Respiration

Physiology

1833191 Biomedical Press

(1977) 31,

@El sevier/North-Holland

VENTILATORY CHEMOREFLEX DRIVE IN THE TORTOISE, TESTUDO HORSFIELDI

G. BENCHETRIT’, Laboratoire

de Physiologie

Respiratoire,

J. ARMAND Centre National

67087 Strasbourg,

and P. DEJOURS

de la Recherche

Scientjfique,

23 rue Becquerel,

France

Ventilation was recorded by pneumotachography in lightly anesthetized tortoises Testudo placed at 25, 30 or 35°C and breathing air or an hypoxic mixture containing 10 or 5% 02. Hypoxic tortoises hyperventilated. In these nine environmental conditions the animals were switched onto pure oxygen for one tidal volume. In normoxic tortoises at 35 “C and in all hypoxic animals, this led to a transient fall of ventilation, a response abolished by cutting both vagus nerves. The injection of NaCN-containing saline into one of the main vessels from the cardiac ventricle led to a transient increase of ventilation in tortoises breathing air, a response absent in animals breathing pure oxygen. By comparing the delay and the magnitude of the ventilatory reaction to injection of small doses of NaCN in the right or left aortic arches, or in the right or left pulmonary arteries, it appears probable that the putative chemoreceptor structures are perfused by blood of both pulmonary arteries. In conclusion, ventilatory chemoreflexes exist in the tortoise, Testudo horsfieldi; the chemoreceptor structures are presumably perfused by the pulmonary arteries and innervated by branches of the right and left vagus nerves. Abstract.

horsfieldi,

Arterial chemoreceptors Chemoreflexes in breathing Control of breathing

Testudo

Ventilatory response to cyanide Ventilatory response to hypoxia

Chelonian breathing is affected by hypoxia ; this phenomenon has been observed in freshwater turtles, Chelydra serpentina (Boyer, 1963), Chelys fimbriata (Lenfant et al., 1970), Pseudemys scripta (Randall et al., 1944; Frankel et al., 1969; Jackson, 1973). The differing ventilatory responses to hypoxia in these studies may be speciesrelated or due to different experimental conditions, in particular regarding temperaAccepted for publication

28 April 1977.

’ Present address and reprint request: Laboratoire de Physiologie, Universitt Scientifique et Medicale de Grenoble, Domaine de la Merci, 38700 La Tronche, France. 183

184

G. BENCHETRIT et a/.

ture. Few data are available on land turtles or tortoises. Lumsden (1924) observed ventilatory changes resulting from the alteration of the inspired gas composition, but the species and experimental conditions were not specified. The present study on Testudo horsfieldi was undertaken (1) to describe the effect on breathing of changes in temperature and in oxygenation of the surrounding, (2) to search for hypothetical peripheral chemoreceptive areas sensitive to blood oxygen tension.

Methods

Eighteen male or female tortoises (Testudo horsfieldi) weighing 500 to 850 g were studied. All the experiments were performed under anesthesia obtained by subcutaneous injection of 50 mg . kg-’ of Nembutal. As a rule the anesthesia began half an hour after the injection, persisted for a few hours, and was never deep enough to suppress limb reflexes to pinching. Ventilation A few days prior to the study a base-plate of dental cement with a central opening of about 5 mm wide was built up around the nares (fig. 1). Eating and drinking were not prevented by this experimental device which the animals wore during the 2- to 3-week experimental periOp. For the actual experiment, a tube holding a Fleisch pneumotachograph (No. 0000, or No. 000 for the larger animals) was fixed in the opening of the base-plate, the mouth being firmly closed. The inspired gas mixture

Fig. I. Tortoise with base-plate of dental cement fixed around the nares to hold a tube attached to a pneumotachograph.

RESPIRATION

IN THE TORTOISE

185

flowed in through a T-tube on the distal end of the pneumotachograph and the pressure difference across the pneumotachograph was sensed by a differential strain gauge (Statham PM 197). The pneumotachogram and/or spirogram, obtained by integration of the instantaneous gas flow, were recorded, along with the intrapulmonary pressure measured with a PM 5TC Statham strain gauge set in a hole drilled in the dorsal carapace. Oxygen tests

Six normal, lightly anesthetized animals breathed air or a hypoxic mixture (FI,, = 0.05 or 0.10) for at least one hour before starting a series of ‘02 tests’ (Dejours, 1957). The animal breathed one tidal volume of pure O2 and was switched back to the previous inspired gas. Ventilation was continuously recorded and subsequently analyzed over short periods of time. Oxygen tests were performed at intervals of 10 minutes or more, to allow the return to the reference level of breathing. The experiments were carried out at 25, 30 or 35 “C, the animal being placed in a heated box and the cloaca1 temperature monitored continuously. The procedure was then repeated one or more days after bilateral vagotomy in the middle part of the neck. Sodium cyanide injections

In twelve anesthetized tortoises, a 2.5 cm hole was drilled in the plastron over the cardiac area. Through the hole, catheters were inserted downstream either into the three main arteries originating in the heart ventricle, the truncus arteriosus or the right aorta, the left aorta and the pulmonary artery trunk (5 animals), or in seven other animals into the right and left pulmonary arteries. The catheters were fixed on the pericardium by small stitches and passed through a rubber stopper which was fixed in the hole of the carapace. Through the catheters, filled with heparinized saline, small volumes of saline containing 25 or 50 pg of NaCN were injected. Sodium cyanide was injected in animals breathing air or pure oxygen. In control experiments, saline alone was injected. In the experiments with catheters indwelling in the right and left pulmonary arteries, cyanide was injected unilaterally before and after section of the ipsilateral vagus. At the end of the experiments the precise location of the catheters in the three main arteries originating in the cardiac ventricle was checked.

Results

The inhalation of hypoxic mixtures led to an increase of ventilation (fig. 2) resulting itself from the increase of the respiratory frequency and/or tidal volume. At a given temperature, the lower FIN,, the higher the ventilation. At a given F102, the higher the temperature, the higher the ventilation.

186

G. BENCHETRIT i/E.

“‘II

et a/.

STPS.min-'

75 -

50 -

25 -

“0

Fig. 2. Ventilatory

0.10

flow rate, VE, as a function

and cloaca1 temperatures of ventilation

0.05

of the 02 fraction

25, 30 and 35 ’ C in a tortoise

for a given inspired

values of ventilation

0.21

or apnea

02 fraction recorded

FIN,

in the inspired gas (no,)

weighing

and a given cloaca1 temperature. after inhalation

at three ambtent

0.60 kg. Closed symbols,

of one tidal volume

steady

Open symbols,

values lowest

of pure oxygen.

Oxygen tests The inhalation of one breath of pure oxygen led to a decrease or an arrest of ventilation (fig. 3). Figure 2 (open symbols) shows at each level of ambient oxygenation the lowest value of ventilation consecutive to oxygen breathing. It is clear that the lower the value of FtoZ, the greater the ventilatory reaction to oxygen breathing.

Y

Pr,*WTorr&“re 02 I_37 I-

Torr

l. 5 set

Fig. 3. Pneumotachogram, spirogram and pulmonary pressure as functions of time in a tortoise (0.55 kg) breathing a 5% 02 in Nz mixture at 28°C. At the first vertical line, the animal was abruptly switched on to a pure oxygen for one tidal volume, and then at the second vertical line was switched mixture.

I, E: respectively

inspiration

and expiration.

back to the original

RESPIRATION

187

IN THE TORTOISE

VE , ml 6rPs. min-’

I I I

20 *~.‘,*******.*.~* *.....: bivagotom~zed~

I

10 -

O-

:“....*...: Z’.....*...* ..*..**..*.: : : i ,...... *..*.. L......... ..,..*.,....: ;..*. . .. . . ..:

:. . ..I...... i

, 5

t 4

3

, 2

I 1

I 0

1

2

3

4

5 min

Fig. 4. Ventilatory flow rate us time in a tortoise of 0.56 kg breathing a hypoxic mixture interrupted by

a single breath of pure oxygen at time 0. Temperature, 27°C. Continuous line: intact animal. Dashed line: vagotomized animal. Vagotomy suppresses the hyperventilation in hypoxia and the reaction to pure 02 inhalation observed in intact animals.

Fxo, - 0.21

NaCN,(SOpg)

1 5ml[ -2

cm 40

0 +2

/P FIEF= 1.00

NaCN )5Ot&~9)

Fig. 5. Spirogram and pulmonary pressure us time in a tortoise of 0.55 kg breathing air (top) or pure oxygen (bottom). Temperature 27 “C. At the arrow, 50 pg of NaCN was injected into the trunk of the pulmonary artery. Hyperoxygenation suppressed the hyperventilatory response to NaCN injection.

188

G. BENCHETRIT

et Ui.

Presumably, there exists also a ventilatory oxygen drive in normoxia, at least when, at high temperature, the ventilatory activity is relatively intense. Bivagotomy suppressed (1) the ventilatory reaction to continuous hypoxia, (2) the transient fall of ventilation following inhalation of pure oxygen (fig. 4). Cyanide injections The injection of NaCN-containing saline into the right or left pulmonary arteries induced ventilatory changes. The most obvious change was the increase of the first inspired volume following the injection (fig. 5, top). The same injection in an animal which had been breathing pure oxygen for one hour did not induce any ventilatory changes (fig. 5, bottom). No control injection of pure saline was followed by any change of ventilation. Injections of 25 or 50 pg of NaCN solution in the left aorta or in the truncus arteriosus did not result in an increase of ventilation. When more NaCN was injected, a ventilatory effect was sometimes observed but after a delay longer, i.e. on the second or third cycle following the injection, than that seen after injection into the pulmonary artery.

NaCN (25pg) left pulm. art. r-l-

NaCN (25 pg) right pulm. art.

I

NaCN (25 pg) left pu/m. art.

cmH20

bivagotomized

-5

0 +5 5 set

Fig. 6. Spirogram

and pulmonary

pressure

vs time in a tortoise

right vagus nerve was cut before the beginning monary

artery

(top, second

arrow)

was without

of the recording effect. Injection

of 0.53 kg, at 25 “C, breathing and injection of NaCN

of NaCN

air. The

in the right pul-

in the left pulmonary

with the left vagus intact (top, first arrow) led to an increase of the ventilation, an effect abolished subsequent section of the left vagus nerve (bottom, third arrow).

artery after

RESPIRATION

IN THE TORTOISE

189

Figure 6 shows that the NaCN injected into the left pulmonary artery while the left vagus nerve was intact caused an increase of ventilation ; whereas NaCN into the right pulmonary artery after section of the homolateral vagus nerve did not lead to any change of breathing. IIowever, when the left vagus was then cut, the ventilatory reaction to NaCN injection in the left pulmonary artery was abolished.

Discussion At 25 and 30 “C the ventilation of the tortoise only slightly increased when a 10 % O2 mixture is breathed. In humans also, acute hypoxia of the same magnitude does not much affect the ventilation (Rahn and Otis, 1947). In tortoises at 35 “C breathing 10 % O2 mixture, ventilation clearly increased. Temperature-dependent ventilatory effects of hypoxia have also been seen by Jackson (1973) in the fresh water turtle Pseude~ys scripta efegms, but at a different temperature range. Possibly, higher temperatures, by increasing the oxygen demand of poikilothermic animals, make them more sensitive to the change of the ambient oxygenation. Also Frankel et al. (1969) showed in Pseudemys scripta at 28 “C that prolonged oxygen breathing depressed respiration, increased arterial blood PO, from 30 to 34 Torr and decreased blood pH from 7.64 to 7.59. Our results suggest a possible role of oxygen in the control of the tortoise’s ventilation, and the existence of an oxygen drive. Withdrawing the hypoxi’c stimulus by allowing the animal to breathe one tidal volume of pure oxygen decreased ventilation, and the fall of the ventilation was the greatest when the hypoxic conditions were the most stressful, i.e. at 35 “C and 5 % oxygen. The hypoventilation following pure oxygen breathing resembles the response in mammals (see Dejours, 1962; Bouverot et& 1965; Leitner etal., 1965)and the fowl (Bouverot and Leitner, 1972), in these animals a response due to a decrease of the activity of arterial chemoreceptors. In homeotherms whose arterial chemoreceptors have been surgically removed, ventilation in normoxia is depressed, hypoxic hy~~entilation is no longer observed and 02 inhalation does not lead to a fall of ventilation (Bouverot et al., 1965). These are the reactions seen in tortoises after section of both vagus nerves (fig. 4). They suggest that tortoises have chemoreceptors and that their ventilatory reactions to changes of blood oxygenation are chemoreflexes as in higher vertebrates, the chemore~ptive areas being innervated by the vagus nerves. But in the tortoises, the chemoreceptive area does not seem to be located in the vicinity of the carotid bifurcation which is located high in the neck (Adams, 1958), that is much above our section of the vagus nerves. The chemoreceptors of mammals and birds respond to stimuIation by sodium cyanide (see Heymans and Neil, 1958; Bouverot and Leitner, 1972; Dejours, 1976) and the tortoise’s ventilation is also affected by this agent. We tried to localize chemoreceptive areas by selectively placing injections of NaCN in the main vessels

190

G. BENCHETRIT et Uf.

leading from the heart and analyzing breath by breath the pneumotachographic record. The absence of a regular breathing pattern in the tortoise makes it difficult to predict when the next breathing will occur and how deep it will be. However, we observed that NaCN injection into either pulmonary artery elicited an increase of the first inspired volume of air following the injection. Injected elsewhere - in the aortic arches - the dose of NaCN had to be higher to obtain this effect. Vagotomy on the side of the injection abolished the response without affecting the response on the contralateral side. These results indicate that there are hypoxia-sensitive chemoreceptive areas in the tortoise, that they are perfused by pulmonary arterial blood, and that they are bilateral. They do not support the suggestion of Frankel et al. (1969) that there are peripheral chemoreceptors at the bifurcation of the common carotid artery. The finding of chemoreceptor structures innervated by branches of the vagus nerves is not surprising. Most mammals have vagally innervated aortic bodies. The carotid bodies of birds are inside the thorax and are innervated by vagal branches (Adams, 1958; Jones and Purves, 1970; Bouverot and Leitner, 1972). According to Adams (1962), in the sea turtle Chebniu my&s, the tissue situated between the truncus arteriosus, the left aorta and the pulmonary artery contains small nests of epithelioid cells and is profusely innervated; Adams suggested that this tissue may subserve a chemoreceptor function. But its very rich vascularization by small vessels seems to arise only from the left aorta. However, in some organisms chemoreceptor structures may be perfused by pulmonary artery blood. It is the case in the human fetus, where the inferior aortic-pulmonary glomus receives its blood supply from the pulmonary trunk (Boyd, 1961); and Nonidez (1936) has described a small arterial branch to the presumably chemoreceptive aortic paraganglion of kittens, arising directly from the pulmonary trunk or of one ofthe pulmonary arteries. No anatomical evidence of the disposition in tortoise is yet available.

References Adams, W. E. (1958). The Comparative Morphology of the Carotid Body and Carotid Sinus. Springfield, Ill., Charles C. Thomas, 272 p. Adams, W. E. (1962). The carotid sinus-carotid body problem in the Chelonia (with a note on a foramen of Panizza in Dermochelys). Arch. Int. Pharmacodyn. 139: 28-37. Bouverot, P.. R. Flandrois, R. Puccinelli and P. Dejours (1965). Etude du role des chemorecepteurs arteriels dans la regulation de la respiration pulmonaire chez le chien tveillt. Arch. IN. Pharmacodyn. 157: 253-271.

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Boyd, J. D. (1961). The inferior aortico-pulmonary glomus. Brir. Med. BUN. 17: 127-I 31. Boyer, D. R. (1963). Hypoxia: effects on heart rate and respiration in the snapping turtle. Science 140: 813-814. Dejours, P. (1957). Inter&t mtthodologique de I’ttude d’un organisme vivant a la phase initiale de rupture d’un Cquilibre physiologique. C. R. Acad. Sci. 245: 1946-1948.

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