blood-pressure, heart rate and web circulation on the other. Spinal frogs were used. The blood-pressure was recorded by means of

Transcription

1 6I2.59:6I2.I3 THE INFLUENCE OF TEMPERATURE IN THE FROG. (I) On the circulation, and (2) On the circulatory effects of adrenaline and of sodium nitrite. BY W. C. CULLIS AND E. M. SCARBOROUGH. (London (R.F.H.) School of Medicine for Women.) WITH a view to testing tbe effects of temperature on the action of histamine upon the circulatory system of the frog, a series of experiments was undertaken. This consisted of perfusion experiments, in which the bloodpressure variations were recorded from the aorta, and the changes in calibre of web vessels were studied, either by direct measurement or by observing the flow through them of solutions containing dyes or suspended particles. As a result of these investigations it became clear that, before the effects of change of temperature on the action of a drug could be determined, it was necessary to study the effects on the system of temperature variations alone. Also that satisfactory observations could only be expected when working as far as possible with an intact circulation. A preliminary series of experiments was then undertaken to determine the relationship-if any-between temperature on the one hand, and blood-pressure, heart rate and web circulation on the other. METHOD. Spinal frogs were used. The blood-pressure was recorded by means of a water manometer connected with the right arch of the aorta; the heart rate was counted directly through the small incision; and the web circulation observed in the foot attached to a microscope stage. It was found that the natural circulation through the web was not interfered with by the manipulative procedure, provided that bleeding was avoided and that the blood-pressure cannula caused no blocking of the left arch of the aorta. The temperature of the frog was controlled by water dripping onta a cotton-wool jacket surrounding the preparation. The temperature was recorded from a small thermometer inserted into the thoracico-abdominal cavity through the incision. PH. LXxV. 3

2 34 W. C. CULLIS AND E. M. SCARBOROUGH. RESULTS. Figs. 1 and 2 show the results, given in a condensed form, of two such experiments. A brief analysis of the results obtained from twenty-two experiments in which the temperature was the variable factor showed the ollowing: 4t A. 0 ~ E i B K It-I t 40.t E 11 a A. S4e k TX,,b 01TARMEVRAE 17 'Z z W11 *I 60 k sk s-.- - z k k k 40,1 to CA#rT RAtE Q \ --_o * rer' AJ"0 leap. IL.. -. HOURS 1 A I I Fih g. 1. HourS 1 2 Fig. 2. Fig. 1. Effects of fall of temperature on blood-pressure, heart rate and web circulation. Fig. 2. Effects of rise of temperature on blood-pressure, heart rate and web circulation. 1. Confirmatory to the results obtained by Taylor [1930], the relationship between temperature and h e art rate was found to be a definitely linear one, except at high temperature (25 C. and upwards). 2. The b loo d - pr e s s u r e readings were variable both at constant and at changing temperatures, and there was difficulty in establishing any definite relationship between temperature and blood-pressure. Over prolonged periods, however, cold tended to arrest or recover a falling blood-pressure, whereas heat accelerated a fall. Further investigation of this relationship is at present being made, making use of a subsequent improvement of technique.

3 INFLUENCE OF TEMPERATURE IN THE FROG The we b circulation varied chiefly in rate of flow and in the number rather than in the calibre of active vessels. Under the conditions of these experiments the blood-pressure appeared to be a more important determining factor in control of the web circulation than the temperature. Fig. 3. Copper-box apparatus, with outlined position of frog. A, open lid; B, base inset into cork; C, inlet for water (under cork); D, communication between base and lid; E, outlet for water; F, cannula in aorta for blood-pressure; G, thermometer; H, syringe needle in lymph sac; J, web prepared for microscope stage. Changes of temperature between 10 and 200 C., unless accompanied by some gross change in the blood-pressure, produced very slight variations, if any. Between 20 and 250 C. the web circulation often appeared to be brisker and more open (using " open " in reference to the number, not the calibre, of active vessels). Above 250 C. it became sluggish and closed 3-2

4 36 W. C. CULLIS AND E. M. SCARBOROUGH. (i.e. number of active vessels diminished), coinciding with the general failure of circulation due to the breakdown of the temperature and heart-rate relationship and the consequent fall of blood-pressure. Below 100 C. a typical "cold effect" was frequently observed. The web circulation was sluggish and the field closed, with marked congestion and pulsation in the active vessels. This "cold effect" developed even when the blood-pressure was well maintained, and, unlike the circulatory failure produced at high temperatures, it was not a death change, but one from which the animal could be completely recovered by warmth. With the object of controlling the influence of temperature on the circulation more carefully, the experimental method was modified by the introduction into the apparatus of a copper box, within which the frog was enclosed. Through the hollow base and lid of the box flowed a stream of water kept at constant temperature, or varied as required' (Fig. 3). The apparatus was tested by repeating the preceding experiments, with confirmatory results. With this modification it was possible to demonstrate the maintenance of a steady temperature, blood-pressure, heart rate and web circulation over periods up to I1 hours, and the method, therefore, seemed suitable for use in the investigation of effects produced by drugs upon the circulation. EXPERIMENTAL WORK UPON THE EFFECTS OF DRUGS. Control experiments. The introduction of a syringe needle into the lymph space of the thigh, during the course of an experiment, caused an immediate and marked rise in blood-pressure. This rise occurred whether the frog struggled or not. A similar effect was produced by jerking the needle and so pulling on the skin during an injection, or by withdrawing the needle, or merely by stroking the frog's skin. The syringe needle was, therefore, introduced into the lymph space of the thigh during the preparation of the frog, and remained in position throughout the experiment. With this precaution, the careful injection of 0 5 c.c. of saline, during the course of an experiment, produced only a slight and transitory rise in blood-pressure, easily distinguishable from a true drug effect. For example, an injection of 0-5 c.c. of saline produced an immediate rise of 15 mm. aq., whereas a subsequent injection into the same animal of 0 5 c.c. of adrenaline solution, 1: 100,000, produced a rise in bloodpressure of 240 mm. aq. The web circulation, as described by Krogh [1921], was observed 1 We wish to express to Dr Jeanne Eder of Zurich our thanks for suggesting and superintending the making of this apparatus.

5 INFLUENCE OF TEMPERATURE IN THE FROG. 37 to react to various local stimuli such as direct touch, or the removal of a wet pad. When the web was kept moist with water dropped from a pipette, however, no local reaction was observed. Method. In the subsequent series of experiments the same method as before was employed, with the modification of the copper-box apparatus. The precautions suggested by the control experiments were observed throughout. Drug solutions were injected in doses of 0 5 c.c. into the lymph space of the thigh. Observations of the blood-pressure, heart rate and condition of the web circulation were taken at frequent intervals, varying with the conditions of the experiment but averaging about five readings an hour. Histamine injections were tried in a few experiments, but the results obtained showed considerable variation. Calcium chloride injections produced unexpectedly little effect, compared with the markedly stimulating effect produced by the local application of calcium chloride solution to a sluggish web circulation. The study of these drugs was, therefore, temporarily abandoned, in favour of an investigation of the effects of temperature on two drugs, adrenaline and sodium nitrite, whose influence upon the vascular system is consistent and marked. The doses which were found convenient, and were used throughout with a few exceptions, were: adrenaline hydrochloride 0*5 c.c. of 1: 100,000 sol.: sodium nitrite 0 5 c.c. of 2p.c.sol. RESULTS OBTAINED WITH ADRENALINE. Figs. 4 and 5 show characteristic results in experiments in which adrenaline was injected at low and high temperature. The following results were obtained from 18 such experiments, 4 at 100 C., 6 at 150 C. and 8 at 20 C. (approx.). The blood-pressure was raised in every case by adrenaline, on an average by 45 p.c. The average time taken to reach maximum pressure was 35 min. at low, 16 min. at medium and 8 min. at high temperature; and the average duration of the blood-pressure effect was 2 hours at low, 1 hour 30 min. at medium and 40 min. at high temperature. The h e a r t rate was accelerated except in 3 cases, all at high temperature, in which the rate was slowed. The we b c irc ulation in 6 cases (2 low, 1 medium, 3 high) began and ended brisk and open, but was temporarily less good during the adrenaline rise of blood-pressure, when the field became closed, the flow in the active vessels was slowed, and in some cases the calibre of the vessels was diminished. In 5 cases (1 low, 1 medium, 3 high) the web circulation

6 M 38 W. C. CULLIS AND E. M. SCARBOROUGH. X a 1- te W. 4'C o t Q 4; a! Z 4. rn. ItS vc-,,,," 30 t _ / T-E g-rt ' EPRA7URE. 20 HQLi,QS 1' 1 2 HOuPqS A Fig. 4. Effects, at 100 C., of injection, at A, of adrenaline HCI 0 5 c.c., 1:100,000, on blood-pressure, heart rate and web circulation. 1.0 W. 0,i X. t0 Id t~ co v/ s I/ / - \&Aer RAra N Cw W AO I '-_ 60 4+c 20 7kmArmarvRE : 'P Fig. 5. OW 1' OUR5 t AL 1 o A2 Z Effects, at 200 C., of injection, at A1, repeated at A2, of adrenaline HCI 0 5 c.c., 1:100,000, on blood-pressure, heart rate and web circulation.

7 INFLUENCE OF TEMPERATURE IN THE FROG. 39 began less than good and in each case was improved by the injection of adrenaline. In 3 other cases the circulation began and remained good throughout, showing no closing during the rise in blood-pressure, which was, however, relatively slight in each case. The remaining 4 cases cannot be classified, as various external factors interfered with the condition of the web circulation during the experiment. DISCUSSION OF ADRENALINE RESULTS. The percentage rise in blood-pressure caused by adrenaline is similar at high and low temperatures, but the rise develops and passes off more quickly at 20 than at 100 C. This difference (in rate but not in quality of the effect) may, therefore, be related to a difference in rate of absorption of the drug at high and low temperatures, rather than to a modification in the action of the drug itself. Evidence that the former explanation is the more probable was obtained from the results of six experiments in which the frog's vessels were perfused with adrenaline solution at 10 and 200 C. Under these conditions the difference in temperatures produced no detectable difference in the action of the drug. The failure of the heart to accelerate with adrenaline as consistently at high as at lower temperatures does not necessarily indicate any modified action of the drug. Since the average heart rate is 26 beats per min. at 100 C., 40 at 150 C. and 60 at 200 C., the possibility of transitory sympathetic stimulation failing to produce acceleration seems greater at 200 than at 100 C., even apart from the fact that the duration of the drug effect is shorter at the high temperature. The type of effect produced by adrenaline on the web circulation seems to bear no direct relationship to the temperature, except that the effects which develop during the rise of blood-pressure are more prolonged at low than at high temperatures. The web circulation is ultimately unimpaired or improved by adrenaline, but the transitory closing of the field during the rise of blood-pressure occurs sufficiently consistently (given an initially brisk and open circulation and a good rise in blood-pressure) to be regarded as a characteristic of the drug. Adrenaline produces a similar but greatly exaggerated effect upon the web circulation when applied directly to the foot, as described by Oinuma [1924] and others. It would seem that the rapid initial rise in blood-pressure is brought about not only by arterial constriction and acceleration of the heart, but also in some cases by a temporary diminution in the number of active vessels in the peripheral circulation, the capillaries of the web acting independently of the arterial pressure, as described by Krogh [1922].

8 40 W. C. CULLIS AND E. M. SCARBOROUGH. RESULTS OBTAINED WITH SODIUM NITRITE. Figs. 6 and 7 show characteristic results in experiments in whicb nitrite was injected at low and high temperatures. The following results were obtained from 14 such experiments, 6 at 10 C. and 8 at 20 C. (approx.). 06:~~~~ ARrRArei PE~~~~~~~tATh~~~ ~ ~~~~~femrrae 20, IV~~~~~~~~~~~~~~~~~~~~~~I Fig. 6. Fig. 7. Fig. 6. Effects, at approx. 100 C., of injection, at N, of sodium nitrite 0 5 c.c., 2 p.c., on blood-pressure, heart rate and web circulation; showing breakdown of the heart rate and temperature relationship with rise of temperature. Fig. 7. Effects, at 21 C., of injection, at N, of sodium nitrite 0 5 c.c., 2 p.c., on bloodpressure, heart rate and web circulation; showing failure to arrest these effects by cooling. The blood-pres sure was lowered in every case by nitrite, but the fall was relatively slighter and slower at low temperature. The pressure fell on an average by 30 p.c. in 15 min. at 200C.; and by 10 p.c. in 15 min., by 29 p.c. in 1 hour, at 100 C. The heart rate at 200 C. was slowed by nitrite in 7 cases (in 3 of which there was a preliminary slight rise), and slightly accelerated in 1 case. At 100 C. it accelerated in 4 cases and slowed in 2. In 4 cases at 200 C.

9 INFLUENCE OF TEMPERATURE IN THE FROG. and 3 at 100 C. there was a late exaggerated fall in heart rate, associated with failure of the heart. In these cases the heart frequently became dissociated before arrest in diastole. Upon the web circulation typical effects were produced by nitrite, slow flow, closed field, congestion and marked pulsation in active vessels leading to an oscillating movement of the corpuscles and ultimate arrest of circulation. These changes were produced, to a varying degree, in every 41 Fig. 8.- Effect upon a developing nitrite action (injected 1 hour before the commencement of tracing) of cooling and of injection, at A, of adrenaline; and the subsequent effect of repeated nitrite, at N. case. In some cases increase in the calibre of the vessels was observed, and a condition described as "vessels well delineated" was frequently noted. At 200 C. the arrest of circulation occurred in average time of 40 min.; at 100 C., 1 hour 15 min. At low temperatures the web circulation already tended, in some cases, to be slow, closed and congested, with visible pulsation. The effect of nitrite was thus masked by the "cold effect" and changes in web circulation appeared slighter at low than at high temperatures. The "cold + nitrite effect," however, unlike the "cold effect," was an irreversible change, which ultimately produced arrest of the circulation in every case.

10 42 W. C. CULLIS AND E. M. SCARBOROUGH. Slight temporary improvement of the circulatory conditions was produced in 1 case out of 3 where high temperature was lowered, and in 1 case out of 3 where low temperature was raised, during a developing nitrite action. Injection of adrenaline during a developing nitrite action, at , produced in one case no effect; in a second a temporary improvement; and in a third case, when cooling accompanied the injection of adrenaline, a well-marked recovery (Fig. 8). Repetition of the nitrite injection caused subsequent rapid failure of the circulation in the two latter cases. Injection of adrenaline during a developing nitrite action at 10 C. produced in 4 cases a temporary improvement, but the typical nitrite effect supervened in each case. DISCUSSION OF NITRITE RESULTS. The percentage fall in blood-pressure caused by nitrite is greater at high than at low temperature and the effect develops more quickly. This difference may be related to a difference in rate of absorption, or to a modification of the drug action itself. As in the case of adrenaline, the former explanation seems the more probable. In six experiments in which the frog's vessels were perfused with nitrite solution, at 10 and 20 C., the difference in temperature produced no detectable difference in the action of the drug. The tendency of nitrite to slow the heart at 200C., whereas it produces inconsistent effects at 100 C., may be related to the more rapid development of the drug effect at high temperature, and also to the fact that the heart is already slowed at low temperature. The average heart rate at onset, in this series, is 27 beats per min. at 100 C., and 54 at 200 C. The slowing appears to be associated with actual failure of the heart itself, although perfusion of nitrite solution, of comparable strength, directly through the heart, produces very little effect. With the circulation intact, a possible cause of injury to the heart may be the formation of methaemoglobin and other compounds by the action of nitrite on the hawmoglobin, as described by Haldane, Makgill and Mavrogordato [1896]. The blood from a heart arrested by nitrite gave a metheemoglobin spectrum. The characteristic effect produced by nitrite on the web circulation has been already described. Direct application of nitrite solution to the web produces similar congestion and pulsation in the larger vessels. The change in the web circulation is more marked, however, when the nitrite is in the circulation, as the effects secondary to heart failure and fall of

11 INFLUENCE OF TEMPERATURE IN THE FROG. blood-pressure are added to the direct effect of nitrite on the web vessels. At both high and low temperature nitrite, under these conditions, causes an injurious effect upon the circulation, which it is very difficult to modify; and, though the onset of the fatal termination is more rapid at high temperature, it is ultimately just as inevitable at low temperature. SUMMARY. 1. Preliminary experiments upon the influence of temperature on the blood-pressure, heart rate and web circulation of the frog, when working with the intact circulation, are reported. 2. An experimental method of maintaining these factors constant, over periods suitable for the investigation of drug effects, is described. 3. The characteristic effects of adrenaline and of sodium nitrite upon the web circulation, at high and low temperatures, are described, and their relationship to the other circulatory factors is discussed. 4. It is suggested that the differences in these effects at high and low temperatures are due to a difference in the rate of absorption of the drug. 43 REFERENCES. Haldane, J. S., Makgill, R. H. and Mavrogordato, A. E. (1896)..J. Phy8iol. 20, 18 P. Krogh, A. (1921). Ibid. 55, 412. Krogh, A. (1922). The Anatomy and Physiology of Capillaries, p. 42. Oinuma, S. (1924). J. Physiol. 58, 318. Taylor, N. B. (1930). Ibid. 70, 40 P.