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Keywords:

  • animal;
  • hypercapnia;
  • hypoxia;
  • swallowing;
  • tachypnea;
  • ventilation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References

Background  It has been demonstrated that aspirations could occur during respiratory failure, explained by a lack of co-ordination between swallowing and ventilation. To test this hypothesis, we examined the co-ordination of ventilation and swallowing in a completely unrestrained rat model during different level of hypercapnia, during hypoxia, and during tachypnea.

Methods  A total of 50 male Wistar rats (250–350 g) were studied in a barometric plethysmograph to analyze swallowing and ventilation during swallowing, at different gas concentration [room air (G1), 10% of O2 and 0% of CO2 (G2), 21% of O2 and 5% of CO2 (G3), 21% of O2 and 10% of CO2 (G4), tachypnea (G5)].

Key Results  During hypoxia, there was no difference between G2 and G1 regarding the swallowing parameters and ventilatory parameters. During hypercapnia, there was an increase in swallowing during inspiration in G4 (16 ± 20%< 0.01) compared with G1. The analysis of ventilatory parameters during swallowing showed an increase in tidal volume (VT) and mean inspiratory time (VT/TI) (< 0.001) with no change in respiratory cycle duration (TTOT), inspiratory time (TI), and expiratory time (TE) when compared with G1. During tachypnea (G5), the VT decreased (< 0.05) without any change in VT/TI.

Conclusions & Inferences  Our results on animal demonstrated that hypercapnia increased swallowing during inspiration, which was not the case for tachypnea or hypoxia, and could explain some aspirations during respiratory failure.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References

In chronic respiratory failure, a new conceptual approach is to demonstrate that exacerbations could be partly explained by oropharyngeal dysphagia. This has been first demonstrated in a retrospective study in chronic obstructive pulmonary disease (COPD) patients. In this study, nearly 85% of these patients evidenced some degree of dysphagia, and laryngeal penetrations or aspirations were observed in 44 of them.1 More recently, COPD has been identified as a risk factor of pneumonia aspiration.2 In these patients, oropharyngeal dysphagia is explained by a decrease in maximal laryngeal elevation during swallowing.3 Oropharyngeal dysphagia is defined by alterations of swallowing that could induce laryngeal penetration or bronchial aspiration. It represents a very common disease due to lot of pathological conditions. Its major complications are malnutrition and inhalation pneumonia.4 The main causes are neurological disorders5 or ENT cancer,6 but in chronic respiratory failure, it has also been demonstrated that aspirations could occur during exacerbation. One explanation is that there is a lack of co-ordination between swallowing and ventilation in these patients and that some swallowing apneas occur during inspiration as usually it occurs during expiration. This phenomenon has been demonstrated in chronic obstructive pulmonary disease7 and in myopathy.8

In animal, we have already demonstrated that this swallowing apnea could be compromised during pharyngeal anesthesia9 and after unilateral vagotomy.10 In this case, we also demonstrated that respiratory drive was decrease when aspirations occurred. This suggests that swallowing difficulties could affect the control of ventilation. It has also been demonstrated that an increase in ventilatory drive could also modify swallowing function. For example, hypercapnia and increased inspiratory resistive load modify swallowing and breathing co-ordination and increase laryngeal aspiration.11 However, no study had been carried out to assess if hypercapnia, hypoxia, and an increase in respiratory rate that are frequently observed in respiratory disease, affect swallowing and breathing co-ordination in completely unrestrained conditions. Indeed, all the previous published studies included restrained condition that is known to modify swallowing behavior. To test this hypothesis, we examined the co-ordination of ventilation and swallowing in completely unrestrained rats during hypercapnia, during hypoxia, and during tachypnea.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References

Animals

We used 50 male Wistar rats (250–350 g; Charles River, Arbresle, France). The experiments were approved by the local ethics committee and were performed in the Experimental Surgery Laboratory of Rouen University (license: A76-450-05, surgeon’s license: 76.A.21).

Ventilation analysis

Ventilation analysis has been previously described.9 Briefly, respiratory variables in unrestrained rats were measured non-invasively by whole-body barometric plethysmography (Emka, Paris, France; range 0.1 mb). The differential pressure between the two chambers (transducer; Emka; range 0.1 mb) was converted into a digital signal with a 1000 Hz sampling rate (Chart Ad Instruments, Oxford, UK). Body temperatures were not continuously recorded during the ventilatory measurements, but were measured in the plethysmograph. A video camera was used to monitor the rats during the experiments (60 frames s−1; Logitech Europe SA, Morges, Switzerland) and was synchronized with the chamber pressure prior to each experiment. The plethysmograph was placed in a large box airtight (99 L), to assure stable gas concentrations, the O2 and CO2 concentrations, were modified. The plethysmograph was open into this box and connected to an aspiration system at 2.5 mL min−1 (Emka) which permitted to maintain a stable gas concentration in the plethysmograph. The different gas concentrations were room air, 10% of O2 and 0% of CO2, 21% of O2 and 5% of CO2, and 21% of O2 and 10% of CO2 (Vitalair, Chatillon, France). The gas in the plethysmograph was analyzed in real time with a O2 and CO2 gas analyzer (Chart Ad Instruments).

Experimental protocol

The rats were divided into five groups of 10 rats and were assessed at healthy state, at rest, and during water swallowing after being deprived of water for 12 h. The first group (G1) was studied in room air breathing, the second group (G2) at 10% of O2, the third group (G3) at 5% of CO2, and the fourth group (G4) at 10% of CO2. The fifth group (G5) was studied in room air breathing with an increase in temperature into the plethysmograph obtained with a hair dryer placed at 5 cm from the plethysmograph. The rats were then placed in the plethysmograph chamber where they could drink water ad libitum through a baby bottle. After 20 min of stabilization of ventilation, the baby bottle was introduced into the plethysmograph.

Data analysis and statistical analysis

We chose 10 s periods during swallowing with the help of video recordings to determine the swallowing frequency and type based on the ventilatory cycle phases [inspiration (I − I), expiration (E − E), transition between inspiration and expiration (I − E), and transition between expiration and inspiration (E − I)] preceding and following the apnea. Next, 10 stable, consecutive ventilatory cycles at rest and during swallowing were identified and analyzed. Ventilatory variables [inspiratory time (TI), expiratory time (TE), duration of ventilatory cycle (TT), tidal volume (VT), and mean tidal volume (VT/TI)] were measured or calculated for each cycle and the means of the ten cycles were calculated for each variable. Swallowing frequency and type (I − I, E − E, I − E, and E − I) and the means of the ventilatory variables at rest and during water swallowing were compared between the different groups by an analysis of variance (GraphPad Prism, La Jolla, CA, USA). Differences were considered significant when the probability (P) of a type I error was 0.05 or less. Results are expressed as means ± standard deviation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References

All rats performed the protocol without any problem. During hypercapnia, we had to perform the protocols two times in two rats because they did not drink. In any groups during the different conditions we did not observed I–E or E–I apnea.

In G1 (room air), at rest, the swallowing frequency was 12 ± 4 min−1, and 98 ± 4% of swallows were during expiration and 2 ± 4% during inspiration. During swallowing, there was an increase in mean inspiratory time (VT/TI) and a decrease in respiratory cycle duration (TTOT), inspiratory time (TI), and expiratory time (TE) as previously described (Table 1).

Table 1. Tidal volume (VT), inspiratory time (TI), expiratory time (TE), total duration of respiratory cycle (TTOT), and mean tidal volume (VT/TI) (means ± SD) in rats exposed to room air (G1), hypoxia (G2), and hypercapnia 10% (G4) at rest and during swallowing
 Rest G1Rest G2Rest G3Rest G4Swallowing-G1Swallowing-G2Swallowing-G3Swallowing-G4
  1. *< 0.05 when compared with G1 at rest.

  2. < 0.001 when compared with G1 at rest.

  3. < 0.001 when compared with G1 during swallowing.

VT (mL kg−1)9.7 ± 3.78.5 ± 1014.12 ± 1.3421.16 ± 9.608.52 ± 1.47*8.80 ± 1.1712.69 ± 1.7815.91 ± 1.64
Tl (s)0.24 ± 0.040.16 ± 0.020.20 ± 0.020.15 ± 0.030.18 ± 0.09*0.15 ± 0.010.17 ± 0.010.18 ± 0.01
TE (s)0.49 ± 0.110.27 ± 0.050.22 ± 0.040.23 ± 0.040.28 ± 0.08*0.23 ± 0.030.21 ± 0.020.22 ± 0.02
TTOT (s)0.73 ± 0.140.43 ± 0.060.43 ± 0.060.43 ± 0.150.44 ± 0.06*0.39 ± 0.040.38 ± 0.030.40 ± 0.03
VT/TI (mL kg−1 s−1)36.59 ± 6.7851.69 ± 4.9070.42 ± 11.9125.9 ± 29.1353.11 ± 17.2357.18 ± 8.7074.67 ± 9.1790.41 ± 10.78

During hypoxia (G2) at rest, there was a decrease in respiratory cycle duration (TTOT), inspiratory time (TI), and expiratory time (TE) compared with G1 (room air) (< 0.05), with no change in mean inspiratory time (VT/TI). During swallowing, there was no difference between G2 and G1 regarding the swallowing parameters (Fig. 1) and ventilatory parameters (Fig. 2).

image

Figure 1.  Percentage of swallowing occurring during expiration in rats exposed to room air (G1), hypoxia (G2), hypercapnia 5% (G3), and in hypercapnia 10% (G4) and in hyperventilation (G5). *< 0.05 when compared with G1.

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image

Figure 2.  Variation in mean tidal volume (VT/TI) during swallowing in rats exposed to room air (G1), hypoxia (G2), hypercapnia 5% (G3), hypercapnia 10% (G4), and during hyperventilation (G5). *< 0.05 when compared with G1.

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During hypercapnia, at rest, there was an increase in mean inspiratory time (VT/TI) compared with G1, more pronounced in G4 (10%) (< 0.001) than in G3 (5%) (< 0.001) (Table 1). During swallowing, there was an increase in swallowing during inspiration in G4 (16 ± 20%< 0.01) compared with G1 (Figs 1 and 3). Figure 3 represents raw data of ventilation during swallowing in one animal. The analysis of ventilatory parameters during swallowing showed an increase in tidal volume (VT) and mean inspiratory time (VT/TI) (< 0.001) with no change in other respiratory variables (TTOT, TI, and TE) when compared with G1 (Fig. 2). Unfortunately, there was no episode of aspiration during hypercapnia or hypoxia.

image

Figure 3.  Example of raw data of 12 s of recording of ventilation during swallowing in one animal. This experiment was made during 10% of hypercapnia. Expiratory swallowing and inspiratory swallowing are identified by an arrow. Temperature, inbox pressure, CO2 concentration in the plethysmograph, and tidal volume were always and continuously recorded.

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During tachypnea, at rest, there was a decrease in TI, TE, and TTOT without any change in VT and VT/TI when compared with G1. In this group, swallowing parameters did not change (Fig. 1). During swallowing, only the VT decreased (< 0.05) without any change in VT/TI (Fig. 2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References

This study is the first to systematically analyze the modification in swallowing and breathing co-ordination during hypercapnia, hypoxia, and tachypnea on completely unrestrained animals. The results mainly demonstrated that hypercapnia destabilized swallowing and breathing co-ordination, which was not the case of hypoxia and tachypnea.

The major advantages of the plethysmography combined with video recordings are that swallowing apnea can be identified with confidence and that unrestrained animals can be studied.9 This is not the case of other methods were animals are tracheotomized to analyze ventilation, or when catheters are putted into the pharynx or the esophagus to analyze swallowing pressure.

Regarding the physiology of swallowing apnea, it has been demonstrated that phrenic nerve activity is present during swallowing.12 This pattern is different from an inspiration and did not produce airflow. This aims to ensure that ventilation has stopped before pharyngeal swallowing at a level above FRC, to obtain expiration at the end of swallowing apnea. In fact, swallowing apnea starts before glottic closure and is controlled into the brainstem.13 The end of swallowing apnea correlates with laryngeal opening13 and occurred at the transition from the pharyngeal to the esophageal phase of swallowing. Expiratory airflow after swallowing may help clear the laryngeal inlet from bolus remnants and, thereby, prevent aspiration. This explained why swallowings occur mainly during expiration.

The swallowing center is a complex organization of neural elements in the cortex and brainstem. The neurons in the brainstem involved in swallowing lie in the nucleus tractus solitarius (NTS).14 The two regions are represented on both sides of the brainstem and are extensively interconnected.15 Ventilation is regulated from complex interactions between two functional groups of neurons in the medulla: the dorsal (DRG) and ventral respiratory groups (VRG). The DRG receives inputs from the cranial nerves IX and X, and from the carotid body. The DRG sends fibers to the inspiratory spinal motor neurons and to the VRG.16 The VRG also innervates the larynx and has both inspiratory and expiratory functions. The larger caudal part, the nucleus retroambiguus, has both inspiratory and expiratory neurons innervating motor units of the diaphragm and intercostal muscles. The shared vagal motor innervation of pharynx and larynx via nucleus ambiguus and the close anatomical relationship between respiratory and pharyngeal afferents with potentially overlapping regions of the nucleus of the tractus solitarius17 and the medullary respiratory centers suggest a high level of co-ordination between breathing and swallowing, including the inhibition of breathing during swallow apnea.15 Those different interactions between ventilation and swallowing into the brainstem are also present at the cortical level. In a recent study, we determine whether ventilation and swallowing tasks can modify oropharyngeal cortical motor organization on healthy subjects and demonstrated that the cortical magnitude of the oropharyngeal muscle representation increased after swallowing practice ventilation tasks.18

In the present study, we try to demonstrate that interaction between SPG and VPG is centrally mediated. We therefore used three different ventilatory stimuli. The first one was hypercapnia at 5% and 10%, which is known to stimulate the peripheral and central chemoreceptors, the second one was hypoxia which stimulates only peripheral chemoreceptors, and the last one a rise in temperature which only induce a tachypnea. We therefore demonstrated that hypercapnia modified the breathing and swallowing co-ordination and reduced swallowing apnea, as it was not the case for hypoxia or tachypnea, as previously demonstrated in non-nutritive swallows.19 This highlight the centrally mediated co-ordination of breathing and swallowing into the brainstem and suggested that ventilatory drive could alter the swallowing pattern generator. In our study, the frequency of swallows and expiratory swallows decreases during hypercapnia and is analogous to the observations made in humans that airway protective reflexes are attenuated during hypercapnia.11 Thus, it may be possible that the attenuation of swallowing control during hypercapnia is a common feature of airway protective reflexes, including the swallowing response. It seems to be reasonable to speculate that the automatic respiratory control system prevails over the swallowing responses when the maintenance of ventilation is particularly important. The modification in the swallowing response during hypercapnia is compatible with the finding that hypercapnia enhances the chance of laryngeal irritation during swallowing. Nevertheless, we did not observe any aspiration during the experiments in hypercapnia. This could be explained by the fact that the level of hypercania was not important (5% and 10%) and that the animal did not have respiratory disease. It could be argued that aspirations were observed in human during hypercapnia, but those study need to be interpreted with caution because swallows were induced with a pharyngeal catheter filled with water.11 Our results complete previous studies in which we demonstrated that swallowing difficulties (oropharyngeal dysphagia) in animal affected ventilatory drive with a decrease in the ventilatory control during swallowing that could be interpreted as a centrally protective mechanism in case of aspiration.10

In conclusion, our results on animal demonstrated that hypercapnia modified swallowing pattern and increased swallowing during inspiration, and decreased swallowing frequency, which was not the case for tachypnea or hypoxia. Those results on animal suggest that swallowing and breathing co-ordination is centrally integrated and that hypercapnia strongly modified this co-ordination. This should now be studied in respiratory failure.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. References