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Xsrox University Microfilms 300 North Zoob Road Ann Arbor, Michigan 43106 77-2369 CHIANG, Ming-Jen, 1948- THE EFFECTS OF SULFUR DIOXIDE BLOCK OF AVIAN INTRAPULMONARY CHEMORECEPTORS ON THE VENTILATORY REGULATION OF C02. The Ohio State University, Ph.D., 1976 Physiology
Xerox University Microfilms ,Ann Arbor, Michigan 48106 THE EFFECTS OF SULFUR DIOXIDE BLOCK OF AVIAN 1NTRAPULMONARY
CHEMORLCEFTORS ON THE VENTILATORY REGULATION OF C02
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By Ming-Jen Chiang, B.S.
The Ohio State University 1976
Reading Committeet
Professor Albert L. Kunz Professor E. Keith Michal Professor Charles W. Smith Professor Harold S. Weiss Approved by
(. k ( L ~ t s , Adviser q Department of Physiology ACKNO.;i NDGUiftTo
I wish to express my approei a ticn to my adviser,
Dr. Albert L. Kunz for his guidance throughout this research.
His advice and stimulation, both scientific arid ph: losophical, led me to a better understand.!ng of the physical mode.lr used in the analysis of biological systems. His instruction was invaluable in helping me to logically develop my own original and abstract thinking*
Thai'H;;.-! are also extended to all the faculty, staff and graduate students in the department of Physiolop.y• whose help and stimulation have made this work enjoyable and cor,, .true live. Specifically I am indebted to Dr. Philip f erg or who offered helpful criticisms and suggestions in preparing thi thesis.
Finally, I vrculd like to thank Office of Naval Research Cra nt
(No. 101~?33) and National Heart and Lung Institute Giant (No.
14870-02) for financial support for this project. VITA
Date of Birthi September 2, 19^8
Place of Birth: Taiwan (Formosa), Republic of China Education:
B.S. National Taiwan University Department of Zoology Taipe i, Ta iv/an, 19 70
Teaching and Research Associate, Department of Physiology, The Ohio State University Columbus, Ohio 1972-1976
Academic Experience and Training:
Biological Control System Dr. Albert L. Kunz Feedback and Control System Dr. Pimmel and Dr. Thurston Analysis Dept, of Electrical Engineering Avian Physiology Dr. Harold S. Weiss
Single Unit Neural Dr. Albert L. Kunz and Recording Dr. Philip Berger
iii TABUS OF CONTIS NTS
PAGE ACKNOWLEDGEMENTS...... VITA ...... iii
LIST OF TABLES ...... vii LIST OF FIGURES...... viii
INTRODUCTION ...... 1
METHODS t 11 Unidirectional Flow System Preparation. » . H
S02 Delivery System ...... 12 Surgical Vagotomy ...... 15
Cannulation of Wing Vein and Artery .... 15 Experimental Set-Up ...... ^
Monitoring Device and Calibration ..... ^ Isolation of the Animal ...... 2^ Programming...... 22 Evaluation of Preparation ...... 2 3 Closed-Loop Computer Algorithms...... 23 RESULTSi 25 A. Effects of Low Doses of SOg * ...... 25 1. Open-Loop COp Pacing Experiments and Statistical Analysis ..... 25 2. Closed-Loop Steady-State C02 Regulation...... 3® iv TABLE OP’ CONTENTS (cont.)
Page
3 . Closed-Loop Transients of C02 Regulation. 32
B. Effects of Large Doses of SO2 ...... 3^ 1. Post Vagotomy-Like Breathing (PVLB).'...... 34
2. Statistical Analysis...... * . 39
3* Open-loop Ventilatory Response after Large Doses of S02 ...... ^1 C. Effects of Intravenous Infusion of S02 . A9 1. Closed-Loop C02 Regulation...... il9 2. Open-Loop C02 Pacing...... 51
D. pH Analysis...... 53
DISCUSSION: 55 Experimental Block of IPCs ...... 55
SOg Block of IPCs...... 57 Single Unit Recording...... 59 Mechanism of S02 Blocking Activity of IPCs . 6l Steady-State and Transient-State Analysis in the Closed-Loop Kiode...... 65
Models for S02 Effects Upon the Dynamics of C02 regulation...... 70
Steady-State and Transient Analysis of Open-Loop C02 Forcing...... 73 PVLB and Further Surgical Vagotomy ...... 7^
pH Analysis...... 76
v TABLB OF CONTENTS (cont.)
PAGE
SUMMARY ...... 77 BIBLIOGRAPHY...... 80 LIST or TABLES
Table Page
1. Break of COp Pacing with Low Doses of SO2 * ...... 29
2. Post Vagotomy Like Breathing (PVLB) with Large Doses of SO2 ...... 0
3* pH Measurements...... 5^
vii LIST OF FIGURES
Figure Page 1. Error-Actuated Feedback Respiratory Control System ...... 2 2. Avian Intrapulnonary CO2 Receptor Response (Prom Osborne) ...... ? 3 . Schematic of Bird's Respiratory System (From hunz)...... 13 Determination of S02 Concentration...... 1^ 5 . Schematic of Experimental Set-Up (From Kunz) . . 17
6 . Determination of C02 Concentration...... 18 7 . Break of C02 Pacing with Small Doses of S02. . . 26 8 . The Continuation Record of Figure 7 Showing the Recovery of COg Pacing ...... 27
9. Closed-Loop C02 Regulation after Small Doses of SO2 in the Steady-State...... 31 10. Closed-Loop C02 Regulation after Small Doses of SO2 in the Transient-State...... 33 11. Controlled Closed-Loop Steady-State C02 Regulation and Subsequent Surgical Vagotomy. . . 35 12. Onset of Post Vagotomy Like Breathing (PVLB) . * 36 1 3 . PVLB and Subsequent Unilateral and Bilateral Vagotomy ...... 38
L6. V vs. Time Response of Open-Loop C02 Transient . ^2
1 5 . Respiratory Rate vs. Time Response of Open-Loop ^3 C02 Transient...... 16. V (J, vs. Time Response of Open-Loop C02 Transient.
viii LIST 01’ FlCUJtSG (cont.)
F igures Page
17* V vs. ^ COp Response of Open-Loop C0? Steady-State...... 46
18. Period vs. £ C02 Response of Open-Loop C02 Steady -S late...... 47 19* V(n vc. ?■ COp Response of Open-Loop C0? Steady-State...... 48
20. Intravenous (l.V.) Infusion of S02-Aerated Saline in the Closed-Loop m o d e ...... • 50
21. I.V. Infusion of SO?-Aerated Saline in C0? Pacing...... 52
22. Single Unit Recording of S02 Blocking IPC and its Spontaneous Recovery (Prom Kunz ct al)...... * ..... 60
23* Three C02 Receptors with their Inherent Delays. . 66
24. Possible Respiratory Drive of Multiple Feedback Diagram...... 68 2 5 * Threshold for Instability (Sensitivity or Gain X Delay) ...... 69
26. Analog Simulation Model ... 71
2 7 . Results of Analog Simulation...... 72
ix INTRODUCTION
The primary goal of the entire ventilatory apparatus is to regulate the supply of oxygen to the organism and to eliminate carbon dioxide. For a control system to function in this manner, adequate sensory elements quantitating the concentration of carbon dioxide and oxygen must be present at accessible sampling sites. Grodins (1950) described the respiratory system as a regulated system where arterial PCOg is the controlled quanity. The influence of C02 tension on respiratory drive has long been of interest to investigators. C02 regulation is compatible with either the concept of a direct action of C02 on receptors or an indirect action of CO2 via a pH effect at receptor sites. In the language of control theory, the respiratory stimulus, C02 , and the output re sponse, ventilation, can be conceptualized as an error- actuated feedback control system, (figure 1 ). Neither control of respiration on a breath-to-breath basis nor the hypernea of exercise can be adequately explained by the function of steady-state C02 operating point alone. Since mean arterial blood gas tension remains essentially normal during many types of exercise, the arterial chemoreceptors do not appear to be provided with OPERATNG POINT METABOLIC CO*
ERROR CONTROLLER PLANT SIGNAL MPIASEJ SEC.
RECEPTORS
Figure X. Error-Actuated Feedback Respiratory Control System sufficient stimulus to cause the resulting ventilatory
changes. Therefore, the dynamic C02 signal may make a signi
ficant contribution to the respiratory drive. Yamamoto*s
hypothesis (19^0 ) that the hypernoa of exercise may result
from oscillations of the C02 level of the arterial blood
has opened a new dimension to understanding respiration. Dutton (1968) found that C02 oscillations in the carotid
arteries of the dogs cause a greater elevation in ventilation
than perfusing the same arteries with blood of higher
mean C02 but without oscillations. This supports the hypothesis that oscillating information in arterial blood
can affect ventilation.
Another interesting phenomenon is that vagotomy
diminishes or abolishes the tachypnea which occurs with
the inhalation of C02 in mammals (Bartoli et al. 197**i Von Euler et al. 1972; Weimer & Kiwull, 1973)* Von Euler et al (1972) proposed that the increase of respiratory frequency is due to the increase of discharge from pulmonary stretch receptors; i.e.( C02 stimulates ventilation but the stretch receptors cut off inspiration when a threshold volume is reached so respiratory frequency is increased.
Since the afferent pathway of this feedback information runs up the vagus nerves, vagotomy abolishes this reflex response. An alternative explanation could be that C02 directly excites vagal receptors in the lungs. Support for such an explanation comes from the experiments of Bartoli et al (197*0* They showed that C02 in the airways and alveoli increases respiratory frequency in dogs on cardiopulmonary bypass. Since this C02 does not circulate in the blood they concluded that C02 must be sensed in the lung (i.e., pulmonary C02 receptors). Cervical vagtomy abolishes this reflex. Wasserman et al (1973)> using C02 - equilibrated blood infusion into ascending aorta and superior vena cava of unanesthetized dogs, demonstrated the presence of a ventilatory control component capable of triggering rapid ventilatory response to the changes in arterial PC02 and pH. The carotid and aortic bodies proved not to be the unique chemoreceptions responsible for this rapid response. However, they showed that vagotomy did not abolish this response. Recently, it has been shown that mammalian lung stretch receptors display a distinct C02 sensitivity (Schoener and Frankel, 1972 j Mustafa and Purves, 1972 j
Sant' Ambrogio et al, 197*0* Kunz and Scheid (1975)* using a hyperventilated cat preparation, in which they could distinguish between the stimulus of intrapulmonary C02 concentration and the stretch of lung tissue, concluded that the cat lung does not contain specialized C02 receptors and that intrapulmonary C02 sensitivity in cats is mediated by C02 sensitive stretch receptors. Although the search for mammalian intrapulmonary chemoreceptors is still being pursued, it has been known for many years that birds respond quickly to change in airway 5 COg concentration (Orr and Watson, 1913• Dooley and Koppanyi,
1929, Hiestand and Randall, 19^1* Ray and Fedde, 19^91
Jones and Purvcs, 1970). Recently, intrapulmonary COg-
sensitive receptors (IPCs) have "been identified in birds: the firing rate of IPC is inversely related to airway COg
tension (Fedde and Peterson, 1970j Fedde, 1970i Bouverot
and Leitner, 1972j Molony, 1972; Osborne, 1972). They do
not respond to stretch (Fedde et al, 197*01 nor are their firing rates affected by hydrogen ion concentration of
the blood or by relative hypoxia (Osborne and Burger, 197*^1
Molony, 1972 i Fedde and Scheid, 197*0* Therefore, it appears that the discharge frequency of the receptors is specifically
affected by the COg concentration at the vicinity of the
receptors. King et al, (197*0 have found a number of instances where a granular cell is close to a nerve axon in the
primary bronchus of birds which they suggest might be the
C02 -sensitive receptors. However, no physiological test
had been performed in their study, Fedde et al (197*0 have presented physiological evidence that more than 95% of IPCs lie in caudal half of the lung and that IPCs do not
lie in the trachea or primary bronchus. The exact location
of IPCs is unknown but it is clear that their afferent fibers run up the vagus nerve. ;
The frequency of discharge from the avian IPCs is
maximum at 05?* COg and falls subsequently with increasing
COg concentration. At above 8# most IPCs are silent (Osborne, 1972), Figure 2 has been taken from Osborne's work and shows the stimulus-response relationship between CO2 and discharge frequency of an average receptor. The impulses from IPCs are believed to be inhibitory to medullary respiratory neurons (Kuna and Miller, 1974). It has been proposed (Molony, 1972; Osborne, 1972; Fedde et al, 1974) that this C02~niediated decrease in the frequency of discharge of IPCs results in a net increase of respiratory drive and is responsible for the fast ventilatory response to increase in airway C02. The avian respiratory system provides an excellent model for the study oi the peripheral control of respiration be cause of its unique structural characteristics. The high flow, unidirectionally-ventilated system developed by Kunz et al (1970) offers the advantage of minimizing the effects of the amounts of gases the chicken adds to or removes from the air stream. Two important parts of this technique are 1 (1) to make the air flow high (10 X normal) to wash away the contribution of CO2 from venous return, and (2 ) to keep the resistance of the exit tubes low so that respiratory movements do not blow air back through the lung. Thus the control loop for C02 is opened by making the bird's respiratory movements ineffective in changing C02 concentration in the lung. The input variable, ^SCC^i thus can be independently forced to follow a prescribed temporal pattern. Later, the externally closed-loop preparation coupled with computer feedback was developed; i.e., the loop was reclosed 7
100 From Otborno
< W Z 3 o X til < « 5 0
til Z to in cr o < os x 2 5 Ui o OL CO
CARBON DIOXIDE FRACTION
Figure 2. Avian Intrapulmonary C02 Receptor Response (From Osborne) externally by taking the electrical signal output from the plethysmograph (which monitors ventilatory movements), processing it with an analog computer and causing an electropneumatic transducer system to modulate the C02 concentration in the lung. This preparation allowed
respiratory movemem again to affect the input COg, but in a way that is dependent upon the mathematical program of the computer.
Two major findings were made with the awake, uni directi onally-ventilated preparation) 1) When the COg concentration in the lung was oscillated at a frequency near that of the normal breathing of the animal, the ventilatory rhythm locked onto this frequency, and the animal breathed once each COg cycles this phenomenon has been called pacing (Kunz and Miller, 197*0. They hypothetized that IPCs are most probably the receptors involved in this pacing phenomenon. 2) Placing a delay in the feedback loop for C02 reg ulation produced a profound effect upon rhythmic breathing. Kunz and Miller (197*+) have shown that if the product of gain times delay exceeds a certain value, C02 regulation becomes unstable and oscillates as in Cheyne-Stokes breathing. Since the receptors (i.e., IPCs) in the lung have a shorter delay time than the chemoreceptors in the carotid artery or brain they can exercise the greatest gain and still maintain stability. It has been shown by Magno (1973)» Bouverot and Leitner (1972), Leitner (197*0 that chickens have funct ional systemic shemoreceptors (carotid bodies and central chemoreceptors) in addition to IPCs. They concluded that systemic chemoreceptors are involved in eupneic breathing. It then becomes important to know the relative contribution of each of the three sets of receptors to the respiratory drive? Since the central chemoreceptors are anatomically difficult to approach, it is easier to investigate the contribution to respiratory control of either carotid bodies or IPCs. In the chicken, the vagus nerves carry both the afferents from carotid body chemoreceptors and from IPCs hence any block of the cervical vagus nerve will affect both. This makes the analysis of individual com ponents more difficult. Molony (1972) showed that large doses of S02 could block the firings of IPCs. The uncontrolled doses he used were likely to have been very high and have had systemic effects. However, an agent which selectively blocks IPC activity would be invaluable in the study of the avian ventilation. The goal of this research was to elucidate the role of IPCs in the dynamic regulation of C02 in the awake, uni- directionally-ventilated chicken preparation. The method used to achieve this purpose was to block IPCs with a calib rated pulse of S02i while not affecting the other components. The following questions are pertinent to this worki 10
1) Can a dose of SO2 be optimized in terms of concen tration and duration to block IPCs in the awake, unidirect- ionally-ventilated chicken without disturbing the bird or significantly affecting the other components? 2) What do IPCs do? What is the role of IPCs in the pacing phenomenon and do they contribute to the respiratory drive? METHODS
Unidirectional Flow System Preparation* The experiments were performed on awake► seven to 14-week-old, 1.5-2.5 Kg domestic roosters (Gallus domesticus). The surgical pro cedures used for converting to unidirectional flow were a tracheostomy plus bilateral aerosacostomies (opening of air sacs). Under a local anesthetic (2^ xylocane), a small skin incision was made in the flank behind the thigh, 2 cm ventral to the pelvic bone and 2 cm caudal to the rib cage. Using blunt dissection, the fibers of the obliquous abdom
inous were spread apart with a hemostat and entry was made in the posterior thoracic air sac. The membraneous partition between the anterior and posterior air sacs was obliterated with the finger making the sacs confluent. An endoscope was then inserted to view the interior of the posterior thoracic sac. A passage was made into the abdominal air sac by cauterizing a hole in the membrane where the two air sacs are adherent. A one-quarter-inch internal diameter (I.D.) silastic rubber exit tube with its last 1^ inches containing holes was inserted and sewn in place with a purse string suture. This process was repeated on the other side of the animal. Then, a tracheostomy was made in the lower half of the trachea.
11 12 This procedure allowed air to be forced at a high rate of flow in sequence through the trachea, lung, air sacs, and
exit tubes to the outside. The £ inch internal diameter of the exit tubes offers much less resistance to air flow than the bird's respiratory tubules, hence the bellows
like movements of air sacs are unable to pump air back
through the lung. Also, air is forced into the trachea at a constant rate of 3.5 liters per minute. This high flow of air through the chicken minimizes the effect of the
amount of CO2 the chicken adds to the system. Figure 3 shows the schematic of the bird's respiratory system and
the surgical modification just described. Note specifically the separation between lung and air sacs. This awake
preparation also eliminates the effects of anesthesia which depresses the respiratory control system.
SO? Delivery System. The SO2 doses used in these experiments were made accurately by using a Masterflex
Rollar Pump (Cole-Palmer, 7013 type wich tygon tube size
ID" X 0D" O.CT315 X 0.1625)* This is a constant flow servo- con trol pump with adjustable speed ranging from 0 -3°°° revolutions per minute (RPti). The pump was calibrated by
pumping air or water at different speeds, e.g. 100, 2 0 0 , 300, *f00, 500, etc. RPM, and measuring volume being pumped per minute at each speed. This is the way the calibration curve of figure ^ was obtained. By referring this curve to the air flow rate in the experiments (3 .51,/min) the ©
LUNG ADVANTAGES TUBE t AWAKE 2. UP RIGHT 3. INTACT 4. RECORDS f, VT 5.0PEN OR CLOSED LOOP
Figure 3. Schematic of Bird's Respiratory System (From Kunz) GO
50
4 0 VOLUME (ml.) RPM 30 100
200 0.326
2 0 300 0.430
4 0 0 0.560
500 0.810
AIRFLOW = 3.5 L/MIN.
0 200 4 0 0 600 800 1000 REVOLUTION/ MIN. ( RPM)
Figure k. Determination of S02 Concentration 15 S02 concentration created in the gas stream was determined. The S02 was pumped from a reservoir which w«as kept about
10-30 mmhg above atmospheric pressure. The pumping at any one setting pumped a constant volume per unit time and was not affected by the resistance of the tube in the delivery system. The calibration curve is based upon SO2
being at ambient pressure so this increase in pressure packs more S02 molecules into the volume pumped and prod uces an error. However, a calculation shows that this
error produced in S02 concentration is small, (e.g.
Y&6 " 2 * ^ )
This S02 delivery system provides a constant flow source, or, in terms of the electrical analog, a constant
current generator.
Surgical Vagotomy. During some experiments, bilateral surgical vagotomy was performed as follows; After trach eostomy, the jugular veins on both sides of the neck were located;the vagi run in close apposition to them. After
carefully separating vein and nerve a ligature was tied loosely around each vagus nerve. This permitted surgical vagotomy to be quickly performed later without greatly disturbing the animal.
Cannulation of Wina Vein and Artery. The ulnar vein in the wing was cannulated using 110 lye the lone tube (J*.E. 90 16 type) in come experiments so that blood could be withdrawn for the measurement of blood pH before and after S02 administration.
Experimental Het-Up. Figure 5 is a schematic diagram of the experimental set-up of this work. After surgery, the animal was placed in a whole-body pie thy sinograph, and the tracheal cannula was connected to the input gas tubing through which the gas mixtures entered the bird's respiratory system. The exit tubes in the bird were each connected to tubes in the plethysmograph that conducted the flow-through gas to the outside of the chamber. The air was preheated to 35° by heat from a 25 watt electrical resistor. Condensation of water in the air flow tubes as well as exit tubes was prevented by running a nichrome heater wire in the tubes. In addition, the air was humidified by atomizing O .15 ml per minute of water into the air stream.
This water was injected into the atomizer with an infusing pump {Harvard Apparatus). The air temperature and the humidity levels were designed to keep the losses of heat and water during unidirectional ventilation comparable to those during normal breathing.
C02 was added to the air stream using an electro- pnuematic transducer (EFT) which transformed an electrical signal (eC02 ) into a C02 pressure signal (FCO2 ) at its output (figure 6). This pressure drove a flow of C02 gas Feedback r
COMPUTER LB-1, CO* %coa ANALYZER RECORDER ~ ~ r ~ RESR ELECTRO t FT PNEUMATIC TRANS TRANSDUCER DUCER COi
HEATER HUMIDIFIER
MR
PLETHYSMOGRAPH
Figure 5- Schematic of Experimental Set-Up (From Kunz) EPT CO,
PR
AIR
Figure 6 Determination of C02 Concentration 19 through the fixed linear resistance R^. This C02 flowed to the constant air stream and produced a C02 concentration e proportional to the input electrical signal, C02 . A pressure regulator (PR) maintained the pressure (P2 ) constant independent of the varying C02 inflow. Therefore, the signal produced in C02 concentration of the gas supplied to the bird was not accompanied by changes in pressure or flow.
The C02 signal serves as input during the experiments. This system has a rise time for step change to go from 10^ to 905S of 0.2 second and an absolute time delay of
0.3 second.
Monitoring Devices and Calibration Temperature. A YSI thermister (type 401) was used to measure the tracheal temperature throughout the exper iments. The thermister was connected to a YSI telether mometer (model 8423) and temperature read off the meter. Flethysinograph. A volumetric pressure transducer (Grass model PT-5A) monitored the bird's respiratory movements. The transducer output was displayed on an eight channel pen recorder (Gould Brush Instrument Model 481) and also fed into an analog computer. The piethys- mograph was calibrated to measure tidal volume by either injecting a known volume of air into the chamber with a 20
syringe or pumping given volumes; of air back and forth into
the chamber with a email animal respirator (Harvard Apparatus)
Dual phase control).
A small calibrated look was introduced into the
chamber to enable the measurement of rate and volume of ventilation while eliminating the troublesome effects of
temperature and pressure changes and of inboard gas leaks from the procuration. The size of the leak could be assessed by pushing a known volume of air from the syringe into the chamber and observing the exponential decay of the volume to the initial state. The leak was so tuned that even at lower respiratory frequency (e.g., 10 cycles per minute), the measured tidal volume was still at least 90# of the I’eal value. A small reference chamber was connected to the reference side of the pressure transducer to make the system independent of minor pressure changes in the laboratory, such as those caused by opening and closing doors. COg and 0g Recording. The COg concentration of the input gas mixturewas continuously monitored with a Beckman LB-1 infrared COg analyzer. A sampling flow rate of 5°0 per minute was drawn from the gas flow tube just before it entered the plethysmograph. The C02 concen tration signal was linerarized to provide a linear record of percent COg. Known gas mixtures (e.g., 2, 5 and 7^) were passed through the system for calibrating the COg analyzer. The 0g concentration was also continuously 21
monitored with a Beckman Ow-ll Og analyzer. The sampling
pick-up head of the 01.;-11 w a s placed in series with the LB-1 sampling head. A vacuum was used to draw the sample
of the pas stream thru LB-1 and Oi-i-11 pick-ups. The Og analyzer was calibrated using compressed air (assumed to be 2 0 .85? 02) and with 100# Og. Tracheal Pressure. A Crass transducer (model PT-5A) was connected via a small polyethelene tube to the junction of the tracheal cannula and the input gas tubing. Since the flow-through gas rate is constant, resistance change of the bird’s respiratoi’y tract results in pressure fluctuations. The tracheal pressure normally stayed between 10 and 20 cm HgO.
pH meters. A pH microelectrode unit Radiometer (type
27 Copenhagen) was calibrated before experiments and was then used for measuring the blood pH during experiments. Data Recording on Tape. The signals of the time- varying dependent variables were recorded on FI«i magnetic
tape (Model C-4( Vetter Company) and were used for making figures and for analysis of data. Isolation of the Animal
The plethysmograph in which the experiments were con ducted was itself in a much larger chamber to prevent the animal from seeing the experimenter. The animal*s posture and movements were monitored via a closed circuit TV camera (Raytheon 605). The chamber was illuminated with a single red light, since it has been the experience of this laboratory that chickens have a more regular respiratory 22
pattern in the dark. The large door to this chamber was
covered with cork to prevent the noises in laboratory from reaching the bii'd. Soft music was played in the chamber from an 8 tract tape recorder, in the belief that this mufjic not only mask the laboratory noises, but had a general calming effect on the bii’d. This isolation from outside disturbances helped to keep the bird's ventilation regular so that changes in z*espiratory pattern caused by exper imental conditions could be judged against this more regular background, i.e., improved the signal to noise ratio.
Programming. The analog computer (E.A.I. I6 -3I) was used to control and manipulate the variables being measured. It is a general purpose analog computer with forty amplifiersj also, individual digital circuits (Schmitt triggers and monostable multivibrators) were added to the system. The basic measurements recorded were:
1) time varying input COg concentration
2 ) tidal volume from pie thy sinograph or from the
integrated inspiratory flow 3 ) respiratory period measured from the integrated
period between breaths
4) A synchronized channel with the input C02 signal
accompanied by a pulse which corresponded to the
beginning of each inspiration.
5 ) tracheal pressure
6 ) temperature measurement of inspired air
7 ) time varying input 02 concentration 23 Evaluation of Preparation. Jo me criteria were used to judge the quality of the preparation.
The following procedures were followed during all
experiment:; to establish criteria for an adequate pre paration:
1) The normal resting breathing rate of the animal
was measured prior to surgery. If after operation, the
animal's ventilatory frequency differed by more than 20$
from the conti~ol, the preparation was then discarded.
2) The average value of tracheal pressure was about
10-20 cm HgO. If the pressure increased above 30 cm HgO, this indicated the possibility of an obstructed airway, and the trachea was then aspirated to remove any clotted blood or mucous. 3) In the closed-loop preparation where the animal's breathing is allowed to affect COg concentrations in the lung, the animal usually maintains a steady-state COg concentration at about 3*5 * ^*0$ providing Q, the analog of metabolic CO2 production, is appropriately set by the experimenter. If the animal did not maintain this steady- state equilibrium, then the preparation was discarded.
Closed-Loop Computer Algorithms. The externally closed-loop chicken preparation used the pie thy sinograph output to continuously set the level of input COg concen tration via the algorithm: $C0g&<^(Q - KV^Jdt , where Q is the analog of metabolic COg production and 24 % VT is inspiratory flow rate. K is a scaling constant. This equation was continuously solved on-line by the analog computer. The pattern of COg produced was that when the animal was not breathing the COg drifted up with a slope proportional to Q, With each breath the COg was brought down an increment proportional to the tidal volume of that breath. This externally closed-loop preparation achieves a stable steady-state in which the intrapulmonary COg fluctuates about a constant mean value of about 4.0f-. RESULTS
The experimental results fell into three studies. The first concerned the effects of low doses of SOg. The second was of S02 doses high enough to cause Post Vagotomy
Like Breathing (PVLB). The third was a study of the effects of intravenous infusion of SOg aerated saline solution. A* Effects of low doses of SOg
i* Open-loop COg pacing experiments. Figures 7 and 8 are from a typical experiment. These two figures constitute a continuous record showing that input C02 oscillations could drive the bird's respiratory frequency i.e., pace ventilation; that pacing was broken by admin istering o.l6?5 of S02 for 1.5 minutes; and that pacing spontaneously returned seven minutes after the apparent break with this dose of SOg. The first channel is a record of the C02 concentration of the gas forced into the trachea. COg concentration in this figure was sinusoidally oscillated from a low of yfc to a high of 5# with a mean of The second channel shows the bird's ventilatory response as measured by a whole body plethysmograph. The third channel labeled "SYNC" is a composite of the COg concentration signal and a pulse marking the beginning of each inspiration (When the plethysmograph tracing
25 % C 0 2 /wwwwwvwwvwvwwwwvwvwwvwwwvwwwvwwv^ I SEC. mfoule* ohiir' ttort o f so2 ->6 Hull Wm PLETH.
/U/UA/U/lW/W/UA/U/U/U/l/W/U/i/UH/U/tl/W/W/W/i/W^K)\/W\K^i\J\/i^^ SYNC. t BREAK
PERIOD 0.16 % S 0 2
Figure ?. Break of C02 Pacing with Small Doses of S02 w %C02 : i
\/WVWWWWWWWWWI^^
t ■------— 1---—S------1------*------8------
1 1 * 1 PLETH.
SYNC. ♦ RECOVER
PERIOD CHANGE PAPER SPEED
Figure 8. The Continuation Record of Figure 7 Showing the Recovery of C02 Pacing 28
passes through a minimum). The last channel is a record of the period of each breath recorded as the height of a ramp. In channel 3 ("SYNC") of figure 7. before the SOg was given, the animal's ventilation was locked onto the COg oscillations, with the beginning of each inspir ation occurring at about the same instant in each COg cycle. S02 (0.16^) was added to the inflow gas for 1.5 minutes as indicated by the solid bar pacing broke at the time indicated by the arrow in the figure; synchrony was lost, and respiration became slower. Pacing returned spontaneously after seven minutes as shown in figure 8 . Note that tidal volume stayed nearly fixed and rate changes occurred gradually. There was no evidence of discomfort or respiratory reaction to any irritant effects of S02 . When birds experience an irritant effect of SOg at high doses they usually thrash about and the respiratory move ments become irregular with large explosive expiratory gasping. Table 1 tabulates the results of the effects of low doses of S02 upon open-loop C02 pacing. This study encompasses 22 trials in 12 different chickens. Chickens in which pacing could not consistently be achieved over long periods of time are not included. In twelve out of 22 trials pacing broke when SOg was administered. The mean
effective dose of S02 which caused pacing to break was (0.17 - 0.07^) X (100 - 68 seconds). The mean latent period of onset of breaking pace was 106 - 86 seconds. 29
TABUS 1
Break of COg pacing with Low Doses of SOg
Animal SO? Pulses Latent Period Duration of (Trial) (cone ‘ x sec) of Onset (sec) Effects (min) c - 312( 1) 0 .0*i x 210 c - 312( 2 ) 0.08 x 150+ 30 ? c - 313( 1) 0.16 X 85 45 7.0 c - 314(1) 0.08 x 210 280 4.0 c - 315(1) 0.08 x 230 — - -- c - 315(2 ) 0.12 x 50 25 7 C - 315(3) 0 . 14 x 240 200 ? c - 320(1) 0.13 x 90 540 6.0 c - 321(1) 0.13 x 60 -- ___ c - 321(2 ) 0.20 x 90 120 5.0 C - 324(2) 0.24 x 35 -- C - 324(3) 0.30 x 80+ 65 ? C - 330(1) 0.08 X 30 — — — C - 330(2 ) 0.16 x 30+ 70 ? c - 330(3) 0.16 x 30 — — C - 334(1) 0.16 x 75 -- -- C - 334(2) O.25 x 70 -- -- C - 334(3) 0.32 X 50 -- -- C - 338(1) 0.16 x 90 150 3.2 C - 344(1) 0.24 x 40 20 4.5 C - 346(1) 0.25 X 75 -- C - 346(2) 0.32 X 30 20 7.0
Statistics Mean SOg Pulse Mean Latent Mean Duration of trials (Mean - S.D.) Period of + of Effects (Animals) (?) x (sec) in Onset (Mean - (Mean - S.D.) in which which Pacing S.D.) (sec) in (min) in Pacing was was broken which Pacing was which Pacing broken broken was recovered
12/22 (11/ 12) (0 .17*0 .0?) x 106-86 5.4*1.4 (100-68) (12)++ (7)++ (12 )
+The ventilatory response showed interrupted expiratory gaspings after breaking CO2 Pacing
++Number of measurements 30
In seven -trial:; paci ny spontaneously returned. When pacing returned, it cai.ic hack in Jen:-: than ten minute a (mean 5. *!*!_']_. T minutes). The duration of PUg pulses in these experiments was usual.ly determined by how soon the animal broke pace because the COg was discontinued as soon as it was apparent that paci.ir; had stopped. Thus, the duration oi' oOg pnls.es was not uni term in tJiesc experiments. Most of the animals did not shew any si pm; of discomfort or disturbance al though in throe of the 12 trials expiratory pa;'.pa were soon am one, the slower, deeper brvathiny pattern:; observed after
SOg had boon administered.
2* r LOiTdJr.ijl0!'1 'b'arjlis:'’- C O g Ifor-'u J at ion. Pipure 9 is an example of stoady-s fate COg regulation in the closed- loop pro pa. rati or i. This f i pure comprises two strip-s of record showlm; the COg concentration in the Inn,;; and the resultinr piethysrjot raph outxm'i;. lanxmber that in the closed-loop mode, a.:; described in methods, the animal's respiratory movers nts arc monitored by a plcfbysmopraph and * fefbrct thru a computer circuit to set the percent COg in the insufflating pas stream thereby externally closing the
COg control loop. In figure 9 the COg concentration was regulated at about 3*5^ in "the control period. When a pulse of COg was piven as indicated by the solid bar, the regulated COg level drifted up within twenty seconds and then levcled-off at a mean of 6.5/-; after eleven minutes, the COg level decreased and regulated a&ain at the previous %co*
0 .1 3 % SOg I SEC minute* after start of to ^ O
PLETH.
M N m r :
PLETH.
Figure 9* Closed-Loop CCU Regulation after Sira.ll Doses of SO2 in tne Steady-State 32 level. Ventilation became slower and deeper immediately after ^02 ; at the time of recovery, the breathing pattern returned to that seen during the control phase i.e.,
the rate was faster and the tidal volume was smaller. Both the time until onset and the duration of the effects of
SOp were similar to those in the experiments involving breaking of COg pacing in the open-loop. Though the return to the previous COg level after recovery is sudden in this figure, in other experiments, the
# COg returned to the previous steady-state level more gradually after a period of "apparent searching".
3* Closed-loop Transients of COg Regulation. Figure 10 shows an experiment on the closed-loop preparation in which
COg regulation was intentionally disturbed by pulses of high COg to examine the stability of the system as indicated by its transient response. This technique is similar to the one developed by Kunz and Miller (1974). The first
channel presents # COg, and the second channel ventilation. The inital control record shows transient responses to
sudden changes in COg level. Although the responses after the two controlled pulses label (1 ) & (2 ) in the figure were not identical, they both showed a stable damped pattern in returning the COg to the steady-state level. After giving
0.16# S02 for 60 seconds, the next C02 pulse (3) was followed by undulations in the regulated C02 level. These undulations appeared to bo self-perpetuating in this preparation. %C02
* *tt>M W
(2 0 SEC)
O .I6 % S O , PLETH. 2 .
% C 0 2
Mni; aMf1 M j; ^ m m w . PLETH
Figure 10, Closed-Loop C02 Regulation after Small Doses of S02 in the Transient-State
U) D . Effects of largo dor,or: of SO?
1. Pont Varotomy Like Breathing (PVLB). Figure 11 chows
two strips of record. The upper one depicts the controlled closed-loop steady-state COg regulationt the level at which CO^. is regulated was maintained at about U-tOfo. The lower one illustrates the regulated COg level as well as
■ ventilation after bilateral surgical vagotomy. The Q,
the analog of COg production, had been decreased to one third of the controlled setting. Note that breathing
became slower and deeper and the COg level was elevated to about 8.0^,
Figure 12 is the record showing the onset of PVLB in response to a large dose of SOg, 0.40£ for 80 seconds. This is about two and half times the concentration used in
the low dose study. The figure shows three strips of record of COg concentration vs. ventilation. The upper
strip is the controli note that the direction of inspiration
is dovrn. In this set of experiments the closed-loop feed back caused COg to drift up during inspiration and to be brought down by each expiration proportional to the tidal volume of that breath. This phase relationship is the reverse of that intended, but the low amplitude of the COg oscillations makes the phase relationship of little import ance. The middle strip of the figure shows that, after giving a large dose of SOg during the time marked in the figure, the breathing pattern changed but not as quickly CONTROL
o/#C0 •
I SEC p l e t h . imuMmmmMM
BILATERAL VA60T0MY
%CO*
PLETH.
Figure 11. Controlled Closed-Loop Steady-State C02 Regulation and Subsequent Surgical Vagotomy ©/
1 SEC
PLETH. fg M W W * ( insp. 4 )
% CO2
0 .40 % S 0 2
. PLETH.
% c o « 3 MIN. LATER Itlti
PLETH,
Figure 12. Onset of Post Vagotomy Like Breathing (PVLB) as in most other experiments. From the experiments or many trials, one feels that repeated exposure of the animal to S02 will make him less responsive to SOg. This long latency thus may have been due to the fact that this animal had been conditioned by his four previous S02 exposures.
The bottom strip of the figure illustrates that three minutes after S02 administration, PVLB occurred and the C02 level then rose to 8% (the maximum value permitted by the computer)\ hence there was no longer any self-regulation of C02 . The analog computer used in the feedback loop for C02 allows the experimenter to throw a switch to reset the initial conditions (starting value) of COg concentration. This changes the value in the integrator of the computer back to the initial one (kfa) with which the experiment started. In the bottom tracing of figure 12 the % C02 signal was reset three times when the bird’s inadequate ventilation had led to the C02 level drifting above the maximum value allowed (8^). These three resets proved to be in vain because the breathing movements of the animal could not match the analog of C02 production. Hence it could no longer maintain C02 in a steady-state equilibrium. Figure 13 shows the same preparation after Q has been decreased to one fourth of the previous setting. The bird even with PVLB was then able to regulate CO^ in a stable
manner, but the regulation occurred at a higher level of C0 2 * This is shown in the upper strip of the figure. Breathing =%C0h:Vi/UVVVl/l/VV ISEC S O g is-tttittnmn,
%co,
S 02 +RIGHT VAGOTOMY pleth- nnfin
%C02
SO2 + BILATERAL VAGOTOMY N, ri PL£TH. M T ff]
Figure 13. PVLB and Subsequent Unilateral and Bilateral Vagotomy was slow, deep and regular* similar to the pattern after surgical vagotomy. The middle and bottom part of the figure show the effects of subsequently cutting first the
right and then the left vagus on the regulated COg level and breathing pattern. Note that the regulated COg level
was further elevated from 4.8# to 5*6# and then to 6.1# with first unilateral and then bilateral surgical vagotomy, but that there was little change in breathing pattern or
respiratory period. No spontaneous recovery of breathing pattern was observed,
2. Statistical Analysis. Table 2 shows the responses
of seventeen animals to large doses of SOg in the range 0.20- 0.49# for 35 to 180 seconds. Fourteen animals showed a PVLB pattern after the administration of this size dose of SOg. Only those animals whose breathing was both slow and deep were considered to be breathing in a PVLB fashion. The
duration of these large doses of SOg was determined in two
ways: first, SOg was discontinued as soon as it was apparent that PVLB would occur* second, in no event was the duration continued for more than three minutes, no matter what con
centration of SOg was used. The SOg concentration used in
this study never exceeded 0.49# because at higher doses the animal became restless and moved wildly about in the plethve- mograph i.e., the resting ventilatory pattern was disrupted.
From the table, one sees that eleven out of 14 animals with PVLB showed some signs of explosive expiratory gasps TABLE 2
Post Vagotomy-Like Breathing (PVLB) with Large Doses of SO2 Expiratory Gaspings Occurred SO2 Pulses Latent Period After SO2 Animal_____ (conn j- x sec) of Onset (sec) Administration c - 317 0.32 x 180 600 + C - 318 0.40 x 120 90 0 C - 319 O .45 x 120 — — — c - 320 0.49 x 120 — — — c - 322 0.49 x 120 ? + c - 325 0.49 x 90 180 + c - 328 0.40 x 80 150 + C - 335 0.20 x 60 130 0 c - 337 0.35 x 35 30 0 C - 338 0.49 x 90 C - 339 0.45 x 90 240 + c - 340 0.49 X 110 180 + c - 347 0.25 X 40 30 + c - 350 0.40 x 40 20 + c - 358 0.35 x 60 60 + C - 359 0.49 x 70 100 + C - 360 0.32 X 65 105 +
Statistics Mean SO2 Pulse Mean Latency Expiratory of Animals (Mean ±S. D.) (l.iean*S.D.) Gaspings After in which in which PVLB in which PVLB S02 PVLB occurred occurred occurred
14/17 (0.39*0.09) x 106*65 11/14 (72*25). (14)* ______H i l l ______
^Number of Measurements 41 for^sfrort period;: during the test. The onset of PVLB occurred within minutes of the Loginning of S02 administration
except in two animals (C-317 and C-339)* The average latent
period of onset was 106 - 65 seconds and the average dose
of S02 was (O.39 - 0.09^) X (72 - 25) seconds.
3* Open-loop Vcnti latory Kc r:ponse after Large Doses of
S02. Figures 14, 15, & 16 show the open-loop transient response to a step change in % C02 from 0?> to about 4. 5?* and back. V, used here as the dependent variable, is defined
as breaths per minute times tidal volume. This is not to
be confused with the rate that air is flowing through these
unidirectionally-ventilated birds which is artifically held
constant at 3*5 liters per minute. These data are averaged from four animals to show the transient responses between
control and SOg-induced PVLB preparations. Figure 14 • depicts the time response of V to step changes in # C02 from to 4.5^ and back as shown in the figure. The control groups showed a fast ON response as compared to the slow,
lagging response ( > 5 seconds) elicited by PVLB groups. Furthermore, the fast "OFF" transient of the control groups
(latency less than one second) was different from the slow return of PVLB groups.
Figure 15 shows that the instantaneous frequency which was calculated as the reciprocal of respiratory period varied between the control and PVLB groups. Both
ON and OFF transient responses wore similar to V vs. time CONTROL I MEAN + S.D. PVLB I MEAN -S.D. 1000 4.5
8 0 0 % C ° *
600 ♦ V imls /nmin) iifi 4 0 0
200 T
30 40 50 60 TIME (tee.)
Figure 14. V vs. Time Response of Open-Loop CO2 Transient
& to I MEAN + S.D. CONTROL PVLB I MEAN -S.D.
4 .5
30
20
.r
T1ME (sec.)
Figure 15. Respiratory Rate vs. Time Response of Open-Loop CO^ Transient •----- *CONTROL * *pvi n MEAN t S.D.
4.5
4 0 %CQ
- • L
20
10
30 4 0 60 TIME (secj
Figure 1 6 , Vj vs. Time Response of Open-Loop COg Transient ^5 response. Between the ON and OPT for each group, the rates were regular at about 22 breaths per minute for the control
and 10 breaths per minute for PVLB; therefore, the respi ratory rates wore stabilized at the first ventilatory response after the ON transients in both groups. Figure 16 illustrates the transient response of V,ji vs.
time response. It is evident, from the figure, that Vfj, fluctuated a lot from the mean values in both control and
PVLB groups.
Figure 17, 18 & 19 present data from two animals to
show steady-state C02-sensitivity cui’ves in open-loop preparations. The ventilation parameters shown in the figures were obtained during stepwise increase and decrease of the C02 concentration of the insufflating gas. Hysteresis, a difference between the steady-state values obtained from increasing and decreasing steps of C02 concentration, accounts for some of the variation in the values for each group. Figure 17 shows the V vs. f C02 response. The re gression analysis showed that the slopes of the sensitivity curve (V-f C02 ) of PVLB and control group differed sign ificantly (P< 0.05). The horizontal intercept of both curves, however, did not differ significantly (P>0.05)» Figure 18 depicts respiratory period vs. % COg. One sees that PVLB group .had longer periods (mean -S.D., + 9 0 0 - 1*27 seconds) compared to the control (mean - S.D.* 1000
8 0 0 CONTROL PVLB
(mL- C - 317 im n) 8 0 0 upstep o
A 4 0 0 C-319
✓ ups tap a 2 0 0 downstep .
%co,
Figure 17. V vs. # COg Response of Open-Loop COg Steady-State 10 A • CONTROL PVLB
C-317 8 upstep o •
downstepa A PERIOD 6 (•ec.) C-319 I O B upstep a * i : ' dcwistep V ▼
T lo %coa
Figure 18. Period vs. # COg Response of Open-Loop COg Steady-State 50
CONTROL PVLB
4 0 C-317 T (ml.) upstep o 30 downstep A
C-319 20 upstep o
downstep 7 10
% C02
Figure 19. Vj vs. % COg Response of Open-Loop COg Steady-State 49 *4* i 3.76 - O .39 seconds). However, regression analysis showed that the two slopes of control and PVLB did not differ significantly ( F > 0 .0 5 ).
Figure 19 shows VT vs. % C02 for control and PVLB animals. Regression analysis showed that the slopes differed
significantly (P<0.05)/but the intercepts were not different. Thus analysis indicates that the minute ventilation and the tidal volumes of control animals increase in re
sponse to COg to a significantly greater degree than animals with PVLB. The period between two groups are different, however, the slopes of two groups do not vary signigicantly. C- Effects of IV. infusion of S02. The PVLB pattern in duced by large doses of SOg is assumed to be due to the effects of S02 upon the lungs, since the dominant effect when S02 goes into solution is a decrease in pH and in mammals a decrease in pH speeds rather than slows ventil ation. In order to test this assumption, it is necessary to test the systemic effects of SOg. 1. Closed-loop COg Regulation. Figure 20 compares the response to thel.V. infusion of SOg-aerated saline with the response of the same bird to insufflated SOg in closed- loop C 02 regulation. The upper strip of this figure shows the control recordj C02 was regulated at about in this preparation. The middle strip of the figure shows that, immediately after I.V. infusion of SOg, the respiratory rate increased (unlike the decrease seen in PVLB). C02 , %COt I SEC : p.leth. \0iiismm04mMsM,
S 0 2 AERATED SAUNE (1 0 ml I.V.)
PLETH. \N0k
SOg INSUFFLATION ( 0.3 % X I min )
PLETH.
Intravenous (I.V.) Infusion of S02-Aerated Saline in the Closed-Loop mode 51 however, was still able to be regulated at about the same level. The bottom strip of the figure, again, shows the typical PVLB pattern after S02 insufflation. Note that the paper speed was increased near the end of the tracing. The high air flow presumably is able to protect IPCs from being damaged by SOg-aerated solution. In early preparations in which the unidirectional ventilation stream was stopped during I.V. S02 infusion, PVLB was produced.
The effect of I.V. SOg infusion is not due to the distur bance of the injection technique. This was shown by in jecting saline as a control and getting no effect.
2* Open-loop CO^ Facing. Figure 21 presents an exper iment to show the effect of I.V. infusion of SOg-aerated saline upon open-loop C02 pacing. The first channel, again, is a record of C02 concentration of the gas forced into the trachea. COg concentration was sinusoidally oscillated from a low of 2# to a high of 5# with a mean of 3* 5#* The second channel is the bird's ventilation. The third channel labeled "SYNC" is a composite of the C02 concentration signal and a pulse marking the beginning of each inspiration. At left side of the figure, one sees that pacing was stable with the beginning of each inspiration occurring at about the same instant in each C02 cycle. When I.V. infusion of 3ml S02-aerated saline (equivalent to S02 insufflation of 0.40# for one minute at an air flow of 3.5 L/min.) was administered as indicated by a solid bar, C02 pacing % C Q a
.TIME (seel
f i i i i p PLETH.(insp.*)
\ r li SYNC. 0.40 %S02 1 *' ’ T ' 11
Figure 21. I.V. Infusion of S02-Aerated Saline in C02 Pacing 53 broke immediately. Ventilatory frequency was increased (unlike PVLB). The resumption of pacing, however, occurred soon after changing to a faster frequency (e.g. from 0.28 Hz to O .36 Hz) of C02 oscillation as indicated by an arrow in the figure. This is indeed true pacing as evidenced by the fact that the respiratory frequency followed the period of
the C02 oscillation over a range of different frequencies,
e.g., O .36 Hz to 0,30 Hz.
D. pH Analysis■ In order to gain some insight into the acid-base status of the blood during SO^ administration, pH measurements were performed immediately after S02 pulses had been added to the insufflating gas and after
I.V. infusion of S02 solution. Table 3 summarizes the results. pH changes were measured from either the ulnar vein or the brachial artery of the animals. Low doses of S02 when insufflated did not significantly change the blood pH (P>0.05); howeverr large doses of S02 when insufflated did change the blood pH significantly (F<0.005). TABLE 3
PH Measurements
After OL CO2 After small After large Pacing & dose of SOg dose of SO 2 Vein or Animal Control Transient incuff .1 at ion insufflation artery
C - 3 2 9 7 . 4 6 — M ^ 7 . 2 1 vein C - 3 3 0 7 . 4 4 ------7 . 2 5 vein C - 3 3 1 7 . 3 9 ------7 . 2 2 vein C - 3 3 2 7 . 3 3 ------?.20 vein C- 3 ^ 3 ? . 4 2 7 . 2 1 ----- — --- vein c - 3 ^r 7 . 4 3 ?.'+5 7 . 4 1 7.31 vein ----- c - 3 4 7 7 - 4 9 7 - 3 9 7 . 3 0 vein c-348 7 . 3 9 7 . 1 6 7 . 1 4 ----- vein C ~ 3 ^ 9 7 . 4 3 7 . 4 6 7 . 4 4 7.32 vein C- 3 5 C 7 . 50 7 . 3 7 7.29 7.35 vein C - 3 5 1 7 . 5 1 7 . 3 7 ---- vein
Animal Control After s o ? After SO2 Vein or aerated• r ■saline insufflation artery infusion (0.2-0.3)^ X 1.0 min (0.3-0.5)??
C-353 7.42 7.22 ---- vein C-361 7.44 7.22 7.37 artery .C-362 7.61 7.58 7.54 7.46 artery
Control After 0L COg After small dose After large transient & pacing of SOg dose of SO2
7 .^50*0.067 7.373*0.126t+ 7.333-0.104+ 7.278-0.062++ (8 )* (7)* (7)*
+ Statistically non significant (P>0.05)
++ Statistically significant (P<0.005)
* Number of measurements DISCUSSION
The results of this work indicate that it is possible to give a pulse of S02 of low enough concentration and short enough duration to block Intrapulmonary C02-Sensitive Receptors (IPCs) without greatly affecting central or arterial chemoreceptors and without disturbing the behavior of the bird. Block of IPCs demonstrates that they are essential for COg pacing and that they contribute to the ventilatory drive. Furthermore, the breathing pattern after large doses of SO^ is similar to the breathing pattern after surgical vagotomy. This effect is due to
S02 acting upon receptors or other components in the lung and not upon systemic chemoreceptors. Experimental Block of IPCs i Surgical vagotomy has been used in studies of respi ratory control in birds (Fedde, 19&3; Richards, 1969)* While restriction of vagotomy to the pulmonary branch of the vagus nerve is possible, the procedure requires open- chest surgery and anesthesia. This proves not to be an easy task. The procedure of cervical vagotomy interrupts information not only from IPCs but also from afferents of carotid chemoreceptors and from pulmonary stretch receptors. It also blocks motor neurons to the region. Therefore,
55 a specific block of JlC'c information to the brain can not
be achieved by cutting the cervical vagus nerve.
High COg concentration blocks IPCs but it also gets into the blood to increase systemic PC02 and CH+J . Therefore,
the block of I P C s activity by high C02 is masked by the respiratory drive from other sets of C02 receptors. There is a technique which uses a cardiopulmonary bypass
which allows the C02 concentration in the lung to be raised without affecting systemic C02 levels. This method
has been used in mammals (Bartoli e_t aJL, 197*0* However, the technique requires anesthesia and uses an oxygenator to
maintain a satisfactory arterial PC02 level. Such a technique would almost certainly disturb the preparation and
make analysis of results difficult. Molony (1972) showed that conduction velocities between neurons from IPCs and from stretch receptors are very
similar. Since cold block of a nerve can only block conduct ion in one or more of a group of fibers with differing conduction velocities, this technique can not be used to block the activity of IPCs without affecting stretch receptors at the same time. The present work began as an attempt to use halothane to block IPCs. Marley and Stephenson (1968) indicated that respiration was depressed by halothane and that respiratory slov/ing was due to an action of halothane on vagal afferents. However, the results indicated that halothane is also a strong central depressant 57 and disturbs the central components of the respiratory controller. Following a brief study of the usefulness of halothane as an agent to block IPCs, the action of SOg was investigated. S02 Block of IPCs t Molony (1972) showed that large, uncontrolled doses of SOg could block the firing of IPCs in decerebrate chickens. However, the dosage of SOg used by Molony was not controlled quantitatively and could have been high enough to have had systemic effects. In addition, a lot of physiological
parameters or variables could also have been affected. In order to study respiratory control in awake birds, it is necessary to minimize the major physiological effects accompanied by administering SOg. Since SOg could block IPCs, it suggests that it may be an invaluable agent to study the control of avian ventilation. The present work was initiated to see whether a well-controlled,small dose
of SOg could block IPCs without disturbing the awake birds and without affecting other systemic chemoreceptors in the unidirectionally-ventilated high flow chicken preparation. Most previous investigations of the respiratory effects of SOg were related to environmental pollution. Thus the effects of exposures to low concentration of SOg lasting from days to months. The effects have been tested on many animal species and human subjects. SOg is a common pollutant of the air that arises mainly from industrial and domestic combustion of fossil fuels. The doses used in long-term experiments a m approximately
0.05 to 700 Parts Per million (PPM) (Aindur, 1966) and the effects have been reported elsewhere {Frank, 1962} Balchum,
I96O 1 Loong, 1965: Salem, 1961; etc.)* Chan gets of lung and respiratory tract mechanics are mainly cited in their work.
Inhalation of SCh, produces all grades of respiratory tract irritation, sometimes with pulmonary edema. The concen
tration probably determines the mode of death; e.g.,
suffocation from reflex respiratory arrest (very high concen tration), pulmonary edema (moderate concentration) or
systemic acidosis (low concentration). It is difficult to compare most previous work to this study because both the methods and the animals are different.
Recently, Widdicombe et al (19?5) reported the acute effects of S02 on pulmonary mechanics, breathing patterns and pulmonary vagal afferent receptors discharge in rabbits. The animals were anesthetized and a dose of 200 PPM (0.02?5) of S02 for 5 minutes was given through a tracheal cannula. Twenty-three out of twenty-six stretch receptors were blocked by this procedure} the receptors then eventually recovei’ed (mean recovery time* 15 minutes). At the same level of SOg, eight out of sixteen lung irritant receptors Showed changes in firing pattern during SOg administration.
However, no irritant receptor was blocked after 5 minutes of
SOg administration. From these studies, it was concluded that exposure of rabbits to 200 PPM of S02 for 5 minutes could specifically block the brcucr-Hering reflex and pul monary stretch receptor activity, while leaving lung irritant receptor activity intact. Also, in studies using anesth
etized geese, iJiddicombc et al (197*0 suggested that SOg acts on nervous receptors in the respiratory system.
Single Unit rdi_n r;. Single unit recordings verify that
SOg could exert its actions on COg pacing and closed-loop
COg regulation by abolishing the activity of IPCs. Kunz
et al (1975) conducted single unit recordings of vagal afferents from IPCs in chickens. Figure 22 is a typical record showing the firing pattern of a single XPC in re
sponse to changes in COg concentration. This figure shows
step changes in the COg concentretion of the ventilating gas stream and the resulting discharge reported in a single vagal fiber, The COg was alternated between 0 and remaining at each value about 2 seconds. The time bar
indicates one second. The IPC fires when COg is low and
stops firing at high COg concentration. This is character istic of IPCs. One might say that they sense "fresh air",
or the absence of COg. The solid bar indicates that when SOg was given (O.O87S X 30 sec.) the unit continued to fire for three COg cycles after the SOg was started. The fiber then stopped abruptly and was silent for the next 11 minutes, and came back spontaneously to almost the initial firing pattern after 15 minutes. Of six fibers which resumed firing after the SOg block a mean period of 6.5 minutes %co2 ~V / V_ / "X__
so2 (0.08 %X. 30 Sec) l“V r~\_jy—\__ a *
A / V / V after II min
sec!
after 15 min
Figure 22, Single Unit Recording of SO? Blocking IPC and its Spontaneous Recovery (From Runz et al) 61 elapsed between block and resumption of firing, This period is similar to the duration we have found for which SOg breaks COg pacing in awake preparation (mean ^ S.D.,
5.4 - 1.^ minutes, Table 1). Some of the results from Kunz and burger (1975* personal communication) are tabulated as follows:
one 0 H % SOg (X minute) 0.03 0.08 • 0.23 0.30
Fibers block 2/14 6/14 7 / n 3/? V 3 — fTbers^studied
In the open-loop COg pacing experiments, the average dose of SOg which broke COg pacing was approximately (0.17*0.07^) X (100 - 68 sec,). The fact that some units stopped firing below this dose and others above it, suggests that pacing in these experiments by blocking a certain population of IPCs.
Mechanism of CCU Blocking Activity of IPCs: This study interprets the concept of receptors in a broad sense; i.e., it implies that receptors meet the defin ition of the sensors which detect input COg concentrations and cause the output ventilatory response. First, one should know what happens before one can actually say how and why it happens. The following is a discussion of the possible mechanisms by which COg may block IPCs.
(1) COg affects IPCs via the effects of (H+J.
It has been shown that firing of IPCs is not affected 62 by (H+J injected into the pulmonary circulation (Molony, 1972 i Osborne, 1972), However, it should be noted that
IPCs might not be accessible to this kind of administration
because (.H"*"} docs not easily cross the membrane barrier to
roach the receptors. Since SOg is very soluble (>(0 times greater than COg at '!0°c), the possibility that SOg is
easily dissolved in the liquid phase of the tissues of the
lungs to become sull’urous acid (HgSO^) which affects IPCs can not be excluded. This speaks to the theory that SOg block of IPC activity is secondary to damage of lung tissues by the toxicity of ^ H+J . The resumption of firing of IPCs, however, suggests that SOg action, if mediated by CK*3. is not caused by destroying of lung tissues by hydrogen ions.
(2) SOg affects IPCs via the following secondary effects: (A) SOg constricts the airway smooth muscles, i.e., increases airway resistance and causes local bronchoconstriction (Frank et al. 1961) and thereby reduces or prevents air flow to the vicinity of the receptor sites. Hecently, Molony (1976) showed that the smooth muscle around medio- ventral secondary bronchus in ducks were affected by COg} i.e., increased resistance. Whether or not SOg has the same effect is not known. (B) SOg constricts pulmonary veins which causes immediate bronchodilation (Salem et al, 196l),or SOg stimulates the release of Histamine which acts upon trachobronchial muscles causing bronchoconstriction. All those mechanisms 63 may operate by blocking parts of airflow to the IPCs. However, these physiological changes may not account for the block of IPCs with S02. The reason is that IPCs are located in different regions of the paleopulmo and neo- pulmo of the lungs in birds (Fedde et al, 197^)• Therefore, not all IPCs could be affected by these changes. (3) SC>2 affects IPCs via the mechanism of S02 binding to protein portion of plasma (Bystrova, 1959)* This hypothesis seems almost impossible because this is an effect of long term exposures (days to months) to low SOg. Frank et al (1965) reported that after administering 200- 500 PPI.S of SOg ior 3-5 minutes through tracheal cannula, most of the lungs in dogs still appeared normal. The strongest argument against the pathological effects of S02 upon the lungs is that IPCs resume firing after a mean of 6.5 minutes after S02 insufflation (Kunz et al, 1975 personal communication). (4) S02 may affect IPCs as a competitive or noncompetitive inhibitor of C02 at the receptor sites. It is reasonable to believe that because of the similiarity between C02 and
S02 in structure they may facilitate one another's activity at the receptor( I.e.,they occupy sites which are the same or in close proximity on the receptor. Suppose 5% C02 stops the receptor activity within one to two seconds
(Fedde, 1970)* According to the assumption of receptor binding, then 0.2^ of S02 should stop firing of the 61* receptors within 25 to 50 seconds. The experimental results did support this hypothesis to coma extentj i.e> ■ COg pacing was broken with a mean latent period of onset of 106-86 seconds. After a mean COg pulse of (0.1?-0.0?) 0 X (100-68)
seconds. (See table 1)
Although there is no clear-cut answer a^ this moment, it is considered likely that a direct action of SOg (either
C. J or SOg itself) upon the receptors is the most likely explanation especially considering the small doses of SOg
which break COg pacing in the open-loop and increase the
regulated COg levels in the closed-loop. If the action of
SOg was pathological and damaged the lung tissues in the dose administered it is difficult to explain the spontaneous recovery of the activity of IPCs.
Break of COg Facing with Small Doses of SOg 1
It has been hypothetized that COg pacing in the awake, open-loop preparation is mediated thru IPCs since the small
COg oscillations of the inflowing gas are probably greatly attenuated by the time they reach systemic COg receptors
(Kunz and Miller, 19?*0* As shown in figures ? and 8 , when a small dose of SOg broke pacing, the breathing fre quency became slower but ventilation remained regular. At the time when pacing was re-established, ventilation returned to the previous pattern indicating that administration of
SOg at this small dose did not irreversibly harm the IPCs. The smooth and regular breathing pattern after breaking 65
COg pacing also indicate that this dose of administration did not disturb the birds or cause any reflex response to
SOg irritation, both the time before onset of breaking pace and the time for which pacing remained broken are similar to the onset and duration of the SOg block of single IPC afferents. At the concentration of SOg which breaks
COg pacing,the IPCs also cease firing; at the time of re covery of pacing, firing of IPCs resumes. This supports the hypothesis that COp pacing is mediated thru IPCs. As shown in Table 1, in ten of 22 trials pacing was not broken by SOg. Careful observation of the data indicates that this variation may be attributed to the fact that the duration of SOg pulses was not long enough to break pacing.
For example, C-33^ did not break COg pacing in three trials. However, the duration of SOg administration was less than
75 seconds; more prolonged exposure to SOg might have led to a break of pacing. Steady-St ate and Transient-State Analysis in C losed-Loop.
A second type of experiment in which IPCs are believed to be involved is in the closed-loop regulation of CO2 . Figure 23 shows schematically three COg sensitive receptors with their inherent delays. This diagram illustrates the control of ventilation with different sets of COg receptors. The experimental results (figure 9) in this mode of regulation can be interpreted as SOg blocking IPCs and momentarily decreasing the respiratory drive. Ventilation during that ARTERY LUNG CONTROLLER
CAROTID BODY
MEDULLA
Figure 23. Three C02 Receptors with Their Inherent Delays 67 time falls behind U (analog ol‘ COg production), and COg
drifts up until it reaches a level where the COg drive from the remaining receptors compensates for the loss of the drive from IPCs* At recovery, the IPCs return, ventilation is momentarily greater than Q, and COg comes down. Figure 2** shows the feedback diagram of possible respiratory drive in this multiple-feedback scheme. Both receptors HI (IPCs) and R9 (systemic chcmoreceptors) involve a negative feed back and e -ST represents the circulation delay of receptors; note that the controller and lung-blood-gas exchanger have been presented as a single integrator element. The error signal, 6, is the difference between the reference
COg setting and the respiratory drive of R1 and R2 in COg regulation. In the steady-state condition, V is a function of this error signal which in turns depends upon the drive from and Rg and the reference point of COg. The "a" marked in this figure indicates the reciprocal of the time constant of the respiratory controller and Kg are in dividual gain components of the system.
Figure 25 is a sensitivity vs. delay curve. It indicates the stability of the system is determined by thishyperbolic curve; i.e., either increased delay or increased sensitivity will make the system become unstable (Kunz and Miller, 197**) •
It is evident from the closed-loop transient study (figure
10) that the COg level maintained by the bird becomes LUNG BLOOD-GAS CONTROLLER EXCHANGER Ki £ Ki CO. - v . S+a V _ y S
I PC
-&r R«
SYSTEMIC DELAY CHEMORECEPTORS
Figure 24. Possible Respiratory Drive of Multiple Feedback Diagram UNSTABLE SENSITIVITY
DELAY
Figure 25. Threshold for Instability (Sensitivity or Gain X Delay)
ON NO 7 0 unstable and undulating after S02 administration. This is interpreted mostly to be due to the block of IPCs which increases the transportation delay for the system to regulate C02 level through other sets of COg receptors. Models for S02 Effects Upon the Dynamics of COg Regulation. One of the goals of this study was to informationally dissect the respiratory control loops. It was the purpose of this work to investigate what happens in the regulation
of C02 when IPCs are blocked and the C02 level must then be regulated by the other two sets of C02 receptors. The experimental results confirm that IPCs are important and responsible for 002 pacing and contributed to the respiratory drive in the closed-loop. This can further be designed in the model of analog computer simulation experiments. Figure 26 shows the analog simulation diagram; *'R^" and
"R^' represent IPCs and Systemic chemorecepters respectively. As shown in Figure 2? after "R^" was blocked as by opening the switch, the baseline (analog of C02 steady-state regulation) was elevated and oscillated. Note that before "R-^" was turned off the system showed a damped, stable response; after "R^" was off, then the response underdamped and oscillated. The real time delay was set at will by a
PDP-12 computer to simulate the circulation delay of systemic chemoreceptors. REAL TIME DELAY IPDP-I2)
INTEGRATOR PULSE
Figure 26. Analog Simulation Model a = 5 .0 R»=I.O R( = 0 Ri=Q.5 DELAY 5 sec. = 1.0 sec.
j
a =5.0 R. =0 R,=i5 5 sec. Ri=Q7 DELAY = 4.0 sec.
Figure 27. Results of Analog Simulation 73 Steady-State and Transient Analysis of Open-loop COg forcing.
The results may be difficult to interpret meaningfully due to the problem that the awake preparations may change their initial steady-state condition. The steady-state analysis shows that the slope of V - C02 sensitivity
curve is decreased after SOg administration. In additiont the slopes between two groups differ significantly ( K 0 .05) suggesting that the minute volume of PVLB in response to steady-state CO2 level was greatly depressed as compared to control. Also, the control group and SOg-treated
animals do not show a significant (P>0»05) change of the intercept which implies that the set-point of the COg controller has not changed. In transient analysis, as expected, the ON time delay is significantly prolonged after
S02 administration and after further surgical vagotomy. This may be interpreted as due to the fact that IPCs are blocked, and the systemttherefore, has to depend more upon other sets of C02 receptors which have longer transmission delay. Finally, one thing needs to be considered. Whether this variation in respiratory drive can be attributed to the block of IPC activity alone is questionable! because at present there are no experimental results to exclude the possibility that other lung receptors or other parameters are also affected by these large doses of SOg. 74 PVT id and further Surg.icat Vagotomy. The irreversibj lity after FVIJi suggested tha t either
a large amount of vulnerable IPCs wore affected, or lung mechanoreceptor were damaged or both. Since the COg level
could be regulated after PVLB providing: ft, the analog of metabolic COg production, was decreased from the previous
setting. This implies that one of the IPC functions may be to react to nn increase in Q by increasing the respiratory drive proportionately in order to maintain a steady-state equilibrium. The further elevation of the level at which
COg is regulated after unilateral and then bilateral vagotomy implies that bilateral vogotomy further eliminates the activity from carotid chemorcceptors. This also implies that the SOg block of IPC activity does not block systemic chemoreceptors. It is possible that SOg also affects other parameters in the lung of chickens. For instance, during SOg admin istration, an increase of tracheal pressure in the uni- directionally-ventilated awake preparation was sometimes observed. In addition, an elevation of central arterial blood pressure occurred in anesthetized chickens during SO2 insufflation (Kunz and 13erger, 1975* Personal communication). These results suggest that SOg affect other physiological variables even though there is no apparent change in the breathing pattern or behavior of the animals. Molony (1972) described and classified IFCs according to the way in which their firing patterns related to the phase
of breathing. Type Ia fired only in inspiration, type fired only in expiration and type lc fired a burst both in inspiration and expiration. These different typos of IPCs
nay play different roles in res]>iratory control; e.g., only
one tyjxi might be responsible for pacing, PVLB may result
from block of another type of IPCs or from block of receptors which are sensitive to expansion of the thorax or
abdomen (i.e., stretch receptors). It is conceivable
that small doses of SOg block a vulnuerable set of IPCs while larger doses of SOg block an additional less vulnerable set of IPCs or perhaps pulmonary stretch receptors.
SOg-aerated intravenous infusion in closed-loop COg regulation shows that the effect of SOg injected systcmically is to increase ventilatory frequency. This is in contrast to PVLB in which ventilation is slow and deep. Thus PVLB
must be brought about by an action of SOg in the lungs. Intravenous infusion of SOg in open-loop COg pacing
experiments indicated that pacing could be elicited again
immediately even though the systemic SOg effects were still operative. This clearly shows that COg pacing is mediated thru IPCs; although the frequency range of pacing is influenced by the activities from other systemic chemorcceptor
From both data in closed-loop and open-loop I.V. COg infusion studies, it is concluded that PVLB after SOg 76 insufflation is not caused by S02 circulating in the blood to arterial chemoreceptors. pH Analysis. pH measurements before and after S02 admin istration show that blood acidity was not significantly altered by small doses of S02 (P>0.05). Large doses of S02 did alter blood acidity significantly (P<.0.005). It is questionable whether these pH changes were due to a direct effect of S02 upon the system: i.e., increase H+ of the blood; or secondary to the respiratory acidosis brought about by the slow, deep breathing of PVLB. The systemic absorption of S02 is probably negligible (Strandberg, 196A) in experiments in which S02 was added to the air inspired. Richards (1969) reported that pH drop was parallel to the temporal event of surgical vagotomy i.e., respiratory acidosis. SUMMARY
Intrapulmonary CC^-sensitive receptors (IPCs) have recently been demonstrated in chickens. This study used the awake, unidirectionally-ventilated preparation to in vestigate the roles of IPCs in respiratory control as deduced from blocking their activity with a pulse of SO2 . Air flow through the preparation was set at approximately ten times normal minute volume and the COg concentration of the inflow gas could be varied independently of the chicken's ventilatory effertsj i.e., the respiratory control loop could be opened. The loop could be reclosed externally using an analog computer to set the intrapulmonary COg at a level related to the ventilation of the animal. Breathing movements in this chicken-computer combination are seen
to lock onto an open-loop administered COg oscillation i.e., pacing. In the closed-loop mode the animals can breathe accordingly to maintain a steady-state C02 equilibrium i.e., regulating intrapulmonary C02 at about k.0%. The results show that by administering a controlled
pulse of SO2 to the insufflating gas it is possible to block IPCs without greatly disturbing the bird or affecting systemic chemoreceptors. Two different ranges of S02
were studied* A) Low doses of S02 (0 .17$ X 1.5 minute) caused*
1) a break of C02 pacing in the open-loop mode. This
77 78
effect was cone rally reversible j spontaneous recovery
usually occurred within ten minutes after pacing was
broken.
2 ) an increase in the steady-state concentration at
which COg was regulated in the closed-loop mode.
3 ) instability of COg regulation as indicated by
oscillations in the closed-loop COg transient response. B) Large doses of SOg (0.39? X 1.0 minute) caused* 1 ) the breathing pattern to resemble that after surgical vagotomy; i.e., Post Vagotomy Like Breathing (PVLB).
This effect was irreversible. The level at which COg was regulated in the closed-loop was elevated indicating a
reduction of respiratory drive. The PVLB pattern v/as unaltered by subsequent surgical vagotomy. 2 ) reduction of minute ventilation as compared to
the control in the open-loop. Intravenous infusion of SOg-aerated saline solution
indicated that PVLB was not due to SOg acting upon systemic chemoreceptors but must be due to an action upon receptors
or other components in the lung. The SOg pulses at the low concentration which breaks j pacing, stopped the firing of single IPC afferents running c in the vagus nervei after some time firing began again spontaneously. The time of onset of the block of firing
and its duration wore similar to those observed in COg pacing experiments. This indicates that there is a positive
correlation between IPC*s activity and COg pacing. 79 In conclusion, the roles of IPCs can be established. COg pacing is mediated thru IPCs and IPCs also contribute to the respiratory drive. 80
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