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Intermittent positive pressure and the curarized rat: Implications for cardiovascular conditioning
Physiology & Behavior, Vol. 16, pp. 735--743. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Intermittent Positive Pressure and the Curarized Rat: Implications for Cardiovascular Conditioning ETHEL EISSENBERG 3 AND JASPER BRENER 4
Department o f Psychology, The University o f Tennessee, Knoxville TN (Received 2 July 1974) EISSENBERG, E. AND J. BRENER. Intermittent positive pressure and the curarized rat: implications for cardiovascular conditioning. PHYSIOL. BEHAV. 16(6) 735-743, 1976. - Arterial blood gas analyses performed on noncurarized rats suggest that artificial respiration parameters specified in the literature for maintaining curarized rats in a normal physiological state are inadequate. These procedures were standardized on the assumption that the arterial PCO= for rats is approximately 25 mm Hg. The present analyses yielded a mean PCO2 of 33.3 mm Hg. ' a value more consistent. with . . . . pubhshed data. Moreover, a steady decline m heart rate and tidal volume was observed m two groups of rats resplrated using reported constant pressure values, supporting reported effects of curare on lung elastance. A consideration of important hemodynamic effects of intermittent positive pressure suggests more adequate parameters for the artificial ventilation of this preparation. The technique reported herein maintains tidal volume constant and compares heart rate and temperature measures under three different ratios of inspiration and expiration. This procedure, with an I:E Ratio of 1:2, is determined to be preferable to the traditionally used constant PIP respiration with an I:E Ratio of 1:1. Arterial blood gas Intermittent positive pressure respiration Operant conditioning of heart rate Cannulation
IT has recently become evident that the operant cardiovascular effects reported for curarized rats are not readily amenable to replication. Attempts to demonstrate the effects initially reported by Trowill [23 ], Miller and DiCara [5, 6, 15 ], Hothersall and Brener [ 10] and Slaughter et aL [21] have, without exception been unsuccessful [3, 9, 161. Those investigators reporting failure to replicate the operant cardiovascular effects previously obtained have suggested a variety of possible factors which may account for this major experimental discrepancy. Suggestions have included species changes, varied pharmacological properties of curare and factors associated with the artificial respiration of the curarized rat. Our experience has led us to suspect respiratory parameters to be the major source of error in experiments using this preparation, and as a consequence we have instigated a series of experiments aimed at defining the influence of various respiratory parameters on the state of the curarized rat. The experiments were initially prompted by the observation [ 14] that the use of the respiration technique reported by Miller and DiCara [6,15] produces a state indicative of hyperventilation in the curarized rat. Animals respirated by this procedure display pronounced invariant tachycardia at the
Curare
Cardiovascular conditioning
inception of the session followed by a systematic but small decrease in heart rate as a function of time. Such animals furthermore, remain unresponsive to peripheral stimulation throughout the experimental session. Investigators employing curarized rats have consistently ventilated the preparation using a positive pressure system to deliver room air to the animal through a face mask. Three critical parameters are involved in this method of artificial respiration: respiratory rate (RR), the ratio of time spent in inspiration and expiration during each respiration cycle (I:E Ratio) and the peak inspiration pressure (PIP). The values assigned to these parameters in most of the experiments reported by DiCara and Miller and their colleagues were, respectively, RR = 70 cycles per minute (cpm); I:E Ratio = 1:1 ; PIP = 20 crn H20. Only two papers reporting methods for respirating curarized rats have appeared in the literature. Hahn [8] derived his respiratory parameters on the basis of heart rate data. This method of assessing the adequacy of respiration meets with obvious objections where the experimental goal is the manipulation of cardiovascular activity. Ideally, the adequacy of respiration should be assessed on the basis of gas transport within the curarized preparation. DiCara [4]
t This research was supported by Grant NH 17061 from the National Institute of Mental Health. In part, the data reported herein appears in a dissertation submitted by the first author to the University of Tennessee in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2Our appreciation is extended to Freda Eidson of the University of Tennessee Memorial Research Center for performing the blood gas analyses. 3Reprints may be obtained from the first author at The Rockefeller University, New York, New York 10021, U.S.A. 4Now at the Department of Psychology, The University, Hull, HU6 7RX, Yorkshire, England. 735
736
EISSENBERG AND BRENER
performed such an investigation. He employed arterial blood gas analyses to substantiate the respiratory parameters utilized by him and his associates in previous successful autonomic conditioning experimentation. DiCara reported a PCOz value of 25.4 mm Hg and PO2 value of 75.1 mm Hg for the arterial blood of normal noncurarized rats and then manipulated respiratory parameters for curarized rats so as to produce these blood gas values in curarized rats weighing between 4 0 0 - 5 0 0 g. A respiratory rate of 70 cpm, with an I:E Ratio of 1:1 and peak respiratory pressures of 1 7 - 1 9 cm H20 were required. Although use of these parameters did produce arterial PCO2 values in the curarized preparation that approximated those obtained from noncurarized animals in DiCara's study, the value of 25.5 mm Hg does not accord with other published data for normal, noncurarized rats. The references cited by DiCara [2, I1, 12] do in fact indicate that this value is abnormally low, as do the normative data presented by Spector [22] and Altman and Dittmer [1 ]. Such evidence supports our observation that the use of these respiratory parameters leads to hyperventilation in the curarized rat; therefore a reexamination of the arterial blood gas composition of the noncurarized rat was initiated.
to the cannula to permit blood samples to be taken from outside the animal chamber. Samples between 0.5 and 0.6 cc of arterial blood were withdrawn into a I c c heparinized tuberculin syringe and sealed with mercury-filled caps, sequential samples taken within a 15 minute period with at least two and not more than four samples from each rat. All samples were maintained in anaerobic conditions and refrigerated until they were analyzed within two hours of withdrawal. RESULTS The first samples drawn from each rat were discarded since several proved to be diluted with the heparinized solution in the cannula. Final samples were discarded for those rats from which 4 samples had been obtained, providing one sample for 3 animals and 2 samples for the remaining 4 animals. These data are presented in Table 1, with the values for the latter 4 animals representing the mean of two analyses and the data for the remaining 3 animals representing the results of a single blood gas analysis. qABLE 1 RESULTS OF BLOODGAS ANALYSES FOR 7 RATS
EXPERIMENT 1 METHOD
Animals Seven male rats of the Long-Evans strain, weighing 326 to 422 g, were used.
Apparatus Polyethylene cannulae were fashioned according to the method of Popovic and Popovic [ 19]. Blood samples were collected in disposable 1 cc heparinized tuberculin syringes and sealed with mercury-filled Luer Lok caps. Analysis was performed on an Instrumentation Laboratories Inc. Blood Gas Analyzer, Model No. 13. Animals were restrained in a plastic tube (E and M Instrument Company, Model No. 4) which rested in a sound attenuating chamber, effectively shielding subjects from procedural events.
Procedure For several days prior to cannulation animals were gentled and then individually habituated to the restraining device for 20 min periods. On the day surgery was performed, and following the habituation procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg, 50 mg/ml solution) and a cannula introduced into the external iliac artery of the right rear limb for a distance of approximately 1 cm, in the direction of the abdominal aorta. The cannula was fastened to surrounding fascia, tested for patency, filled with a 2% heparin solution and heat-sealed, after which it was directed to the dorsal side of the animal and led up the back, to exit at the nape of the neck. The cannula was then sutured to the skin and the animal permitted to recover. Habituation to the restraining device was resumed 24 hr post surgery, at which time the cannula was tested for patency, flushed with saline, refilled with heparin and heat-sealed. Forty-eight hours post surgery the cannula was retested, flushed, filled with a heparinized solution, and a polyethylene extension added
Animals
Weight
ph
Po2
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B C N* O* T* U* V
326 326 417 390 363 352 422
7.47 7.48 7.49 7.50 7.47 7.47 7.48
80.0 61.5 81.0 78.5 83.25 93.4 94.0
35.0 34.0 33.5 31.5 32.0 33.9 33.3
370.86 36.94
7.48 .01
81.66 10.09
33.32 1.12
Mean SD
*Blood gas values reported for these subjects represent the mean of two samples. DISCUSSION The mean PO2 value of 81.66 mm Hg is within the range reported by other investigators [2, 11, 12]. The wide variability of this measure has been observed previously [2], and is accountable on the basis of the interanimal variability in hemoglobin concentration in rats [ 11 ]. The observed Pco2 value of 33.32 mm Hg supports our contention that the Pco2 value reported by DiCara [4] for noncurarized rats is abnormally low and indicative of hyperventilation. This low PC02 may be attributed to the fact that the animals in DiCara's experiment had previously experienced traumatic electric shock in the experimental situation and were visibly emotional (DiCara, personal communication). Artificial respiration parameters standardized to produce abnormally low Pc02 values will lead to hyperventilation in the curarized preparation with the ensuing cardiovascular effects. Middaugh [14], for example, has observed that curarized rats respirated according to the parameters proposed by DiCara displayed profound tachycardia and were not amenable to operant heart rate conditioning. Similar factors may well account for other reported failures to replicate operant cardio-
ARTIFICIAL RESPIRATION IN CURARIZED RAT vascular phenomena reported by DiCara and Miller and their associates. This possibility prompted a more complete investigation of the parameters of artificial respiration employed in the maintenance of curarized rats. EXPERIMENT 2 In all reported studies employing curarized rats, animals have been artificially ventilated at constant pressure settings throughout the conditioning procedure. Miller and DiCara and their associates report having respirated all animals in a given experiment at identical pressure settings; whereas other investigators [7, 8, 10] have varied pressures during an adaptation period in order to arrive at a value which was optimal for each individual subject and which was then maintained throughout training for that rat. While the latter technique allows for individual differences in resistance to air flow it does not take into account intraanimal changes in airway resistance as a function of time under curare. That such changes occur is suggested by observations of heart rate decrement over time under curare [7, 14, 20]. This fact has virtually been ignored, even though such a phenomenon would serve to minimize the effects of procedures designed to produce heart rate acceleration, and would confound the interpretation of heart rate changes in the opposite direction. This heart rate deceleration can be explained in terms of the reported substantial changes which curare produces in the elastic properties of the lung and thorax. Following intravenous injections of curare in dogs, Massion [13] demonstrated a 42.2% decrease in the compliance of the chest cage. Thus, as the lungs become more rigid and resistance to air flow is increased, constant pressure respiration leads to diminished alveolar gas turnover. Therefore, a pressure setting which initially serves to maintain the animal in a normal physiological state will eventuate in hypoventilation if adequate compensation for this increase in resistance is not made. Since the rate of air flow is equal to the source pressure divided by the resistance to flow offered by the preparation, it follows that as the lungs become more rigid due to curarization, the pulmonary air turnover will decline. The purpose of the present experiment was to examine this process and its effects on rats respirated at the PIP suggested by procedures reported by Miller and DiCara and their associates (18 cm H20) and at the PIP suggested to be optimal by the experiments of Middaugh (14 cm H2 O). METHOD
A nimals Fifteen male rats of the Long-Evans strain, weighing 412 to 558 g (mean = 453 g), were used. Animals were randomly assigned to Group A (7 animals) or Group B (8 animals).
Apparatus Animals lay, ventral side down, on a small wooden platform which was housed within the same sound attenuating chamber used in Experiment 1. An adjustable moulded nylon face mask attached to the platform delivered room air from a small animal respirator (E and M Instrument Company, Model V5KG) which rested outside of the chamber. Subdermal electrodes in the Lead 1 position were employed to record EKG. The amplified
737 signal was taken from one of the polygraph's J6 output jacks and fed through a pulse shaping circuit which converted the EKG wave form to a square wave pulse of fixed duration. The pluse provided the input to a BRS solid state counting circuit which recorded the n u m b e r of R-waves for consecutive 60 sec intervals. A second polygraph channel provided a beat-to-beat heart rate display. A closed loop mercury strain gauge encircled the thorax of each rat, posterior to the forelimbs, and provided the input to a Model 270 Parks Electronic Company Plethysomograph and an indirect measure of volume change. Amplification of the Plethysomograph signal was achieved via a Grass low level DC preamplifier, Model 7P1. Body temperature was read from a Yellow Springs Instrument company Single Channel Telethermometer for which input was provided by a small animal rectal thermistor probe (Scientific Products T2605), previously calibrated with a NBS thermometer. A thermometer within the chamber was employed to measure the ambient temperature for all animals; this was maintained within the range 7 8 - 8 0 ° F.
Procedure Animals in Group A were respirated at a PIP of 14 cm H2 O, whereas animals in Group B were respirated at a PIP of 18 cm H20. In all other respects the procedures for the two groups were identical. All animals were respirated at a rate of 70 cpm, with an I:E Ratio of I : 1. Following an intraperitoneal injection of d-tubocurarine chloride (3.6 mg/kg) and upon the first signs of paralysis (usually within 10 sec) the face mask was fitted to the animal's snout. The mercury strain gauge was then placed about the animal's upper thorax, pin electrodes placed in the Lead 1 position and the thermal probe inserted. Continuous recordings of heart rate, rectal temperature and circumferential chest movements were made throughout the procedure. The experimenter monitored the PIP at regular intervals and made necessary adjustments to maintain the pressure at 14 cm H 2 0 for animals in Group A and 18 cm H 2 0 for animals of Group B. This procedure was maintained until animals recovered from curare or for a maximum of 180 min following the administration of curare. RESULTS The mean heart rates, circumferential chest movements and rectal temperatures displayed by the two groups of animals during the first 90 min of curarization are presented in Fig. 1. In this and subsequent figures, experimental periods are differentiated as either adaptation periods (A) or pressure change periods (P). Since pressures were predetermined for all animals in Groups A and B, the distinction is not directly applicable to these groups but facilitates comparisons with subsequent groups in which PIP was manipulated to maintain constant chest circumference. It will be observed that animals respirated at a PIP of 18 cm H 2 0 (Group B) display significantly higher heart rates and greater chest movements than do animals respirated at 14 cm H20. It will also be noted that in both groups of animals, heart rate and chest movements decline systematically as a function of time. The temperature data indicate that at the inception of the procedure both groups display subnormal body temperature. Whereas the temperatures of animals respirated at 18 cm H 2 0 tend to increase monotonically as a function of time under curare, those of animals respirated at 14 cm H 2 0 increaseduring the first 30
738
EISSENBERG AND BRENER
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DISCUSSION These results clearly indicate that as a function of time under curare, the tidal respiratory volume, as measured by circumferential chest movements, declines. This decrement in pulmonary air turnover is accompanied by correlated decreases in heart rate which are assumed here to be secondary to the respiratory effect. This effect in turn is attributed to the curare-induced loss of lung compliance. The temperature data support this interpretation. Although the 18 cm H 2 0 group commences the session with a body temperature approximately 1° C. lower than the 14 cm H 2 0 group, these subjects display an increase in body temperature over the 90 min period of approximately 2 ° C. Since the cardiac output is reduced by abnormally high intrathoracic pressures which reduce venous return, it is to be expected that as lung compliance decreases and the pulmonary volume declines that the venous return and hence the cardiac output will increase, leading to parallel increases in body temperature. Although this mechanism operates in the case of the 14 cm H 2 0 group as well, leading to an increase in body temperature over the first 30 min of the procedure, from the 40th through the 90th minute, the alveolar gas turnover provided by the inter-
action of this PIP with the increased pulmonary resistance becomes insufficient to maintain the vital functions. As a consequence the body temperature drops precipitously. Although all animals in the 18 cm H 2 0 group recovered from curare, 5 of 7 animals in the 14 cm H 2 0 group died before the 180th minute of curarization. We may therefore conclude that a PIP of 14 cm H 2 0 which is sufficient to support a curarized rat in a healthy condition (as indicated by rectal temperature and mean heart rate) at the inception of curarization will, if maintained, lead to fatal hypoventilation. On the other hand, a constant PIP which is sufficient to obviate this process will lead to pronounced hyperventilation at the inception of curarization. In view of these considerations, the method of constant pressure ventilation is to be discouraged. These data furthermore suggest that constant volume respiration is a preferred technique for the maintenance of curarized rats since this technique does not permit reductions in alveolar gas turnover as a function of curare-induced loss of lung compliance. The examination of such a method forms the substance of the final experiment in this series. EXPERIMENT 3 Just as the curare-elastance relationship has been overlooked, so it seems have the abnormal effects of intermittent positive pressure respiration on cardiovascular
A R T I F I C I A L RESPIRATION IN CURARIZED RAT activity. The differences between normal and positive pressure respiration are described in detail by Mushin et al. [ 18]. Of particular relevance to procedures in the operant cardiovascular conditioning literature are the following factors: (a) Normally, in the human, alveolar pressure falls to 1 to 2 cm H 2 0 below atmospheric pressure during inspiration and rises 1 to 2 cm H 2 0 above atmospheric pressure during expiration. In contrast, during positive pressure respiration alveolar pressure is greatest during inspiration and fails to approximately atmospheric pressure during expiration. The pulmonary pressure differences are therefore not only out of phase with those produced b y normal respiration, they are also greatly exaggerated (often 20 cm H 2 0 for rats) during positive pressure respiration. (b) The decreased intrathoracic pressure contingent upon normal inspiration causes an augmentation of venous return by drawing blood into the great thoracic veins. Because the intrathoracic pressure during inspiration in positive pressure artificial respiration is greatly increased, this important mechanism is disturbed and venous return to the heart is attenuated. (c) Given the relationship between intrathoracic pressure and venous return, if follows that if the positive pressure inspiration phase is unduly prolonged, the cardiac output will be severely diminished. Moreover, high intrathoracic pressure decreases cardiac output not only by decreasing venous return but also by a direct mechanical compression of the heart. In this connection Mushin et al. [18], p. 13, note: "The higher the peak pressure and the longer the time during which it acts, the greater is the cardiac tamponade and the interference with cardiac o u t p u t . " It has been reported by Morgan et al. [ 17] that when an I:E Ratio of 1:1 is employed with a PIP o f 20 cm H2 O, the cardiac output is decreased by 22% in dogs. Since the reduction in cardiac output that is associated with positive pressure respiration is a direct function of the length of time the lungs are inflated, it follows that the inspiration phase of the respiratory cycle must be kept as short as possible. That is, the I:E Ratio should be reduced to that minimum which will permit adequate pulmonary air turnover in the animal. The present experiment investigates the influence of different I:E Ratios on heart rate and body temperature in curarized rats. Because of the reported effect of curare on lung elastance, PIP was individually adjusted to maintain a constant circumferential chest movement ascertained as optimal for each subject. The effects of this procedure are compared with the effects of traditional constant pressure procedures described in Experiment II. METHOD Animals
Twenty-three rats of the Long-Evans strain, ranging in weight from 384 to 560 gms (mean = 456 gms) were used. Animals were randomly assigned to Group D, Group E or Group F. Apparatus
The apparatus employed described in Experiment 1.
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Procedure
Procedural events were identical with those reported in Experiment 2, with three exceptions. Whereas each animal
739 in the previous experiment was maintained using an invariant pressure setting (14 or 18 cm H 2 0 ) , all animals in this experiment were respirated b y altering peak inspiration pressure to maintain chest movements of a constant amplitude. Secondly, respiration rate for all animals was reduced to 60 cpm to facilitate manipulation of I:E Ratios. This change was justified on the basis of Middaugh's [14] demonstration that diffferences in rate within the range of 5 0 - 7 0 cpm results in little heart rate change. Finally, I:E Ratio was manipulated to enable additional comparisons among groups differentiated by this variable: 8 animals in Group D were maintained on a 1 : 1 Ratio, 7 Ss in Group E represent a Ratio of 1:1, and 8 animals in Group F were maintained on a 1:3 Ratio. During a 30 min adaptation period (A) an optimal chest circumference value was selected for each animal on the basis of heart rate (a mean value between 3 8 0 - 4 6 0 bpm) a n d cardiotachograph records ( 1 0 - 3 0 bpm bidirectional variation). Thereafter, chest circumference was maintained by manipulating PIP during six 10-min pressure change periods (P). Changes in chest circumference were noted every 10 min (1 mm change in gauge circumference = pen deflection of 15.7 mm) and checked and recorded even more frequently following adaptation, in order to maintain a constant value. Temperatures were read every ten minutes and heart rate recorded for each one-minute interval. Sessions extended over 120 min or until subjects became reflexive, indicating recovery from drug effects. RESULTS Figure 2 illustrates the effects of varying I:E Ratio on heart rate, chest movements and b o d y temperature in a procedure where peak inspiration pressure is adjusted to maintain chest movements of a constant amplitude. The relative merits of this respiratory procedure may be assessed by making direct comparisons of its physiological effects with those produced by the constant pressure procedure described in Experiment 2. F Ratios computed to evaluate group differences in heart rate show no significant difference in dispersion between groups (A and B) respirated by the constant pressure procedure and no significant difference between the 1:2 and 1:3 Ratio groups (D and F) respirated by maintaining a constant tidal volume, Group D, however, is significantly different from Groups A and B (pC0.001), as is Group F (p~<0.01 and p~<0.05 respectively). Group E is significantly different from Groups A and D (p~<0.05) suggesting that regulating respiration by maintaining a constant tidal volume and a 1:1 Ratio produces greater heart rate stability than constant pressure respiration at that ratio, and significantly more variability than the same procedure (constant chest circumference) at a reduced I:E Ratio of 1:2 or 1:3. Thus, constant chest circumference respiration with a 1 : 2 Ratio results in the greatest heart rate stability and the same procedure with a 1:3 Ratio is not significantly different. Group A, at the low end of the temperature range, trending downward, but capable of only limited variability is significantly different (p~<0.05) from Group B which exhibits the greatest variability in temperature. Group D is significantly different from Group B (p<~0.05). Thus, aside from Group A, which registered subnormal temperatures, the 1:2 Ratio group evinced the greatest temperature stability, with no significant difference between Groups D and F.
740
EISSENBERG AND BRENER
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FIG. 2. Mean heart rate, chest circumference and temperature measures for groups of animals respirated by maintaining chest circumference constant at three different I:E Ratios. F Ratios performed to evaluate chest circumference variability indicate that Group D is significantly different from Group B (p<0.001). All groups maintained on a constant tidal volume are significantly different from group B (pC0.01) and Group E is significantly different from Group D (p~0.05). The minimal variability demonstrated by Group A is easily understood in view of the low PIP values used in a constant pressure procedure. Moreover, mean variance was calculated from P1 through P6 when chest circumference had already diminished, leaving little opportunity for further variability. DISCUSSION
Although attempts were made to maintain constant chest movements during the last 60 minutes of the session, it will be seen in Fig. 2 that this index does vary within a range of approximately 1.5 mm in the 1:1 Ratio group to approximately 0.65 mm in the 1:2 Ratio group. It will be noticed that the heart rate means show a tendency to increase over the last 60 min of the session with this trend being most marked from the 40th to the 80th minute. However, it will be noted that the mean heart rates for all three groups of animals are considerably more stable than those observed in the previous experiment. During the initial 30 min of the procedure body
temperature is an inverse function of the I:E Ratio, suggesting again that as ratio is attenuated cardiac output is facilitated. Although the temperatures of all groups display a systematic decline during the last 90 min of the session, the total temperature decrement does not exceed 0.5 ° C in any group. A comparison of these data with those of Groups A and B in Fig. 2 indicates that all groups display consistently higher body temperatures than subjects respirated at a constant PIP value with an I:E Ratio of I: 1. GENERAL DISCUSSION The relative merits of this respiratory procedure may be assessed by making direct comparisons of its physiological effects with those produced by the constant pressure procedure described in Experiment 2. Figure 3 graphically displays the clear differences between groups of animals (Groups A and B) respirated under invariant PIP values and animals maintained by a constant measure of chest circumference (Group D, 1:2 Ratio). The chest movements of the latter group remain relatively stable from the 40th through the 90th minute of the session. During this period it will also be observed that the heart rate measure remains relatively stable and does not display the same systematic decrement as is evident in the constant pressure groups. This occurs despite the fact that pressure was adjusted to
ARTIFICIAL RESPIRATION IN CURARIZED RAT
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FIG. 3. Mean heart rate, chest circumference and temperature measures for groups of animals respirated at constant PIP values or by maintaining constant chest circumference. maintain constant chest movements, not invariant heart rate, indicative of the importance of the relationship between these variables. Note that the temperature recorded for Group D, although displaying a slight decrement over the session, are at a considerably higher level than either Group A or Group B. We submit that the higher temperatures displayed by this group is primarily the results of the lower h E Ratio (1:2) employed with these animals. It must be remembered that attenuation of cardiac output is a joint function of the PIP value and the time during which it acts. Cardiac output in Group D was less attenuated because the intrathoracic pressure was elevated for a proportionately shorter period of time than was the case in either Group A or Group B. An important feature of any maintenance procedure is that is should be reliable. In order to ascertain the reliability of the respiratory procedure employed for Group D in Experiment 3 an additional group of 8 rats was run under an identical procedure (Group 1). Figure 4 presents a comparison of mean heart rates, body temperatures and chest movements in these 2 groups of animals. This figure also illustrates the mean PIPs employed in order to maintain optimal chest movements. It will be observed that the temperature changes as well as other measures (excepting pressures) are indeed replicable. It is interesting to note that the group pressure points are parallel although over 2 cm H 2 0 apart throughout the session. This
difference probably is a function of a substantial difference in mean body weight in the two groups: Group D mean body weight = 478 g: Group 1 mean body weight = 394 g. It should also be noted that animals which display similar body weights often require substantially different PIPs in order to maintain the same tidal volume. This is explainable in terms of difference in position, face mask fit and varying effects of curare. It is evident from these data that the selection of appropriate respiration parameters poses a very considerable problem. With regard to peak inspiration pressure, a balance must be struck between excessively high pulmonary pressures which interfere with cardiac output and low pressures which result in a hypoventilated animal. But the reduction of cardiac output associated with positive pressure respiration also varies as a direct function of the time that the lungs are inflated. Therefore, it follows that the inspiration phase of the respiratory cycle must be kept as short as possible, within the limits which permit adequate pulmonary air turnover in the organism. In view of the stability of the heart rate of rats respirated at a 1:2 I:E Ratio and also because of the relative ease with which a constant respiratory volume may be maintained with this setting it is suggested that this ratio and a constant chest circumference procedure is the most efficient of those investigated for maintaining curarized rats in a healthy condition.
742
E I S S E N B E R G AND B R E N E R
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FIG. 4. A comparison of two groups of animals respirated by maintaining chest circumference constant at a 1:2 h E Ratio and 60 cpm.
REFERENCES 1. Altman, P. L. and D. S. Dittmer, editors. Biology Data Book. Washington: Federation of American Societies for Experimental Biology, 1964, p. 260. 2. Blood, F. R., R. V. Elliott and F. E. D'Amour. The physiology of the rat in extreme anoxia. Am. J. Physiol. 146: 319-329, 1946. 3. Brener, J. M., E. M. Eissenberg and S. J. Middaugh. Respiratory and somatomotor factors associated with operant conditioning of cardiovascular responses in curarized rats. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press. 4. DiCara, L. V. Analysis of arterial blood gases in the curarized artificially respirated rat. Behav. Res. Instrum. 2: 67-69, 1970. 5. DiCara, L. V. and N. E. Miller. Changes in heart rate instrumentally learned by curarized rats as avoidance responses. J. comp. physiol. Psychol. 65: 8 - 1 2 , 1968a. 6. DiCara, L. V. and N. E. Miller. Transfer of instrumentally learned heart rate changes from curarized to noncurarized state: implications for a mediational hypothesis. J. comp. physiol. Psychol. 68: 159-162, 1969. 7. Goesling, W. J. The effects of prior skeletal conditioning on the conditioning of heart rate changes in curarized subjects. Unpublished doctoral dissertation, University of Tennessee, 1969. 8. Hahn, W. W. Apparatus and technique for work with the curarized rat. Psychophysiology 7: 283-286, 1970.
9. Hahn, W. W. The learning of autonomic responses by curarized animals. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press. 10. Hothersall, D. and 1. M. Brener. Operant conditioning of changes in heart rate in curarized rats. J. cornp, physiol. Psychol. 68: 338-342, 1969. 11. Jones, W. E., B. G. MacGrath and H. H. Aculthorpe. Pathological processes in disease II. Blood of the albino rat. Approximate physico-chemical description. Ann. trop. Med. Parasit. 44: 168-186, 1950. 12. King, T. K. C. and D. Bell. Arterial blood gas in specific pathogen free and bronchitis rats. J. appl. Physiol. 146: 319-329, 1966. 13. Massion, W. H. Effects of curare on elastic properties of chest and lungs of the dog. J. appl. PhysioL 11: 309- 312, 1957. 14. Middaugh, S. J. Operant conditioning of heart rate: the curarized artificially-respirated rat as a source of variability. Unpublished doctoral dissertation, University of Tennessee, 1971. 15. Miller, N. E. and L. V. DiCara. Instrumental learning of heart rate changes in curarized rats: shaping and specificity to discriminative stimulus. J. comp. physiol. Psychol. 63: 12-19, 1967. 16. Miller, N. E. and B. R. Dworkin. Visceral learning: recent difficulties with curarized rats and significant problems for human research. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener, and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press.
A R T I F I C I A L R E S P I R A T I O N IN C U R A R I Z E D R A T 17. Morgan, B. C., W. E. Martin, T. F. Hornbein, E. W. Crawford, and W. G. Gunteroth. Hemodynamic effects of intermittent positive pressure respiration. Anesthesiology 27: 5 8 4 - 5 9 0 , 1966. 18. Mushin, W. W., L. Rendell-Baker, P. Thompson and W. W. Mapleson. Automatic Ventilation o f the Lungs. Oxford: Blackwell Scientific Publications, 1969, pp. 2 - 2 5 . 19. Popovic, V. and P. Popovic. Permanent cannulation of aorta and vena cava in rats and ground squirrels. J. appl. Physiol. 15: 7 2 7 - 7 2 8 , 1959.
743
20. Ray, R. Classical conditioning of heart rate in restrained and curarized rats. Unpublished doctoral dissertation, University of Tennessee, 1969. 21. Slaughter, J., W. Hahn and P. Rinaldi. Instrumental conditioning in the curarized rat with varied amounts of pre-training. Z comp. physiol. Psychol. 72: 356-395, 1970. 22. Spector, W. E., editor. Handbook o f Biological Data. Philadelphia: W. B. Saunders Company, 1956, p. 270. 23. Trowill, J. A. Instrumental conditioning of the heart rate in the curarized rat. J. cornp, physiol. Psychol. 6 3 : 7 - 1 1 , 1967.
Intermittent Positive Pressure and the Curarized Rat: Implications for Cardiovascular Conditioning ETHEL EISSENBERG 3 AND JASPER BRENER 4
Department o f Psychology, The University o f Tennessee, Knoxville TN (Received 2 July 1974) EISSENBERG, E. AND J. BRENER. Intermittent positive pressure and the curarized rat: implications for cardiovascular conditioning. PHYSIOL. BEHAV. 16(6) 735-743, 1976. - Arterial blood gas analyses performed on noncurarized rats suggest that artificial respiration parameters specified in the literature for maintaining curarized rats in a normal physiological state are inadequate. These procedures were standardized on the assumption that the arterial PCO= for rats is approximately 25 mm Hg. The present analyses yielded a mean PCO2 of 33.3 mm Hg. ' a value more consistent. with . . . . pubhshed data. Moreover, a steady decline m heart rate and tidal volume was observed m two groups of rats resplrated using reported constant pressure values, supporting reported effects of curare on lung elastance. A consideration of important hemodynamic effects of intermittent positive pressure suggests more adequate parameters for the artificial ventilation of this preparation. The technique reported herein maintains tidal volume constant and compares heart rate and temperature measures under three different ratios of inspiration and expiration. This procedure, with an I:E Ratio of 1:2, is determined to be preferable to the traditionally used constant PIP respiration with an I:E Ratio of 1:1. Arterial blood gas Intermittent positive pressure respiration Operant conditioning of heart rate Cannulation
IT has recently become evident that the operant cardiovascular effects reported for curarized rats are not readily amenable to replication. Attempts to demonstrate the effects initially reported by Trowill [23 ], Miller and DiCara [5, 6, 15 ], Hothersall and Brener [ 10] and Slaughter et aL [21] have, without exception been unsuccessful [3, 9, 161. Those investigators reporting failure to replicate the operant cardiovascular effects previously obtained have suggested a variety of possible factors which may account for this major experimental discrepancy. Suggestions have included species changes, varied pharmacological properties of curare and factors associated with the artificial respiration of the curarized rat. Our experience has led us to suspect respiratory parameters to be the major source of error in experiments using this preparation, and as a consequence we have instigated a series of experiments aimed at defining the influence of various respiratory parameters on the state of the curarized rat. The experiments were initially prompted by the observation [ 14] that the use of the respiration technique reported by Miller and DiCara [6,15] produces a state indicative of hyperventilation in the curarized rat. Animals respirated by this procedure display pronounced invariant tachycardia at the
Curare
Cardiovascular conditioning
inception of the session followed by a systematic but small decrease in heart rate as a function of time. Such animals furthermore, remain unresponsive to peripheral stimulation throughout the experimental session. Investigators employing curarized rats have consistently ventilated the preparation using a positive pressure system to deliver room air to the animal through a face mask. Three critical parameters are involved in this method of artificial respiration: respiratory rate (RR), the ratio of time spent in inspiration and expiration during each respiration cycle (I:E Ratio) and the peak inspiration pressure (PIP). The values assigned to these parameters in most of the experiments reported by DiCara and Miller and their colleagues were, respectively, RR = 70 cycles per minute (cpm); I:E Ratio = 1:1 ; PIP = 20 crn H20. Only two papers reporting methods for respirating curarized rats have appeared in the literature. Hahn [8] derived his respiratory parameters on the basis of heart rate data. This method of assessing the adequacy of respiration meets with obvious objections where the experimental goal is the manipulation of cardiovascular activity. Ideally, the adequacy of respiration should be assessed on the basis of gas transport within the curarized preparation. DiCara [4]
t This research was supported by Grant NH 17061 from the National Institute of Mental Health. In part, the data reported herein appears in a dissertation submitted by the first author to the University of Tennessee in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2Our appreciation is extended to Freda Eidson of the University of Tennessee Memorial Research Center for performing the blood gas analyses. 3Reprints may be obtained from the first author at The Rockefeller University, New York, New York 10021, U.S.A. 4Now at the Department of Psychology, The University, Hull, HU6 7RX, Yorkshire, England. 735
736
EISSENBERG AND BRENER
performed such an investigation. He employed arterial blood gas analyses to substantiate the respiratory parameters utilized by him and his associates in previous successful autonomic conditioning experimentation. DiCara reported a PCOz value of 25.4 mm Hg and PO2 value of 75.1 mm Hg for the arterial blood of normal noncurarized rats and then manipulated respiratory parameters for curarized rats so as to produce these blood gas values in curarized rats weighing between 4 0 0 - 5 0 0 g. A respiratory rate of 70 cpm, with an I:E Ratio of 1:1 and peak respiratory pressures of 1 7 - 1 9 cm H20 were required. Although use of these parameters did produce arterial PCO2 values in the curarized preparation that approximated those obtained from noncurarized animals in DiCara's study, the value of 25.5 mm Hg does not accord with other published data for normal, noncurarized rats. The references cited by DiCara [2, I1, 12] do in fact indicate that this value is abnormally low, as do the normative data presented by Spector [22] and Altman and Dittmer [1 ]. Such evidence supports our observation that the use of these respiratory parameters leads to hyperventilation in the curarized rat; therefore a reexamination of the arterial blood gas composition of the noncurarized rat was initiated.
to the cannula to permit blood samples to be taken from outside the animal chamber. Samples between 0.5 and 0.6 cc of arterial blood were withdrawn into a I c c heparinized tuberculin syringe and sealed with mercury-filled caps, sequential samples taken within a 15 minute period with at least two and not more than four samples from each rat. All samples were maintained in anaerobic conditions and refrigerated until they were analyzed within two hours of withdrawal. RESULTS The first samples drawn from each rat were discarded since several proved to be diluted with the heparinized solution in the cannula. Final samples were discarded for those rats from which 4 samples had been obtained, providing one sample for 3 animals and 2 samples for the remaining 4 animals. These data are presented in Table 1, with the values for the latter 4 animals representing the mean of two analyses and the data for the remaining 3 animals representing the results of a single blood gas analysis. qABLE 1 RESULTS OF BLOODGAS ANALYSES FOR 7 RATS
EXPERIMENT 1 METHOD
Animals Seven male rats of the Long-Evans strain, weighing 326 to 422 g, were used.
Apparatus Polyethylene cannulae were fashioned according to the method of Popovic and Popovic [ 19]. Blood samples were collected in disposable 1 cc heparinized tuberculin syringes and sealed with mercury-filled Luer Lok caps. Analysis was performed on an Instrumentation Laboratories Inc. Blood Gas Analyzer, Model No. 13. Animals were restrained in a plastic tube (E and M Instrument Company, Model No. 4) which rested in a sound attenuating chamber, effectively shielding subjects from procedural events.
Procedure For several days prior to cannulation animals were gentled and then individually habituated to the restraining device for 20 min periods. On the day surgery was performed, and following the habituation procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg, 50 mg/ml solution) and a cannula introduced into the external iliac artery of the right rear limb for a distance of approximately 1 cm, in the direction of the abdominal aorta. The cannula was fastened to surrounding fascia, tested for patency, filled with a 2% heparin solution and heat-sealed, after which it was directed to the dorsal side of the animal and led up the back, to exit at the nape of the neck. The cannula was then sutured to the skin and the animal permitted to recover. Habituation to the restraining device was resumed 24 hr post surgery, at which time the cannula was tested for patency, flushed with saline, refilled with heparin and heat-sealed. Forty-eight hours post surgery the cannula was retested, flushed, filled with a heparinized solution, and a polyethylene extension added
Animals
Weight
ph
Po2
Pcoz
B C N* O* T* U* V
326 326 417 390 363 352 422
7.47 7.48 7.49 7.50 7.47 7.47 7.48
80.0 61.5 81.0 78.5 83.25 93.4 94.0
35.0 34.0 33.5 31.5 32.0 33.9 33.3
370.86 36.94
7.48 .01
81.66 10.09
33.32 1.12
Mean SD
*Blood gas values reported for these subjects represent the mean of two samples. DISCUSSION The mean PO2 value of 81.66 mm Hg is within the range reported by other investigators [2, 11, 12]. The wide variability of this measure has been observed previously [2], and is accountable on the basis of the interanimal variability in hemoglobin concentration in rats [ 11 ]. The observed Pco2 value of 33.32 mm Hg supports our contention that the Pco2 value reported by DiCara [4] for noncurarized rats is abnormally low and indicative of hyperventilation. This low PC02 may be attributed to the fact that the animals in DiCara's experiment had previously experienced traumatic electric shock in the experimental situation and were visibly emotional (DiCara, personal communication). Artificial respiration parameters standardized to produce abnormally low Pc02 values will lead to hyperventilation in the curarized preparation with the ensuing cardiovascular effects. Middaugh [14], for example, has observed that curarized rats respirated according to the parameters proposed by DiCara displayed profound tachycardia and were not amenable to operant heart rate conditioning. Similar factors may well account for other reported failures to replicate operant cardio-
ARTIFICIAL RESPIRATION IN CURARIZED RAT vascular phenomena reported by DiCara and Miller and their associates. This possibility prompted a more complete investigation of the parameters of artificial respiration employed in the maintenance of curarized rats. EXPERIMENT 2 In all reported studies employing curarized rats, animals have been artificially ventilated at constant pressure settings throughout the conditioning procedure. Miller and DiCara and their associates report having respirated all animals in a given experiment at identical pressure settings; whereas other investigators [7, 8, 10] have varied pressures during an adaptation period in order to arrive at a value which was optimal for each individual subject and which was then maintained throughout training for that rat. While the latter technique allows for individual differences in resistance to air flow it does not take into account intraanimal changes in airway resistance as a function of time under curare. That such changes occur is suggested by observations of heart rate decrement over time under curare [7, 14, 20]. This fact has virtually been ignored, even though such a phenomenon would serve to minimize the effects of procedures designed to produce heart rate acceleration, and would confound the interpretation of heart rate changes in the opposite direction. This heart rate deceleration can be explained in terms of the reported substantial changes which curare produces in the elastic properties of the lung and thorax. Following intravenous injections of curare in dogs, Massion [13] demonstrated a 42.2% decrease in the compliance of the chest cage. Thus, as the lungs become more rigid and resistance to air flow is increased, constant pressure respiration leads to diminished alveolar gas turnover. Therefore, a pressure setting which initially serves to maintain the animal in a normal physiological state will eventuate in hypoventilation if adequate compensation for this increase in resistance is not made. Since the rate of air flow is equal to the source pressure divided by the resistance to flow offered by the preparation, it follows that as the lungs become more rigid due to curarization, the pulmonary air turnover will decline. The purpose of the present experiment was to examine this process and its effects on rats respirated at the PIP suggested by procedures reported by Miller and DiCara and their associates (18 cm H20) and at the PIP suggested to be optimal by the experiments of Middaugh (14 cm H2 O). METHOD
A nimals Fifteen male rats of the Long-Evans strain, weighing 412 to 558 g (mean = 453 g), were used. Animals were randomly assigned to Group A (7 animals) or Group B (8 animals).
Apparatus Animals lay, ventral side down, on a small wooden platform which was housed within the same sound attenuating chamber used in Experiment 1. An adjustable moulded nylon face mask attached to the platform delivered room air from a small animal respirator (E and M Instrument Company, Model V5KG) which rested outside of the chamber. Subdermal electrodes in the Lead 1 position were employed to record EKG. The amplified
737 signal was taken from one of the polygraph's J6 output jacks and fed through a pulse shaping circuit which converted the EKG wave form to a square wave pulse of fixed duration. The pluse provided the input to a BRS solid state counting circuit which recorded the n u m b e r of R-waves for consecutive 60 sec intervals. A second polygraph channel provided a beat-to-beat heart rate display. A closed loop mercury strain gauge encircled the thorax of each rat, posterior to the forelimbs, and provided the input to a Model 270 Parks Electronic Company Plethysomograph and an indirect measure of volume change. Amplification of the Plethysomograph signal was achieved via a Grass low level DC preamplifier, Model 7P1. Body temperature was read from a Yellow Springs Instrument company Single Channel Telethermometer for which input was provided by a small animal rectal thermistor probe (Scientific Products T2605), previously calibrated with a NBS thermometer. A thermometer within the chamber was employed to measure the ambient temperature for all animals; this was maintained within the range 7 8 - 8 0 ° F.
Procedure Animals in Group A were respirated at a PIP of 14 cm H2 O, whereas animals in Group B were respirated at a PIP of 18 cm H20. In all other respects the procedures for the two groups were identical. All animals were respirated at a rate of 70 cpm, with an I:E Ratio of I : 1. Following an intraperitoneal injection of d-tubocurarine chloride (3.6 mg/kg) and upon the first signs of paralysis (usually within 10 sec) the face mask was fitted to the animal's snout. The mercury strain gauge was then placed about the animal's upper thorax, pin electrodes placed in the Lead 1 position and the thermal probe inserted. Continuous recordings of heart rate, rectal temperature and circumferential chest movements were made throughout the procedure. The experimenter monitored the PIP at regular intervals and made necessary adjustments to maintain the pressure at 14 cm H 2 0 for animals in Group A and 18 cm H 2 0 for animals of Group B. This procedure was maintained until animals recovered from curare or for a maximum of 180 min following the administration of curare. RESULTS The mean heart rates, circumferential chest movements and rectal temperatures displayed by the two groups of animals during the first 90 min of curarization are presented in Fig. 1. In this and subsequent figures, experimental periods are differentiated as either adaptation periods (A) or pressure change periods (P). Since pressures were predetermined for all animals in Groups A and B, the distinction is not directly applicable to these groups but facilitates comparisons with subsequent groups in which PIP was manipulated to maintain constant chest circumference. It will be observed that animals respirated at a PIP of 18 cm H 2 0 (Group B) display significantly higher heart rates and greater chest movements than do animals respirated at 14 cm H20. It will also be noted that in both groups of animals, heart rate and chest movements decline systematically as a function of time. The temperature data indicate that at the inception of the procedure both groups display subnormal body temperature. Whereas the temperatures of animals respirated at 18 cm H 2 0 tend to increase monotonically as a function of time under curare, those of animals respirated at 14 cm H 2 0 increaseduring the first 30
738
EISSENBERG AND BRENER
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DISCUSSION These results clearly indicate that as a function of time under curare, the tidal respiratory volume, as measured by circumferential chest movements, declines. This decrement in pulmonary air turnover is accompanied by correlated decreases in heart rate which are assumed here to be secondary to the respiratory effect. This effect in turn is attributed to the curare-induced loss of lung compliance. The temperature data support this interpretation. Although the 18 cm H 2 0 group commences the session with a body temperature approximately 1° C. lower than the 14 cm H 2 0 group, these subjects display an increase in body temperature over the 90 min period of approximately 2 ° C. Since the cardiac output is reduced by abnormally high intrathoracic pressures which reduce venous return, it is to be expected that as lung compliance decreases and the pulmonary volume declines that the venous return and hence the cardiac output will increase, leading to parallel increases in body temperature. Although this mechanism operates in the case of the 14 cm H 2 0 group as well, leading to an increase in body temperature over the first 30 min of the procedure, from the 40th through the 90th minute, the alveolar gas turnover provided by the inter-
action of this PIP with the increased pulmonary resistance becomes insufficient to maintain the vital functions. As a consequence the body temperature drops precipitously. Although all animals in the 18 cm H 2 0 group recovered from curare, 5 of 7 animals in the 14 cm H 2 0 group died before the 180th minute of curarization. We may therefore conclude that a PIP of 14 cm H 2 0 which is sufficient to support a curarized rat in a healthy condition (as indicated by rectal temperature and mean heart rate) at the inception of curarization will, if maintained, lead to fatal hypoventilation. On the other hand, a constant PIP which is sufficient to obviate this process will lead to pronounced hyperventilation at the inception of curarization. In view of these considerations, the method of constant pressure ventilation is to be discouraged. These data furthermore suggest that constant volume respiration is a preferred technique for the maintenance of curarized rats since this technique does not permit reductions in alveolar gas turnover as a function of curare-induced loss of lung compliance. The examination of such a method forms the substance of the final experiment in this series. EXPERIMENT 3 Just as the curare-elastance relationship has been overlooked, so it seems have the abnormal effects of intermittent positive pressure respiration on cardiovascular
A R T I F I C I A L RESPIRATION IN CURARIZED RAT activity. The differences between normal and positive pressure respiration are described in detail by Mushin et al. [ 18]. Of particular relevance to procedures in the operant cardiovascular conditioning literature are the following factors: (a) Normally, in the human, alveolar pressure falls to 1 to 2 cm H 2 0 below atmospheric pressure during inspiration and rises 1 to 2 cm H 2 0 above atmospheric pressure during expiration. In contrast, during positive pressure respiration alveolar pressure is greatest during inspiration and fails to approximately atmospheric pressure during expiration. The pulmonary pressure differences are therefore not only out of phase with those produced b y normal respiration, they are also greatly exaggerated (often 20 cm H 2 0 for rats) during positive pressure respiration. (b) The decreased intrathoracic pressure contingent upon normal inspiration causes an augmentation of venous return by drawing blood into the great thoracic veins. Because the intrathoracic pressure during inspiration in positive pressure artificial respiration is greatly increased, this important mechanism is disturbed and venous return to the heart is attenuated. (c) Given the relationship between intrathoracic pressure and venous return, if follows that if the positive pressure inspiration phase is unduly prolonged, the cardiac output will be severely diminished. Moreover, high intrathoracic pressure decreases cardiac output not only by decreasing venous return but also by a direct mechanical compression of the heart. In this connection Mushin et al. [18], p. 13, note: "The higher the peak pressure and the longer the time during which it acts, the greater is the cardiac tamponade and the interference with cardiac o u t p u t . " It has been reported by Morgan et al. [ 17] that when an I:E Ratio of 1:1 is employed with a PIP o f 20 cm H2 O, the cardiac output is decreased by 22% in dogs. Since the reduction in cardiac output that is associated with positive pressure respiration is a direct function of the length of time the lungs are inflated, it follows that the inspiration phase of the respiratory cycle must be kept as short as possible. That is, the I:E Ratio should be reduced to that minimum which will permit adequate pulmonary air turnover in the animal. The present experiment investigates the influence of different I:E Ratios on heart rate and body temperature in curarized rats. Because of the reported effect of curare on lung elastance, PIP was individually adjusted to maintain a constant circumferential chest movement ascertained as optimal for each subject. The effects of this procedure are compared with the effects of traditional constant pressure procedures described in Experiment II. METHOD Animals
Twenty-three rats of the Long-Evans strain, ranging in weight from 384 to 560 gms (mean = 456 gms) were used. Animals were randomly assigned to Group D, Group E or Group F. Apparatus
The apparatus employed described in Experiment 1.
were
in all cases those
Procedure
Procedural events were identical with those reported in Experiment 2, with three exceptions. Whereas each animal
739 in the previous experiment was maintained using an invariant pressure setting (14 or 18 cm H 2 0 ) , all animals in this experiment were respirated b y altering peak inspiration pressure to maintain chest movements of a constant amplitude. Secondly, respiration rate for all animals was reduced to 60 cpm to facilitate manipulation of I:E Ratios. This change was justified on the basis of Middaugh's [14] demonstration that diffferences in rate within the range of 5 0 - 7 0 cpm results in little heart rate change. Finally, I:E Ratio was manipulated to enable additional comparisons among groups differentiated by this variable: 8 animals in Group D were maintained on a 1 : 1 Ratio, 7 Ss in Group E represent a Ratio of 1:1, and 8 animals in Group F were maintained on a 1:3 Ratio. During a 30 min adaptation period (A) an optimal chest circumference value was selected for each animal on the basis of heart rate (a mean value between 3 8 0 - 4 6 0 bpm) a n d cardiotachograph records ( 1 0 - 3 0 bpm bidirectional variation). Thereafter, chest circumference was maintained by manipulating PIP during six 10-min pressure change periods (P). Changes in chest circumference were noted every 10 min (1 mm change in gauge circumference = pen deflection of 15.7 mm) and checked and recorded even more frequently following adaptation, in order to maintain a constant value. Temperatures were read every ten minutes and heart rate recorded for each one-minute interval. Sessions extended over 120 min or until subjects became reflexive, indicating recovery from drug effects. RESULTS Figure 2 illustrates the effects of varying I:E Ratio on heart rate, chest movements and b o d y temperature in a procedure where peak inspiration pressure is adjusted to maintain chest movements of a constant amplitude. The relative merits of this respiratory procedure may be assessed by making direct comparisons of its physiological effects with those produced by the constant pressure procedure described in Experiment 2. F Ratios computed to evaluate group differences in heart rate show no significant difference in dispersion between groups (A and B) respirated by the constant pressure procedure and no significant difference between the 1:2 and 1:3 Ratio groups (D and F) respirated by maintaining a constant tidal volume, Group D, however, is significantly different from Groups A and B (pC0.001), as is Group F (p~<0.01 and p~<0.05 respectively). Group E is significantly different from Groups A and D (p~<0.05) suggesting that regulating respiration by maintaining a constant tidal volume and a 1:1 Ratio produces greater heart rate stability than constant pressure respiration at that ratio, and significantly more variability than the same procedure (constant chest circumference) at a reduced I:E Ratio of 1:2 or 1:3. Thus, constant chest circumference respiration with a 1 : 2 Ratio results in the greatest heart rate stability and the same procedure with a 1:3 Ratio is not significantly different. Group A, at the low end of the temperature range, trending downward, but capable of only limited variability is significantly different (p~<0.05) from Group B which exhibits the greatest variability in temperature. Group D is significantly different from Group B (p<~0.05). Thus, aside from Group A, which registered subnormal temperatures, the 1:2 Ratio group evinced the greatest temperature stability, with no significant difference between Groups D and F.
740
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FIG. 2. Mean heart rate, chest circumference and temperature measures for groups of animals respirated by maintaining chest circumference constant at three different I:E Ratios. F Ratios performed to evaluate chest circumference variability indicate that Group D is significantly different from Group B (p<0.001). All groups maintained on a constant tidal volume are significantly different from group B (pC0.01) and Group E is significantly different from Group D (p~0.05). The minimal variability demonstrated by Group A is easily understood in view of the low PIP values used in a constant pressure procedure. Moreover, mean variance was calculated from P1 through P6 when chest circumference had already diminished, leaving little opportunity for further variability. DISCUSSION
Although attempts were made to maintain constant chest movements during the last 60 minutes of the session, it will be seen in Fig. 2 that this index does vary within a range of approximately 1.5 mm in the 1:1 Ratio group to approximately 0.65 mm in the 1:2 Ratio group. It will be noticed that the heart rate means show a tendency to increase over the last 60 min of the session with this trend being most marked from the 40th to the 80th minute. However, it will be noted that the mean heart rates for all three groups of animals are considerably more stable than those observed in the previous experiment. During the initial 30 min of the procedure body
temperature is an inverse function of the I:E Ratio, suggesting again that as ratio is attenuated cardiac output is facilitated. Although the temperatures of all groups display a systematic decline during the last 90 min of the session, the total temperature decrement does not exceed 0.5 ° C in any group. A comparison of these data with those of Groups A and B in Fig. 2 indicates that all groups display consistently higher body temperatures than subjects respirated at a constant PIP value with an I:E Ratio of I: 1. GENERAL DISCUSSION The relative merits of this respiratory procedure may be assessed by making direct comparisons of its physiological effects with those produced by the constant pressure procedure described in Experiment 2. Figure 3 graphically displays the clear differences between groups of animals (Groups A and B) respirated under invariant PIP values and animals maintained by a constant measure of chest circumference (Group D, 1:2 Ratio). The chest movements of the latter group remain relatively stable from the 40th through the 90th minute of the session. During this period it will also be observed that the heart rate measure remains relatively stable and does not display the same systematic decrement as is evident in the constant pressure groups. This occurs despite the fact that pressure was adjusted to
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FIG. 3. Mean heart rate, chest circumference and temperature measures for groups of animals respirated at constant PIP values or by maintaining constant chest circumference. maintain constant chest movements, not invariant heart rate, indicative of the importance of the relationship between these variables. Note that the temperature recorded for Group D, although displaying a slight decrement over the session, are at a considerably higher level than either Group A or Group B. We submit that the higher temperatures displayed by this group is primarily the results of the lower h E Ratio (1:2) employed with these animals. It must be remembered that attenuation of cardiac output is a joint function of the PIP value and the time during which it acts. Cardiac output in Group D was less attenuated because the intrathoracic pressure was elevated for a proportionately shorter period of time than was the case in either Group A or Group B. An important feature of any maintenance procedure is that is should be reliable. In order to ascertain the reliability of the respiratory procedure employed for Group D in Experiment 3 an additional group of 8 rats was run under an identical procedure (Group 1). Figure 4 presents a comparison of mean heart rates, body temperatures and chest movements in these 2 groups of animals. This figure also illustrates the mean PIPs employed in order to maintain optimal chest movements. It will be observed that the temperature changes as well as other measures (excepting pressures) are indeed replicable. It is interesting to note that the group pressure points are parallel although over 2 cm H 2 0 apart throughout the session. This
difference probably is a function of a substantial difference in mean body weight in the two groups: Group D mean body weight = 478 g: Group 1 mean body weight = 394 g. It should also be noted that animals which display similar body weights often require substantially different PIPs in order to maintain the same tidal volume. This is explainable in terms of difference in position, face mask fit and varying effects of curare. It is evident from these data that the selection of appropriate respiration parameters poses a very considerable problem. With regard to peak inspiration pressure, a balance must be struck between excessively high pulmonary pressures which interfere with cardiac output and low pressures which result in a hypoventilated animal. But the reduction of cardiac output associated with positive pressure respiration also varies as a direct function of the time that the lungs are inflated. Therefore, it follows that the inspiration phase of the respiratory cycle must be kept as short as possible, within the limits which permit adequate pulmonary air turnover in the organism. In view of the stability of the heart rate of rats respirated at a 1:2 I:E Ratio and also because of the relative ease with which a constant respiratory volume may be maintained with this setting it is suggested that this ratio and a constant chest circumference procedure is the most efficient of those investigated for maintaining curarized rats in a healthy condition.
742
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FIG. 4. A comparison of two groups of animals respirated by maintaining chest circumference constant at a 1:2 h E Ratio and 60 cpm.
REFERENCES 1. Altman, P. L. and D. S. Dittmer, editors. Biology Data Book. Washington: Federation of American Societies for Experimental Biology, 1964, p. 260. 2. Blood, F. R., R. V. Elliott and F. E. D'Amour. The physiology of the rat in extreme anoxia. Am. J. Physiol. 146: 319-329, 1946. 3. Brener, J. M., E. M. Eissenberg and S. J. Middaugh. Respiratory and somatomotor factors associated with operant conditioning of cardiovascular responses in curarized rats. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press. 4. DiCara, L. V. Analysis of arterial blood gases in the curarized artificially respirated rat. Behav. Res. Instrum. 2: 67-69, 1970. 5. DiCara, L. V. and N. E. Miller. Changes in heart rate instrumentally learned by curarized rats as avoidance responses. J. comp. physiol. Psychol. 65: 8 - 1 2 , 1968a. 6. DiCara, L. V. and N. E. Miller. Transfer of instrumentally learned heart rate changes from curarized to noncurarized state: implications for a mediational hypothesis. J. comp. physiol. Psychol. 68: 159-162, 1969. 7. Goesling, W. J. The effects of prior skeletal conditioning on the conditioning of heart rate changes in curarized subjects. Unpublished doctoral dissertation, University of Tennessee, 1969. 8. Hahn, W. W. Apparatus and technique for work with the curarized rat. Psychophysiology 7: 283-286, 1970.
9. Hahn, W. W. The learning of autonomic responses by curarized animals. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press. 10. Hothersall, D. and 1. M. Brener. Operant conditioning of changes in heart rate in curarized rats. J. cornp, physiol. Psychol. 68: 338-342, 1969. 11. Jones, W. E., B. G. MacGrath and H. H. Aculthorpe. Pathological processes in disease II. Blood of the albino rat. Approximate physico-chemical description. Ann. trop. Med. Parasit. 44: 168-186, 1950. 12. King, T. K. C. and D. Bell. Arterial blood gas in specific pathogen free and bronchitis rats. J. appl. Physiol. 146: 319-329, 1966. 13. Massion, W. H. Effects of curare on elastic properties of chest and lungs of the dog. J. appl. PhysioL 11: 309- 312, 1957. 14. Middaugh, S. J. Operant conditioning of heart rate: the curarized artificially-respirated rat as a source of variability. Unpublished doctoral dissertation, University of Tennessee, 1971. 15. Miller, N. E. and L. V. DiCara. Instrumental learning of heart rate changes in curarized rats: shaping and specificity to discriminative stimulus. J. comp. physiol. Psychol. 63: 12-19, 1967. 16. Miller, N. E. and B. R. Dworkin. Visceral learning: recent difficulties with curarized rats and significant problems for human research. In: Cardiovascular Psychophysiology, edited by P. A. Obrist, A. H. Black, J. M. Brener, and L. V. DiCara. Chicago: Aldine Atherton, 1974, in press.
A R T I F I C I A L R E S P I R A T I O N IN C U R A R I Z E D R A T 17. Morgan, B. C., W. E. Martin, T. F. Hornbein, E. W. Crawford, and W. G. Gunteroth. Hemodynamic effects of intermittent positive pressure respiration. Anesthesiology 27: 5 8 4 - 5 9 0 , 1966. 18. Mushin, W. W., L. Rendell-Baker, P. Thompson and W. W. Mapleson. Automatic Ventilation o f the Lungs. Oxford: Blackwell Scientific Publications, 1969, pp. 2 - 2 5 . 19. Popovic, V. and P. Popovic. Permanent cannulation of aorta and vena cava in rats and ground squirrels. J. appl. Physiol. 15: 7 2 7 - 7 2 8 , 1959.
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20. Ray, R. Classical conditioning of heart rate in restrained and curarized rats. Unpublished doctoral dissertation, University of Tennessee, 1969. 21. Slaughter, J., W. Hahn and P. Rinaldi. Instrumental conditioning in the curarized rat with varied amounts of pre-training. Z comp. physiol. Psychol. 72: 356-395, 1970. 22. Spector, W. E., editor. Handbook o f Biological Data. Philadelphia: W. B. Saunders Company, 1956, p. 270. 23. Trowill, J. A. Instrumental conditioning of the heart rate in the curarized rat. J. cornp, physiol. Psychol. 6 3 : 7 - 1 1 , 1967.