The second sample (chlorazol sky blue FF) was supplied by the British Drug Houses, Ltd., and was specially purified by them for the puirpose of PH. LXXiv.5 PH. LXXIV. 5

2 66 F. W. ROGERS BRAMBELL AND A. S. PARKES. these experiments. Both are believed to have the same constitution, but in aqueous solution the chlorazol blue was of a lighter tint. Both samples were found to have similar properties. Administration of the dye. On Dr Gilding's advice, the dye was injected in 8 p.c. aqueous solution, this being isotonic with blood. The solution was freshly made up and boiled before injection. It was found that after standing some days the solution became very toxic, probably owing to bacterial action. Injection was always into an ear vein, the puncture being carefully clipped afterwards. Adult rabbits, kg., were used. Removal of blood samples. Attempts to obtain the required amount from ear veins sufficiently rapidly were unsuccessful, and it was decided to anaesthetize the animals and remove the blood from one of the large vessels. In the first experiment the animals were lightly ansesthetized with urethane, and then put under ether when the samples were removed. Subsequently, the use of ether was avoided and a heavy dose of urethane given (1.5 g. per kg. followed by 3--8 g. per kg. as required). In the first experiment the blood was removed from the jugular or the external saphenous by means of a syringe. In later experiments the blood samples were obtained by bleeding directly from the carotid artery into the vessel in which the clotting time was tested. The cut end of the artery was ligatured after each bleeding and the ligatured portion cut off before the removal of the next sample. When animals died during the course of the experiment the last sample was taken directly from the heart immediately after death. Approximately 3 c.c. of blood were removed each time from the injected animals. Estimation of clotting time. In choosing a method of calculating the clotting time, two difficulties were immediately apparent: (a) the prolonged time taken by many of the samples to show any signs of clotting, (b) the dark colour of the blood. These points complicated the application of any usual method such as the platinum loop [Gi b b s, 1924] or the shot [D ale and Laidlaw, 1912]. Finally, the method used by Dr Pickering in work on hemophilia was adopted. In the first experiment the blood was drawn into short 14 in. tubes, kept at room temperature, and the endpoint of clotting taken as the time when the blood was sufficientlysolid not to flow on tilting the tube at a steep angle. In the second experiment the samples were taken in test-tubes of approximately uniform bore (1 cm.) which had been carefully cleaned, dried and warmed in an incubator at blood temperature (38 C.). The tube was immediately returned to the incubator after the sample was taken. The blood was considered to be

3 CHICAGO BLUE AND CLOTTING TIME. clotted when the tube could be turned upside down without causing displacement. The normal clotting time of the blood of each animal was taken immediately before the injection of the dye. In vitro experiments. Samples (5 c.c.) were drawn into test-tubes from the carotid of a normal rabbit, 5 c.c. of various solutions of the dye being already present. The tubes were left at room temperature, and the criterion of clotting was as in Exp. 2 below. III. ANTI-COAGULANT EFFECT FOLLOWING INTRAVENOUS INJECTION. Exp. 1. The first experiment was performed on three female rabbits injected with 1, 2 and 3 c.c. per kg. of an 8 p.c. solution of Chicago blue 6 B. The data obtained from this experiment are recorded in Table I. For each sample the time of removal after injection of the dye is given in the first column and the clotting time in the second column. TABLE I. Effect of Chicago blue 6 B on the clotting time of blood. Dose 1st sample 2nd sample 3rd sample No. of in c.c., A A animal per kg. hr. min. hr. min. hr. min. hr. min. hr. min. hr. min. CBC CBC CBC Dose 4th sample 5th sample 6th sample No. of in c.c.,, animal per kg. hr. min. hr. min. hr. min. hr. min. k hr. min. hr. min. CBC CBC CBC Samples failing to clot under 4 hours are shown by a dash. Exp. 2. Ten rabbits were employed, pairs being injected with each of the following doses: 1, 14, 2, 2j and 3 c.c. 8 p.c. solution of chlorazol sky blue FF per kg. body weight. Half the animals were males and half females. The results of this experiment are recorded in Table II (arranged as Table I). Those samples marked with an asterisk were taken from the heart immediately after death. The heavy mortality from 7 hours onwards is to be expected, since the dose of urethane was not much below the lethal point and the animals were suffering from the combined effects of the loss of blood, the operation on the neck and the solution of the dye injected

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5 CHICAGO BLUE AND CLOTTING TIME. The results show clearly that the dye prolongs the coagulation time of blood beyond measurable limits for some time after injection. Both the intensity and the duration of the anti-coagulant effect are directly proportional to the dose given. This conclusion is shown more clearly if the results are tabulated as in Table III, in which the results of Exps. 1 and 2 are combined. The plus signs indicate that the samples clotted to standard under 4 hours after removal and the zero signs that they failed to do so. The results recorded in Tables I and II appear to show that the ~ Initial clotting time -7min a Fig. 1. i 2 8l Time after injection of dye (hours) Anti-coagulant effect of chlorazol blue given intravenously (Animal 1, Exp. 2). clotting time of the blood reaches a maximum immediately or very soon after the injection of the dye. It then falls off continuously, at first rapidly and then increasingly slowly. The general type of curve obtained by plotting the clotting time against the time after injection is represented in Fig. 1, in which the data for Animal 1 are plotted. The data for the other animals, although not so complete, support the assumption that the clotting time after injection falls from an initial maximum in a similar manner. The example figured is noteworthy, in that the clotting tim e after injection falls below the normal clotting time as determined before the dye was injected.

6 7 F. W. ROGERS BRAMBELL AND A. S. PARKES. IV. IN VITRO EXPERIMENTS. The highly effective intravenous dose of 2-5 c.c. of 8 p.c. solution per kg. represents about *4 g. per 1 c.c. blood before diffusion begins. It was anticipated that much less would be required in vitro, and the TABLE IV. Effect of adding chlorazol blue to blood in vitro. Solution of dye of which B c.c. added Dye in g. Saniple to 5 c.c. per 1 c.c. Clotting time N o. blood (p.c.) blood hr. nun. - 3t i j S 1 2 1*25 *125 28j 9 1F5 * Failed to clot in 18 howr is, pi, *S Amount of dye added per 1 c.c. blood (mg.) Fig. 2. Anti-coagulant effect of chlorazol blue in vitro. amounts added to the samples ranged from the equivalent of.1 g. up to the equivalent of -35 g. per 1 c.c. of blood (details in Table IV). To keep constant the amount of fluid (.5 c.c.) added to the blood sample, solutions were made up as shown in Table IV. From this

7 CHICAGO BLUE AND CLOTTING TIME. table it will be seen that '15 g. of dye per 1 c.c. blood inhibits clotting for nearly 2 hours, while *175 g. of dye per 1 c.c. blood prevents clotting indefinitely. Clotting time plotted against amount of dye added (Fig. 2) gives a type of curve which suggests that some critical reaction begins as the amount of dye added rises above.1 g. per 1 c.c. of blood. The smallest intravenous dose given represented about -16 g. per 1 c.c. blood, an amount which 3 min. after injection prevented clotting for an average time of nearly 4 min. Allowing for the 3 mm. interval, it is evident that the intravenous results are strictly comparable with those obtained in vitro and that the efficiency of the dye in the two conditions is similar. V. EFFECT ON OVULATION. In some early experiments in which the dye was injected after copulation, two rabbits failed to ovulate. Further work, however, did not reveal any constant disturbance of ovulation. Details of the eleven animals used are given in Table IV. TABLE IV. Effect of Chicago blue 6 B on ovulation. Time of Dose in c.c. injection Number of Number of per kg. 8 p.c. post coitum follicles animal solution mi. ovulating SOR 1 2*5 2 + SOR 2 2* SOR SOR SOR 6 2X SOR7 2X5 4 + SOR SOR SOR1 2X SORll 2X CBC The dose was large in all animals except SOR 5, and it was administered at various times during the first hour post coitum. Of the four animals which failed to ovulate, one (SOR 9) had definitely immature follicles, and one (SOR 7) exhibited pre-ovulation changes before death at 13 hours post coitum. In view of the fact that about 2 p.c. of normal rabbits fail to ovulate after copulation, these results show conclusively that the dye in the doses given does not inhibit ovulation. V. SUMMARY. 1. Chicago blue 6 B (chlorazol sky blue FF) given intravenously has a marked anti-coagulant effect on the blood of rabbits. 2*5 c.c. per kg. of 71

8 72 F. W. ROGERS BRAMBELL AND A. S. PARKES. an 8 p.c. solution prolongs the clotting time to over 3j hours in samples taken 16 hours after injection. Samples taken earlier than this fail to clot in a significant time. After injection of 1 c.c. per kg. the clotting time returns to normal in about 4 hours. 2. Similar doses failed to show any adverse effect on ovulation. 3. The dye is therefore highly suitable for work on ovulation requiring the use of an anti-coagulant. 4. In vitro, 175 mg. of the dye per 1 c.c. of blood inhibits clotting indefinitely. We are much indebted to Dr Gilding for suggesting to us the use of the dye and for providing us with specially purified material. We are also indebted to Dr F. H. Carr of the British Drug Houses, Ltd., for having the second sample specially purified; to Dr J. W. Pickering for valuable advice on the technique of measuring the coagulation time of the blood, etc.; and to Miss M. Hill who assisted with some of the initial experiments. The expenses of the research were defrayed by a Grant from the Government Grants Committee of the Royal Society to one of us (F. W. R. B.). REFERENCES. Dale, H. H. and Laidlaw, P. P. (1912). J. Path. Bact. 16, 351. Gibbs,. S. (1924). Quart. J. Med. 17, 312. Rous, P., Gilding, H. P. and Smith, F. (193). J. Exp. Med. 51, 87.