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1 Journal of Physiology (1989), 408, pp With 7 text-figures Printed in Great Britain A DYNAMIC ANALYSIS OF THE VENTILATORY RESPONSE TO HYPOXIA IN MAN BY J. F. BERTHOLON, M. EUGENE, THE LATE E. LABEYRIE AND A. TEILLAC From the Laboratoire Central d'explorations Fonctionnelles Respiratoires and U.F.R. Lariboisiere-Saint Louis, Service du Professeur Andre'e Teillac, Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'hopital, Paris, France (Received 24 November 1987) SUMMARY 1. The dynamics of the ventilatory response to isocapnic hypoxia were studied in seven healthy subjects using four different levels of hypoxia, (inspired oxygen pressures, P1I02 equal to 110, 100, 80 and 60 mmhg) successively increasing and decreasing stepwise. 2. Five such progressions were performed for each subject, corresponding to five different durations of the steps (t) ranging between 0 33 and 5 00 min. The overall duration of one test (T) was taken as the sum of the seven successive P10 2 hypoxic steps (t) plus one step t of air breathing. Thus, the values of T ranged between 2-6 and 40 0 min. 3. End-tidal CO2 pressure was maintained constant (±1 mmhg) throughout the test by manipulation of inspired CO2 pressure. 4. We measured, as a function of T, (i) the magnitude of the loops formed by the ventilatory response curves (PAo2-VE) as measured by their surface area (S), (ii) the magnitude of ventilatory response to each rising hypoxic step, and (iii) the difference between resting VE and VE observed at PA, 2 equal to 50 mmhg (AV50). On average, we found one maximum in absolute value of S at T = 8 min and one minimum at T = 12 min, along with two maxima of ventilatory response at T values of 8 and 24 min. 5. The same measurements were made on tidal volume response curves (PA,2 -VT) and ventilatory frequency response curves (PA, 2-f): on average we observed two non-significant peaks in the progression with T of VT and S(VT) and two significant peaks in that of AVT,50 for T = 8 and T = 24 min. No significant peak was observed in the progression with T of f curve parameters. 6. These results are discussed together with the current dynamic model of the ventilatory control system, which includes a central neural controller with no dynamics of its own and a linear response to chemoreceptor inputs. We discuss the physiological meaning of a negative loop area in relation to the previously described depressant effect of hypoxia upon the brain stem. 7. We conclude that the dynamics of the controlling neuronal network are responsible for the observed singularities which result from differential sensitivity

2 474 J. F. BERTHOLON AND OTHERS properties of the controller. We propose the existence of discrete excitatory states of the controller as a possible explanation of the shape of the steady-state response curve to hypoxia and of the loop variations. INTRODUCTION In a prevous paper concerning CO2 regulation, we challenged the assumption made in the classical model of ventilatory control (Grodins, Gray, Schroeder, Noris & Jones, 1954) of a linear neural controller with no dynamics of its own. Our experimental results had shown major discrepancies with the simple dynamic model of Bellville, Whipp, Kaufman, Swanson, Aqleh & Wiberg (1979) and we proposed to explain them by the existence of bimodal sensitivity to the stimulus, originating in the central neural network and revealed by certain dynamics of stimulation (differential sensitivity) or by the crossing of a threshold value of the stimulus (Bertholon, Carles, Eugene, Labeyrie & Teillac, 1988). The present study was made in order to see if, using hypoxia as the chemical stimulus for ventilation, a similar protocol would yield the same qualitative observations. Hypoxia is considered by all authors to increase ventilation only through stimulation of aortic and carotid chemoreceptors, that is, by means of the peripheral chemoreflex. If we only take into account the current dynamic model of the ventilatory control system, a dynamic study with hypoxia as the stimulus should demonstrate the sole dynamics of this peripheral chemoreflex (Robbins, 1984). A review of the literature concerning hypoxic stimulation of the ventilatory control system shows that dynamic studies using Rebuck's rebreathing method (Rebuck & Campbell, 1974) and stable-state studies have both yielded non-linear response curves with the Po2-ventilation curves being best fitted with hyperbolic (Lloyd, Jukes & Cunningham, 1958; Cunningham, Patrick & Lloyd, 1964; Weil, Byrne- Quinn, Sodal, Friessen, Underhill, Filley & Grover, 1970; Rebuck & Campbell, 1974; Hirshman, McCullough & Weil, 1975; Weil & Zwillich, 1976; Slustky, Mahutte & Rebuck, 1979) or exponential (Kronenberg, Hamilton, Graberl, Hickey, Read & Severinghaus, 1972) functions. Apart from rebreathing experiments, specific dynamic studies have used (i) one or two breaths of pure 02 (Dejours, 1962; Downes & Lambertsen, 1966; Dejours, 1968), and (ii) a single hypoxic step (De Goede, Van der Hoeven, Berkenbosch, Olievier & Van Beek, 1983), from which were derived values of delay and time constant for the dynamics of response, or (iii) sinusoidal stimulation (Robbins, 1984). This last study showed that simple models were unable to account for the observed phenomena. As for CO2 stimulation, we decided to study the influence of the rate of rise of the hypoxic stimulus on the morphology of the curves of ventilatory response, a qualitative approach which gets around the problem of the well-known quantitative variability of the responses. If the direction of variation of a dynamic stimulus is reversed, the response curve usually does not follow the on-going path but rather forms a loop. These loops are interesting to study because they can result from the existence of (i) time constants, (ii) non-linearities originating in the controlled system such as delays or nonconstant parameters (both of which have been included in current dynamic models

3 VENTILATOR Y RESPONSE TO HYPOXIA of ventilatory control system (Bellville et al. 1979; De Goede et al. 1983; Robbins, 1984)), or (iii) non-linearities originating from the controller. An example of controller non-linearity could be the phenomenon of central hypoxic depression which was observed by numerous authors with chemodenervated human or animal subjects (Heymans, Bouckaert & Dautrebande, 1930; Davenport, Brewer, Chambers & Goldschmidt, 1947; Wade, Larson, Hickey, Ehrenfeld & Severinghaus, 1970; Vizek, Pickett & Weil, 1972; Lahiri, 1976; Van Beek, Berkenbosch, De Goede, Olievier & Quanjer, 1982). Recently, Berkenbosch (1983) observed a depression of ventilation in anaesthetized cats when the medulla was exposed to normocapnic hypoxia while peripheral Pa,co2 and Pa, 2 were kept at their normal resting values. With normal adult subjects and using a step of systemic normocapnic hypoxia, Chambille, Gue6nard, Loncle & Bargeton (1975), Long & Lawson (1984) and Easton, Slykerman & Anthonisen (1986) showed a progressive decrease in ventilation following a rapid increase lasting 2-5 min. However, some authors did not observe such a central hypoxic depression (Guz, 1966; Lugliani, Whipp, Seard & Wasserman, 1971; Swanson, Whipp, Kaufman, Aqleh, Winter & Bellville, 1978). Others found it at very low levels of hypoxia, such as Morril, Meyer & Weil (1975) with dogs or with anaesthetized animals only, such as Miller & Tenney (1975) and Gautier & Bonora (1980) with cats. Another reported manifestation of controller non-linearity is the central neural after-discharge (Eldridge, Kiley & Paydarfar, 1987). If central hypoxic depression and after-discharge exist, then modifications of the shape of the loops are to be expected as compared with models which only take into account controlled system dynamics, i.e. transfer of the stimulus from the mouth to the chemoreceptors. For the present study, we used a step-by-step stimulation allowing us to vary the duration of each step between 0 3 and 5 min. Considering the dynamics described in the literature the shortest test should be influenced by the dynamics of the controlled system while the others could be thought of as being virtually not influenced by those dynamics and thus be considered as cases of static stimulation. In this paper, we report the progression with the rate of change of the stimulus of the magnitude of ventilatory response and of the loops in the response curves. Then we compare it with the progression that would be expected from current knowledge of the behaviour of the regulator. METHODS Apparatu8 The experimental set-up is shown in Fig. 1. The subject was comfortably seated and listened to soft music with headphones. He breathed through a facial mask and a No. 3 Fleisch pneumotachometer (Gould, Holland) both of which added a dead space of approximately 120 ml to the airways. The gas flow from which the subject breathed exceeded the instantaneous inspiratory flow of any subject during the tests in order to avoid rebreathing of the expired gas. Excess inspiratory and expired gases were ventilated to the exterior. This set-up minimized the mechanical load on the ventilatory system. The stimulation was carried out by increasing the inspired fraction of nitrogen (FIN2) using four different N2 flows sequentially added to the air flow. The sequence of stimulation was driven by an electronic clock which controlled the opening of solenoid valves. End-tidal Pco, variations 475

4 476 J. F. BERTHOLOX AND OTHERS 2 Fig. 1. Experimental set-up: 1, N2 cylinders; 2, CO2 cylinder; 3, facial mask; 4, Fleisch pneumotachometer No. 3; 5, analog integrator; 6, fast 02 and CO2 analysers; 7, electronic clock controlling solenoid valves (EV). with ventilatory response to hypoxia were maintained within 1 mmhg by manually adding pure CO2 in the inspiratory mixture by means of a precise flowmeter (Gauthier, France). A 200 ml min-' flow of gas was sampled in the mask and sent to fast 02 and CO2 analysers (Beckman OM1 1 and LB2, USA). The rise time of the gas analysing system was 230 ms, allowing an accurate measurement of end-tidal 02 and CO2 partial pressures. Instantaneous flow signal was given by the pneumotachometer coupled with a differential manometer (Schlumberger Al 112, France) and a conditioning device (Auxiliaire CA 1065, Schlumberger, France) and then integrated to yield inspired and expired tidal volumes. The analog integrator was reset to zero between inspiration and expiration and, in order to avoid any drift, a check for zero flow was periodically made during long tests by shunting the manometer. The volume measurement system was calibrated, before and after each test, directly in BTPS conditions using a sinusoidal pump with 500 ml capacity and variable period. All volume and flow results are given in the BTPS standard. The electric signals from the analysers and the integrator were continuously recorded with a chart recorder (Siemens Oscillomink 8, FRG). The inspired partial pressures of oxygen (P.O2) were set equal to 110, 100, 80 and 60 mmhg. These values were chosen after preliminary tests in order to obtain roughly equal stimulus increments at the alveolar level, in the hypoxic range to which the regulator is known to be sensitive. Thus, these different PI 2 settings gave the following mean PA,O values on the longest test (T = 40 min) for each step and seven subjects: 70-6(± 5-6), 57-3 ( 3-4), 47-0 (± 2-0) and 37 0 (±2-2) mmhg. During each test we recorded 10 min of air breathing and then successively four steps up in hypoxia and four steps down to air breathing again. The duration of each step (t) was varied for each of the five different tests made with each subject. The values of t were: 0 33, 1-00, 1-50, 3-00 and 5 00 min. T was the total duration of the stimulation, equal to 8 t, and thus had the following values: 2-66, 8-00, 12-00, and min. The slope of the line joining the 1st step and the 4th step was used as a measure of the rate of rise of the stimulus (dp1o,/dt) and had values between 77 and 5 mmhg min-' (Fig. 2). Data analysis Calculation of the ventilatory parameters and of the PA,O was made from the recording chart with a digitizer connected to a microcomputer (Apple II, lisa) in the following way: (i) on the two last cycles of each step t of 0-33 min, (ii) on the five last cycles of each step t of 1-00 and 1-50 min and (iii) on the ten last cycles of each step t of 3-00 and 5-00 min. PA,O was taken as P.O at the end of expiration. The ventilation and alveolar 02 pressure values are given as: VE! VErest and PAO2/ A.02,rest-

5 V7ENTILA TOR Y RESPONSE TO HYPOXIA (min) (min) dpl0o2/dtl 80 b; (Torr min-1) 0 1. " _ ;; (Torr) Rest t T t T z5 Fig. 2. Experimental protocol with seven isocapnic hypoxic steps (t min) of total duration T min and the corresponding values of the rate of rise of the stimulus dpi0o/dt. The magnitude of the loops was quantified by surface area measurement (S) using planimetry after closing the surface by linear interpolation between experimental points. Anticlockwise rotation gave negative areas, clockwise rotation gave positive areas and the total area of the curve's surface was the algebraic sum of elementary areas when the limbs of the curve crossed. This parameter, classically used as a measure of hysteresis in response curves, shows to what extent and in which direction the response of the system is influenced by what happened before. Variations with T of S when the same stimulation is applied to a single compartment model (Robbins, 1984) show a positive maximum corresponding to the time constant of the compartment (maximum phase shift). With such a model, this value is null, both for very fast and very slow dynamics of stimulation. We considered the magnitude of the ventilatory response to each rising hypoxic step (VE!VE.rest (1) to (4)) and the difference between VE at rest before the test and VE at a PA of 50 mmhg (AV50) as measured after linear interpolation between the experimental points. This parameter was chosen after the classical V40 (sensitivity to hypoxia parameter) of Severinghaus, Bainton & Carcelen (1966). We retained the value of 50 mmhg instead of 40 mmhg because, for some subjects and some tests, the ventilatory response was high enough to maintain PA O above this latter value during all steps. The same measurements were made on the PA OVT and PA -f response curves. Statistical analysis Comparisons of the results among tests for the seven subjects were performed using two-way variance analysis without replication and an F test on the remainder of the variance. This allowed elimination of the influence of the great inter-individual variability on the effect of varying the stimulation period T. Multiple comparisons were made using the Student-Newman-Keuls (SNK) test (Zar, 1974). Subjects We studied seven healthy non-smoking subjects (five male and two female) aged The tests were performed in the morning in a random order and with time intervals of at least 1 day to ensure that there would be no influence of the preceding test. They were not informed about the purposes of the experiment but consented to the protocol. RESULTS The progression with t of the mean experimental curves (PA,O2/PA,02,rest)- = 100 min (VE/ VErest) showed, as expected, an increased ventilatory response at t compared to t = 0 33 min, but it also displayed two maxima for the parameters of this response (AV50, VE/VE,rest(4)) at t = 100 min and t = 3 00 min, along with a

6 478 J. F. BERTHOLON AND OTHERS A 2 t (min) PA, 02 /PA, O,. rest 1.5- t(min) PAC, 0,. res C V E (4) _1 0 Vro ~~~~~~~~~~~~-1 V cu Fig. 3. For legend see facing page

7 VENTILATOR Y RESPONSE TO HYPOXIA single minimum for t = 1-50 min. As for the magnitude of the loops, there was a maximum in the absolute value of S (ISI) at t = 1-00 min and a minimum at t = 1P50 min. This is shown in Fig. 3 A for one subject and in Fig. 3B for the mean curves of the seven subjects. At t = 0 33 min, the curves are looping clockwise (positive area) due to delays and damping of the stimulus, but some subjects displayed a steep rise in ventilation from the 4th to the 5th or 6th step. At t = 1-00 min there was a noticeable increase in ventilation even for the 1st step, a fast decrease from the 5th step, and the ventilation frequently remained depressed until the end of the test; thus the response curves are mostly looping anticlockwise with a negative area S. At t = 1-50 min, the ventilation only rose at a high level of hypoxia. On the first return step (5th step), it frequently remained high so the crossing of the limbs of the curves yields very small ISI values. At t = 3 00 min, ventilation increased from the 1st step, decreased rapidly upon lowering of the stimulus and remained depressed until the end of the test. This results in very negative values for S. At t = 5-00 min there was no response to the first steps but, due to the sudden drop in response when the stimulus was lowered and the persistent depression of ventilation, more negative values for S were observed as compared with the test at t = 3-00 min. 479 Figure 3C displays the variations in VE/VE rest (4), A P50 and S as a function of t for the same subject as in Fig. 3A. This subject and two others produced a second maximum of ISI at t = 3 00 min. The significance of these singularities (i.e. maxima and minima) observed in the progression, for increasing values of T, of ventilatory response and loop magnitude was statistically tested. Table 1 shows the results of the comparisons between tests using the SNK multiple comparison test performed after a two-way analysis of variance together with the mean results for each level of the stimulation duration. Ventilatory response as a function of t and stimulus level Figure 4A shows the progression with t of the mean ventilatory response to each rising step of our seven subjects. The mean locations of the peaks were 1-00 and 3-00 min on the t axis for all steps but only the peaks in VE/VE rest (4) are significant. None of these peaks is predicted by the current model of the PA, 2 regulation system. Fig. 3. A shows the results of one subject in the space PA O2/PA. rest X VE/VEret X t. For convenience the curves are equally spaced on the t scale. The highest ventilatory responses to each test are linked by a dashed line. Differences in the morphology of the ascending and descending limbs of the curves according to the value of t are well displayed. B shows the mean results of the stimulation cycles for the seven subjects. Despite the smoothing effect of the averaging process, similar observations can be made. C shows, for the same subject as A, the variations with t of the loop magnitude as measured by S(VE/VE rest) (0@ continuous line) in arbitrary units (a.u.), of the relative ventilatory response VE! VE,rest (4) to the fourth step (O, dashed line) and of AV50 (0). There are two maxima of SI (negative areas), AV50 and VE/VE rest (4) at t values of 1-0 and 3 0 min along with one minimum at t equal to 1-5 min for this subject.

8 480 J. F. BERTHOLON AND OTHERS -C2 4-j- o w 4, -c o. a)o _ E 9 11 o-i -,_ i 6 90 V N~-, V ce o. OQ r- 9._ o ,:~J --o- ovt o V V= I U o U o V oo oc Vf C o V cec >._ 11 <o I M -, v, li- LC CA,o o9 V- 40 ce ce ce 0~I 0 0 X *- I :4 F.Ks :4 -. :.; < C

9 VENTILATOR Y RESPONSE TO HYPOXIA 481 A o lst step nd step 3 t (min) o 3rd step 5 A 4th step B *4 -i Tests Fig. 4. A shows the variations with t of the relative ventilatory response (VE/VE,rest) to each rising step (mean results). The asterisks symbolize the significance of the ventilatory increase at the three first steps as compared with resting ventilation (* P < 005; **P < 0O001). For these steps the peaks were none the less non-significant. At the 4th step, ventilation increased significantly for all values of t and the two peaks of response were statistically significant (mean results+ s.d.). B shows the individual differences in VE/VE,re.t (4) between tests (1: 033 min; 2: 100 min; 3: 1-50 min; 4: 3-00 min; 5: 500 min). We also tested the significance of changes in ventilation as compared with resting ventilation compared with resting ventilation for each of the four increasing levels of hypoxia. We found that in our population there was no significant modification of ventilation with hypoxia for the 1st rising step (PA,o2 = mmhg, mean+s.d.) at all tests and for the 2nd step (PA,o, = mmhg, mean S.D.) at t values of 0 33, 1-50 and 5-00 min (respectively -4%, 2 % and 7 %) whereas 16 PH Y 408

10 482 J. F. BERTHOLON AND OTHERS the ventilation significantly increased for t values of 1 00 and 3-00 min (respectively 22% and 18%) for the 2nd step. For the 3rd step (PA,o2 = mmhg, mean+ S.D.), ventilation increased significantly for all but the shortest test (respectively 3 %, 42 %, 20 %, 40 % and 19 %). For the 4th step (PA,o2 = 44+4 mmhg, mean+s.d.), ventilation increased sig- A B t (min) Tests Fig. 5. A shows the variations with t of the difference between resting ventilation and ventilation at PA = 50 mmhg (AV50). Mean results+ S.D. B shows the individual differences in AV50 between the five tests. nificantly at all tests (respectively 25 %, 105 %, 80 %, 104 % and 80 %). Figure 4B shows the variations in ventilatory response to the 4th step from the shortest to the longest test for each subject. These results suggest the existence of a hypoxic threshold of activation for the regulatory system, the value of which depends on the rate of rise of the stimulus. Figure 5A shows the progression with t of mean AV50, that is, the magnitude of the increase in ventilation from resting state to PA,O2 = 50 mmhg: there was

11 VENTILATOR Y RESPONSE TO HYPOXIA 483 one maximum at t = 1-00 min and one at t = 3 00 min. Figure 5B displays the variations of this parameter from test to test for each subject. As this parameter is classically used to quantify the sensitivity of the system to hypoxia, our results show that sensitivity is rate dependent with two preferential rates of rise for the stimulus. A 2 0 x U) B t (min) 5 0 x Cu U) Tests Fig. 6. A shows the variations with t of the surface area, in arbitrary units (a.u.), of the Mean results+s.d. B shows the fao2/pa,o2,rest). loops (S) in the curves V/ V,=rest=f(P individual differences in S(YVE/YVErest) between the five tests. Three subjects had a second maximum of 1S1 at t = 3 00 min. Loop magnitude as a function of t Figure 6A shows the mean variation in the magnitude of the loops as measured by S(VE/VE rest) with increasing t for our seven subjects. There was one minimum at t = 1-00 min and one maximum at t = 1-50 min. Only three of our seven subjects produced a second peak at t = 3-00 min as shown on Fig. 6B. Known peripheral 16-2

12 484 J. F. BERTHOLON AND OTHERS chemoreflex dynamics predict a single positive peak for short values of t (Bellville et al. 1979; De Goede et al. 1983; Robbins, 1984). Tidal volume (VT) and ventilatory frequency (f) The relative influences of VT and f upon the progression of VE and S were also studied. Two non-significant maxima of response were seen with mean VT curves at t values of 100 and 300 min but not with f curves. The mean frequency stayed A 3 B 5 t (min) 1- g Tests Fig. 7. A shows the variations with t of AVT50 measured in the same way as A V50. Mean results+ S.D. B shows the individual differences in AVT 50 between the five tests. close to the resting values in all tests while mean VT did not exceed 1-6 times the resting value. Two maxima at the same locations on the t scale were observed in the progression with t of VT- VT,rest (AVT,50) (Fig. 7A and B) and Of f-frest (Af50) but they were significant only for the VT response (P < 0025). As for the progression with t of parameter S of the loops in the VT and f response curves, there were two maxima of ISI at t = 1-0 and t = 3-0 min and one minimum

13 VENTILATOR Y RESPONSE TO HYPOXIA 485 at t = 1-5 min for VT curves and a single maximum of ISI at t = 10 min along with one minimum at t = 1P5 min for f curves, but all these peaks were statistically non-significant. Considering the high sensitivity of the system to variations in Pco2, we had to rule out the possibility that a peak in ventilatory response could be explained by a concomitant rise in PA,CO2 (and likewise a trough in ventilation by a drop in this variable); we found no correlation between the two. DISCUSSION The response of the ventilatory control system using dynamic hypoxic stimulation has rarely been studied (Dejours, 1962; Downes & Lambertsen, 1966; De Goede et al. 1983; Robbins, 1984) due to the fact that, classically, 02 regulation only involves the peripheral chemoreflex, whose dynamics have been studied using hypercapnic stimulation. Nevertheless, many studies have been untertaken on 02 regulation using static stimulation (Weil et al. 1970) or rebreathing methods (Rebuck & Campbell, 1974). From these results, the ventilatory response curve has most frequently been modelled by a hyperbolic or exponential function. Using an isocapnic hypoxic stimulus, successively increasing and decreasing stepwise and of varying overall duration T, we made a qualitative study of the curves PA,O2-VE produced by seven healthy subjects. We looked at the variations with increasing T of the magnitude of the ventilatory response to each test (VE/ VE,rest (4) and AV50) and of the loops formed by the curves (S(VE/VE,rest)). We found that the ventilatory response went through two maxima, one for T = 8 min and the second for T = 24 min. These results are similar to those obtained by CO2 stimulation using a very similar protocol (Bertholon et al. 1988). As for the surface area (IS(VE/VE,rest)l) of the loops, which was taken as a measure of their magnitude, there was one minimum at T = 12 min after a first maximum for shorter values of T, as in our CO2 experiments, but the second maximum that had been found with hypercapnia was not seen with hypoxic tests. However, we must stress that a second maximum at T = 24 min was found in S(VE/VE,rest) curves with three subjects, and also on the mean S(VT/VT,rest) curves, which was statistically non-significant. Moreover, the second peak of S(VE/VE,rest) in the CO2 experiments occurred at T > 24 min for certain (four out of ten) subjects. The main difference between CO2 and 02 stimulations was that the surface areas of the loops were positive in hypercapnic tests whereas we found them negative with hypoxic tests (except at T values of 2-6 and 12 min where S was positive; Fig. 6A). There was only a non-significant contribution of the ventilatory frequency (f) to the occurrence of singularities in overall ventilatory response as with hypercapnic stimulation, but the ventilatory levels reached with hypoxia were much lower (maximum mean VE/VE,rest = 2 0) than those reached during hypercapnia (maximum mean VE/VE rest = 3 8). We found that the response curves to hypoxia were non-linear at all values of T; this non-linearity, admitted by all authors, is usually attributed to the hyperbolic or exponential relationship between the activity of the chemoreceptors and Pa 2 (Eyzaguirre & Zapata, 1984). However, although this seems true when going from

14 486 J. F. BERTHOLON AND OTHERS hyperoxia to hypoxia, it is much less obvious in the Pa O2 range of mmhg where there is a steep increase in chemoreceptor activity (Lahiri, Mokashi, Mulligan & Nishino, 1981). The observed slight curvature of the response in this range must, in our view, be attributed to the existence of different characteristics of activation for the chemoreceptive cells. At any rate, this gives no explanation for the ventilatory response which, for certain rates of rise of hypoxia, is indistinguishable from zero down to a very low value of PA 2. Magnitude of the loops in the stimulus-response curves In classical dynamic analysis of the ventilatory control system, most authors have used models comprising two parallel subsystems (central and peripheral chemoreflexes) derived from the hydraulic and electric analog model of Grodins et al. (1954). According to this approach and since hypoxic stimulation is known to involve only the peripheral chemoreflex, the existence of a loop in the stimulus response curve VE = f(pa,o,) can be the result of (i) pure delays, represented by the circulation time between lungs and peripheral chemoreceptors, (ii) damping, caused by the different mechanisms involved in the interreaction of the system with the stimulus, such as chemical reactions concerning 02 and diffusion of 02 to chemosensitive cells, and (iii) neural dynamics as transduction of the chemical stimulus into nervous discharges in the chemoreceptors, integration of the stimuli and generation of the ventilatory drive by the bulbo-pontile centres and spinal motor nuclei. Such dynamics have, up to now, been considered as negligible in dynamic models. The circulation time to the peripheral chemoreceptors has been estimated at 4-13 s in experiments with hypercapnia (Dejours, 1962; Edelman, Epstein, Lahiri & Cherniack, 1973; Gelfand & Lambertsen, 1973; Miller, Cunningham, Lloyd & Young, 1974; Milhorn, 1976; Bellville et al. 1979; Sohrab & Yamashiro, 1980). The damping of the response has generally been evaluated from a simple compartment model using recordings of averaged ventilatory responses to step stimulations by CO2. A review of the literature gives values between 3 and 26 s for the peripheral chemoreflex time constant (Gelfand et al. 1973; Swanson & Beliville, 1975; Bellville et al. 1979; Gardner, 1980). Other values for delay and damping have come from experiments using one or two breaths of pure N2 to create a transitory hypoxia. The delay has been estimated at 3-8 s and the time constant for the response at 6-5 s (Downes & Lambertsen, 1966) which is consistent with Dejours (1962, 1968) experiments. With the same forcing functions we used in our experiments, the classical model of ventilatory control (Bellville et al. 1979; Robbins, 1984) produces one maximum of surface area S(VE) of the loops for a T equal to the time constant of the peripheral chemoreflex system (maximum phase shift). In our hypoxic experiments this maximum should thus be seen at T values smaller than 2-6 min. In fact, we observed a decrease in S for T values greater than 2-6 min, but S had a very negative value at T = 8 min and then went through a slightly positive peak at T = 12 min, followed by increasing negative values for T values of 24 and 40 min (Fig. 6A). These results cannot be produced by a delay or a damping mechanism and we propose, as for our CO2 tests, to explain the plurality of peaks in terms of central neural dynamics.

15 VENTILATOR Y RESPONSE TO HYPOXIA 487 Magnitude of ventilatory response The classical model predicts a smooth rise in ventilatory response as a function of T. The response should stabilize for dynamics of stimulation which are slow compared to the dynamics of the chemoreflex (steady-state conditions). Considering the values of delay and time constant found in the literature, this should be the case for T values larger than 8 min (that is, step duration values (t) exceeding circulation time plus three time constants). Our subjects produced two maxima of response at mean T values of 8 min for the first and 24 min for the second with a minimum of response at T equal to 12 min (Fig. 4). Such responses clearly cannot be obtained with the current singlecompartment model. However, many authors have described a depressant effect of hypoxia on the controller which is attributed by most authors to an increased wash-out of CO2 from medullary tissue due to hypoxic vasodilatation (Berkenbosch, 1983). According to this explanation, the full effect of depression of ventilation should appear for a rather low T value. Weiskopf & Gabel (1975) calculated the time course of cerebral blood flow and subsequent tissue Pco2 and found that a full reduction of local Pco, should occur in less than 2 min. Berkenbosch (1983) has shown that this depressant effect developed in less than 1 min in anaesthetized cats, leading to a plateau in ventilation. De Goede et al. (1983) estimated the dynamics of the central depression to have a time constant of about 90 s in anaesthetized cats. Experiments in man have given conflicting results. Chambille et al. (1975) with an hypoxic isocapnic step (PA,o2 = 60 mmhg) in only one subject showed a maximum of response after 4 min and then a steady decrease until the end of the step at the 20th minute. Swanson et al. (1978) found no depression using hypoxic steps (PA, 2 = 53 mmhg) of 5 min either in normal and carotid-body-resected subjects, but such a hypoxic step during hypercapnia (PA,co2 = 49 mmhg) produced a small decrease in ventilation begining at the 3rd minute of the step and completed 1 min after. Easton et al. (1986) found the depression to take 15 min to occur fully using a hypoxic step (PAo 2 = 50 mmhg). Observations have been made in neonatal animals (Cross & Oppe, 1952; Cross, Hooper, Lord, 1954; Hill, 1959; Lawson & Long, 1983; Long & Lawson, 1984; Bureau, Cote, Blanchard, Hobbs, Foulon & Dalle, 1986) of central hypoxic depression reaching completion after 2-5 min of step stimulation. Some authors have involved a progressive modification of brain neurotransmitter metabolism in the genesis of the respiratory response to hypoxia (Gautier & Bonora, 1980; Lawson & Long, 1983; Neubauer, Posner, Santiago & Edelman, 1987) but the time course of such a phenomenon was not clearly specified. At any rate, these observations cannot account for the existence of two peaks in the progression with T of the ventilatory response to a given hypoxic step. The existence of these peaks could be explained if the sensitivity of the central respiratory controller was rate dependent (differential sensitivity) with two preferential rates of rise of the stimulus. This rate dependence cannot be caused by the differential sensitivity of the carotid chemoreceptors which has been observed for much faster dynamics of stimulation (Dutton, Hodson, Davies & Fenner, 1967)

16 488 J. F. BERTHOLON AND OTHERS or not observed at all (Barnard, Andronikou, Pokorski, Smatresk, Mokashi & Lahiri, 1987). This effect, consistent with what we observed in CO2 experiments, must then be inherent in the central neural controller dynamics. Negativity of loop areas and central hypoxic depression With hypoxic stimulation and using the classical model, a positive loop area is expected for short T values due to circulation delay from the alveoli to the chemoreceptors and damping of several origins. Such a loop should decrease and be null at T values over 8 min and should remain null for slower dynamics of stimulation. However, if a depressant effect of hypoxia on the controller progressively developed during the test, a lower ventilation during the decreasing hypoxic steps and negative areas would be expected. Our results are in favour of the existence of such an effect. Loops of negative area were observed by Weiskopf & Gabel (1975) with a period of triangular stimulation of 10-5 min and were attributed to an increased wash-out of CO2 from medullar tissue. In our study, very negative areas were observed with a T = 8 min. With longer periods, a decrease in the negativity of surface area should occur since the depressant effect of a given level of hypoxia becomes maximal before the end of the step. With high enough T values, the surface area should become null (steady-state conditions). On the contrary, we found that surface area went through a slightly positive peak at T = 12 min then was increasingly negative from T = 12 min to T = 24 min. At T = 40 min it was still very negative. Therefore, some explanation other than the central depressant effect of hypoxia must be found for the progression with t of S(VE/VE,rest) as shown in Fig. 5 and for the shape of the response curves as shown in Fig. 2. From this last figure it can be seen that the larger negativity found at T = 8 min and T = 24 min was partly caused by the fact that the responses to the second and third rising steps were significantly higher for these rates of rise of the stimulus than those yielded by T values of 12 and 40 min. When the sign of dp102/dt was changed, after the 4th step, there was a sudden fall in ventilation which then remained depressed during the rest of the test. This effect was more obvious at T values of 8, 24 and 40 min and was quite spectacular for some subjects. This is different from the post-hyperventilation apnoea described by Lahiri (1977) after hypoxic steps because in our study we maintained PA,CO2 constant and because the values of PA, 2 found on the 4th rising step and on the first two decreasing steps were very close due to the fall in ventilation. A phenomenon of immediate but transitory undershoot of ventilation can be noted after withdrawal of a step of isocapnic hypoxia in the results of some authors (Lawson & Long, 1983; De Goede et al 1983; Long & Lawson, 1984). Such observations rule out the possibility of central after-discharge (Eldridge et al. 1987) in the case of hypoxic stimulation. Concerning the persistent state of depression that we have observed, Easton, Slykerman & Anthonisen (1988), using two equal steps of hypoxia separated by a varying time, concluded that recovery from hypoxic depression could require up to 1 h. In our study, we did not observe recovery even in the longest test (T = 40 min).

17 VENTILATOR Y RESPONSE TO HYPOXIA 489 PropOSed explanation We propose, as a cause of both maxima of loop magnitude and maxima of ventilatory responses, a phenomenon of global excitation of the bulbo-pontile network which can be caused (i) by certain dynamics of stimulation yielding a higher ventilatory response to the same PA,o2 (differential sensitivity of the centres) and (ii) by the crossing of a threshold value of PAO 2' which would also explain the observed non-linearity of the steady-state response curves. Once the process of excitation is triggered by a stimulus, the network activity proceeds until a new equilibrium is reached which we call an excited state. In terms of regulation, this means that the variable is allowed to fluctuate around its mean basal value without much reaction of the controller (low 'gain') in its basal, resting state, but if the variable goes beyond an endangering threshold, or else if it changes at certain rates, the controller goes towards an excited state which results in efficient corrective action (high 'gain'). When the stimulus is lowered, the stimulus threshold for relaxation of the controller is lower than the threshold for activation; such a hysteretic behaviour is the rule for all complex real systems. Such a model of regulation has been introduced as the 'cliff potential well' by Thom (1987) and has qualities of great robustness in face of perturbations from the outside. This concept provides an explanation for the observed progression of the loops in our study of CO2 regulation and for the steady-state 'hockey-stick' response curve. For two values of the rate of rise of hypercapnia, the controller went towards an excited state and commanded an important ventilatory response, even for low values of the stimulus. For other values of dp,0c 2/dt, the controller remained in its basal state and reacted to the stimulus with a low gain. For any rate of rise, if hypercapnia crossed a threshold, the controller proceeded to an excited state of higher gain. When hypercapnia was lowered, there was relaxation of the controller for lower values of PA,CO2 than for excitation (hysteresis) which gave positive maxima in loop magnitude for the values of T which had put the system in an excited state. For steady-state tests the hysteresis phenomenon was no longer observable and the change of slope provided evidence of the change of state of the controller. In this study of hypoxic stimulation, the same excitation for two values of dp1,02/dt and the same changes of slopes in response curves when PAo2 reached threshold values were observed, but when the stimulus was lowered (i.e. change of sign of dp002/dt) without much decrease in PA 2, there was a rapid relaxation of the controller which yielded negative hysteresis areas even for large values of T. The controller remained under hypoxic depression and thus ventilation was persistently lower on the return limb of the curve. At T = 12 min, the system ignored the low stimuli and had not enough time to reach the excited state once the threshold hypoxia was reached. The excitation process thus went on during the first return steps, leading to a positive loop area. This difference in loop progression as compared to the CO2 experiments and the cliff regulation model may be due to the fact that, during hypoxic tests, the controller itself is modified by hypoxia and relaxes as soon as the excitatory input or its rate

18 490 J. F BERTHOLON AND OTHERS of change is modified. With hypercapnia, the controller is stimulated by at least two excitatory inputs, the central and peripheral chemoreceptors, and CO2 has no proper action on neuronal metabolism up to a high level. Thus, positive looping in CO2 response curves is due to slow controlled system dynamics (large CO2 stores) and hysteresis in the relation between stimulus value and the excitatory state of the controller. With 02 response curves, there is positive looping for very low values of T due to the fast dynamics of the controlled system (small 02 stores and no central chemoreflex) and negative looping due to a central depressant effect of hypoxia and instant relaxation of the controller when the stimulus is lowered. In summary, using stepwise increasing and decreasing dynamic stimulation by hypoxia over a long time range in seven subjects, we observed some major discrepancies with the current cybernetic model of the ventilatory control system. These observations are consistent with what was found previously with hypercapnic stimulation using a similar protocol. Our hypothesis is that they are caused by the dynamics of the controller's neural network, which displays differential sensitivity properties along with the probable existence of discrete excitatory states. We want to thank Dr Mark Smith for his help in reviewing and translating our papers on C02 and 02 stimulation and also Mrs Monique Auffredou and I)r Jean Caries for technical assistance. REFERENCES BARNARD, P., ANDRONIKOU, S., POKORSKI, M., SMATRESK, N., MOKAS1II, A. & LAIIIRI. S. (1987). Time-dependent effect of hypoxia on carotid body chemosensory function. Journal of Applied Physiology 62, BELLVILLE, J. W., WHIPP, B. J., KAUFMAN, R. D., SWANSON, G. D., AQLEH, K. A. & WIBERG, D. M. (1979). Central and peripheral chemoreflex loop gain in normal and carotid body resected subjects. Journal of Applied Physiology 46, BERKENBOSCH, A. (1983). Carbon dioxide tension in the CSF and depression of ventilation by brainstem hypoxia. In Modelling and Control of Breathing, ed. WIIII2P, B. J. & WIBERG, D. M., pp New York: Elsevier. BERTHOLON, J. F., CARLES, J., EUGENE, M., LABEYRIE, E. & TEII,IA(C. A. (1988). A (dynamic analysis of the ventilatory response to carbon dioxide inhalation in man. Journal of Physiology 398, BUREAU, M. A., COTE, A., BLANCIIARD, P. W., HOBBS. S., FOULON, P. & DALLE, 1). (1986). Exponential and diphasic ventilatory response to hypoxia in conscious lambs. Journal of Applied Physiology 61, CHAMBILLE, B., GUENARD, H., LONCLE, M. & BARGETON, 1). (1975). Alveostat, an alveolar PO and PC0 control system. Journal of Applied Physiology 39, CROSS, K. W., HOOPER, J. M. & LORD, J. M. (1954). Anoxic depression of the medulla in the new born infant. Journal of Physiology 125, CROSS, K. W. & OPPE, T. E. (1952). The effect of inhalation of high and low concentrations of oxygen on the respiration of the premature infant. Journal of Physiology 117, CUNNINGHAM, D. J., PATRICK, J. M. & LLOYD, B. B. (1964). The respiratory response of manl to hypoxia. In Oxygen in the Animal Organism, ed. DICKENS, F. & NEIL, E., pp Elmsford, NY, USA: Pergamon Press. DAVENPORT, H. W., BREWER, G., CHAMBERS, A. H. & GOLDSCHMIDT, S. (1947). The respiratory responses to anoxemia of unanesthetized dogs with chronically denervated aortic and carotid chemoreceptors and their causes. American Journal of Physiology 148, DE GOEDE, J., VAN DER HOEVEN, N., BERKENBOSCH, A., OLIEVIER, C. & VAN BEEK, J. H. (1983). Ventilatory responses to sudden isocapnic changes in end-tidal 02 in cats. In Modelling and Control of Breathing, ed. WHIPP, B. J. & WIBERG, D., pp New York: Elsevier.

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