INTRODUCTION

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ELECTROMYOGRAPHIC (EMG) recordings from the diaphragm in awake dogs have shown a clear difference in the pattern of activity between the crural and costal portions of the muscle. Specifically, activity of the crural portion persists well after the end of inspiratory flow, whereas activity of the costal portion terminates with the cessation of inspiratory flow (10). This regional difference in postinspiratory activity was recorded both during rest with room-air breathing and during hyperpnea induced by CO₂ or hypoxia; in fact, the regional difference was greater during hyperpnea than during room air breathing (10, 11). Although the mechanism of this difference was not identified, it was suggested that the greater postinspiratory activity of the crural diaphragm was related to the greater spindle content of the muscle (10, 11).

To test the idea that muscle spindles are an important determinant of postinspiratory activity, we have assessed in awake, intact canines the pattern of activation of the two predominant groups of inspiratory intercostal muscles, namely, the parasternal intercostals and the external intercostals in the rostral interspaces. Indeed, histological studies have shown in the cat that the external intercostals in the rostral interspaces contain much larger numbers of muscle spindles than do the parasternal intercostals (8), and this difference is known to play a major role in determining the response of these muscles to increases in inspiratory mechanical load. When the inspiratory airflow resistance is suddenly increased in anesthetized cats (1, 23), rabbits (20), and dogs (3) or when the airway is occluded at end expiration for a single breath (2), the external intercostals in the rostral interspaces show an increased rate of rise of activity. This facilitation of activity is associated with an increased spindle afferent activity (1) and is eliminated by section of the thoracic dorsal roots (1, 2, 20). In contrast, increases in inspiratory airflow resistance or single-breath airway occlusion in anesthetized dogs causes little alteration in parasternal intercostal activity (2, 3). Similarly, when an external force is applied to the ribs to reduce their normal inspiratory cranial displacement, there is a reflex, spindle-induced increase in external intercostal inspiratory activity without any alteration in parasternal intercostal activity (4, 16). Therefore, if spindle density were a significant determinant of the presence or absence of postinspiratory activity, the external intercostals should show greater postinspiratory activity than should the parasternal intercostals.

	METHODS

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The project was approved by the Animal Care Committee at the University of Calgary. Eight adult mongrel canines (mean weight 27.3 kg; range 20-33 kg) were familiarized with the laboratory environment and personnel and were trained over several weeks to breathe quietly in different body positions. After this period of training, bipolar fine-wire EMG electrodes were implanted in the parasternal intercostal and external intercostal muscles of the second to fourth left interspaces. This technique of EMG implantation has been described in detail elsewhere (10, 14, 18). Briefly, with the animals under general anesthesia, the skin was incised and deflected over the sternum, and bipolar EMG wires were implanted in the parasternal intercostal muscle of the second, third, or fourth intercostal space, ~1-2 cm lateral to the edge of the sternum. The external intercostals were exposed by a second incision on the lateral chest wall, and a pair of EMG wires was implanted in the midaxillary line: the two wires of each pair were placed ~1 cm apart along the long axis of a muscle bundle. The implants were secured by fine, synthetic, nonfibrogenic sutures (Prolene, Ethicon); wires were externalized; and the animals were allowed to recover. There was no operative or postoperative complication. The canines were awake and ambulatory within 3-6 h of the operation and were freely active and feeding normally within 1-2 days. When the studies were completed, the animals were euthanized, and the intercostal muscles were examined. The implantation sites showed minimal fibrosis, as noted in earlier studies on the diaphragm by Easton et al. (10).

All measurements were performed with the animals awake and breathing quietly at laboratory temperature of 18-20°C. The animals breathed spontaneously through a snout mask, which was connected through a one-way valve to a low-resistance open-breathing circuit (<1 cmH₂O · l¹s) and a pneumotachograph (Fleisch no. 2) for measurement of airflow. On the expiratory limb, CO₂ was sampled and analyzed continuously (model CD-3A CO₂ analyzer, AMETEK/Thermox Instruments Division, Pittsburgh, PA). EMG signals were amplified (AM Systems, Seattle, WA), band-pass filtered <100 Hz and >700 Hz (Frequency Devices, Haverhill, MA), and then rectified and moving averaged with a time constant of 100 ms (CWE Systems, Ardmore, PA). The time delay imposed on the signal by the moving averager with this time constant is 50 ms, as described in a previous publication (18). By using computer software for data acquisition (DataSponge, Bioscience Analysis Software, Calgary, AB), the signals were monitored in real time on the computer display and simultaneously collected at 100 Hz to hard disk on a microcomputer (PS/2 model 80, IBM, White Plains, NY) equipped with a single-board analog-to-digital system (model MIO-16-H-9, National Instruments, Galveston, TX).

Recordings were made on 2-3 successive days, an average of 17 days after electrode implantation (range 3-41 days). For each recording session, the animal was placed in the right lateral decubitus position, instrumented, and allowed to stabilize for 30 min. While the animal was breathing quietly, recordings of ventilation and EMG activity were made for 20-30 min. Six animals were also instructed to lift and hold the left forelimb up off the platform on which they were lying; this limb-up position was maintained stable for 20-40 s.

After acquisition and storage on disk, data were analyzed by using software programs written by one of the authors (P. A. Easton). These programs provide interactive visual display of the signals during analysis to allow careful examination of each breath. The flow signal was evaluated for respiratory timing and digitally integrated to calculate, breath by breath, inspiratory time (TI), expiratory time (TE), total breath time (T_tot), respiratory frequency, tidal volume (VT), minute ventilation (I), mean inspiratory flow (VT/TI), and inspiratory fraction of respiration (TI/T_tot). The software also calculated whole-breath ("tidal") values of inspiratory airflow and EMG activity for each breath. These calculations have been described in detail elsewhere (10, 14). Briefly, the timing of inspiration was determined from the airflow tracing, and the onset and termination of parasternal and external EMG activity for each breath were measured relative to the beginning and ending of inspiratory airflow; whenever a significant electrocardiographic signal corrupted the EMG trace so as to obscure the timing of inspiratory activity, that breath was omitted from analysis. The onset and termination times of parasternal and external EMG were time corrected to remove the time delay of 50 ms (17) imposed by the moving averager, and then the timing differences were expressed as percentage of TI and percentage of T_tot. Values of all parameters were exported for review to spreadsheet software (Microsoft Excel, Microsoft, Redmond, WA), to graphic software to output Figs. 1-3 (CorelDraw, Corel, Ottawa, ON), and to the personal computer version of SAS (SAS version 6, SAS Institute, Cary, NC) for statistical analysis. Values for tidal breaths were averaged per animal, and then the group mean values for parasternal and external intercostal onset and termination timing were compared by Student's paired t-test. Statistical comparison of the effect of forelimb elevation was also made by using Student's paired t-test.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Traces of inspiratory airflow and raw and moving average EMGs from external and parasternal intercostals from 1 animal breathing quietly in the right lateral decubitus position 14 days after implantation. Vertical dotted lines, onset and termination of inspiratory airflow, respectively. Note prolonged postinspiratory activity in external intercostal.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Traces of inspiratory airflow and moving average EMG from external and parasternal intercostals from a representative animal breathing quietly in right lateral decubitus with the left forelimb at rest (left) and with left forelimb elevated (right). Note prolonged postinspiratory activity in external intercostal with left forelimb elevated.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Example of marked prolongation of postinspiratory activity in external intercostal after elevation of forelimb (right) compared with forelimb at rest (left).

	RESULTS

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Breathing pattern for the eight animals studied was as follows: VT 0.32 ± 0.07 (SD) liter, respiratory rate 31 ± 10 breaths/min, I 9.76 ± 3.4 l/min, TI 0.94 ± 0.26 s, and VT/TI 0.36 ± 0.11 l/s. The parasternal and external intercostal muscles were active in phase with inspiratory flow in all animals, and the onset of activity generally coincided with the onset of inspiratory flow in both muscles. For the group, parasternal intercostal activity could be recognized 2.2 ± 3.7% of TI before the first moment of inspiratory airflow, and external intercostal activity was observed 0.6 ± 4.1% of TI after the onset of inspiratory flow (not significant). However, whereas parasternal intercostal activity terminated simultaneously with the cessation of inspiratory airflow, external intercostal activity commonly carried on after the end of inspiratory airflow into the subsequent expiration. A striking example of this difference is shown in Fig. 1. For the animal group, the duration of postinspiratory activity in the external intercostals averaged 24.7 ± 12.3% of TI or 10.2 ± 5.1% of T_tot; corresponding values for the parasternal intercostals were 3.9 ± 5.7% of TI and 1.2 ± 2.5% of T_tot (P < 0.05).

This difference in postinspiratory activity between the parasternal and the external intercostal persisted when the forelimb was lifted, as shown by the recordings of a representative animal in Fig. 2. In fact, postinspiratory activity in the external intercostal was prolonged with forelimb lifting in all animals, and, in two of them, the change was so prominent that commonly activity was still present at the onset of the next inspiratory burst (Fig. 3). For the six animals, postinspiratory activity in the external intercostal in this position thus lasted for 45.4 ± 16.3% of TI or 17.7 ± 6.5% of T_tot; these values are significantly greater than those in the resting arm position (P = 0.05). In contrast, postinspiratory activity in the parasternal intercostal remained at 4.2 ±5.8% of TI or 1.1 ± 2.4% of T_tot (not significant).

	DISCUSSION

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Previous studies in anesthetized dogs (3, 5, 6) and cats (13, 15, 21) have shown that the parasternal intercostal muscles and the external intercostals in the rostral interspaces discharge during the inspiratory phase of the breathing cycle, and intracellular recordings from respiratory motoneurons in the thoracic spinal cord in cats have clearly established that these rhythmic inspiratory discharges are related to centrally induced alterations in membrane potential. These alterations are usually referred to as "central respiratory drive potentials" (12, 22). Thus the parasternal and external intercostal -motoneurons depolarize during inspiration to reach the threshold of activation and send efferent impulses to the corresponding muscles. This depolarizing phase is then followed by a hyperpolarizing phase during expiration so that the motoneurons remain below the activation threshold and the muscles are kept silent. Because our animals had synchronous parasternal and external intercostal activity at the onset of inspiration, one may therefore conclude that the parasternal and external intercostal -motoneurons in awake dogs depolarize simultaneously.

However, whereas the activity recorded from the parasternal intercostals ended together with inspiratory flow, external intercostal activity continued well after the cessation of inspiration. Postinspiratory activity in the external intercostals was also greater than postinspiratory activity in the parasternal intercostals when animals lifted the forelimb; in some animals external intercostal activity even persisted throughout expiration. This difference between the parasternal and external intercostals is qualitatively similar to that seen between the costal and crural portions of the diaphragm (10, 11), and this is fully consistent with our hypothesis that muscle spindles are a primary determinant of this phenomenon.

The mechanism by which muscle spindles might induce postinspiratory activity cannot be readily demonstrated at this stage but could be explained on the basis of the respiratory changes in muscle length. Studies in which sonomicrometry was used in anesthetized dogs have shown that the external intercostals in the rostral interspaces and the parasternal intercostals shorten in phase with inspiration (3, 5-7). Thus both muscles become gradually shorter as inspiration proceeds, reach their shortest length at peak inspiration, and then lengthen rather abruptly in early expiration. To the extent that muscle spindles are situated in parallel with the (extrafusal) muscle fibers, this muscle lengthening should induce stretching of the central, sensitive portion of the spindles in early expiration. Therefore, the external intercostal muscle spindles should elicit excitatory postsynaptic potentials in the corresponding -motoneurons (22). As a result, the membrane potential of the motoneurons could be maintained above the activation threshold after the end of inspiration, such that efferent -motor activity to the muscles would persist. On the other hand, because the parasternal intercostals are poorly endowed with spindles, the parasternal -motoneurons should be little, if at all, affected by this mechanism. Consequently, efferent -motor activity to these muscles should cease together with the central respiratory drive potential, i.e., at or near the end of inspiration.

It is traditionally considered that postinspiratory activity causes braking of expiratory airflow and plays an important role in the regulation of end-expiratory lung volume (19). Yet, previous studies of Easton et al. (10, 11) and the present observations indicate that, in awake dogs, activity in the costal diaphragm and the parasternal intercostals terminates with the cessation of inspiratory flow. Because these two sets of muscles have the greatest inspiratory effect in the dog, one may question the functional significance of postinspiratory activity. Furthermore, recent studies in anesthetized dogs have suggested that muscle spindles in the external intercostals have relatively little impact on the mechanical behavior of the respiratory system. Although a high inspiratory-airflow resistance or a single-breath airway occlusion at end expiration induces, through the increased spindle afferent activity, a marked increase in external intercostal inspiratory activity (2, 3), the normal inspiratory cranial displacement of the ribs is usually reversed into an inspiratory caudal displacement and the normal inspiratory muscle shortening is reversed into an inspiratory muscle lengthening (3). Similarly, when weights are attached to the ribs caudally, the cranial rib displacement and intercostal muscle shortening during inspiration are reduced, and this also leads to a reflex, spindle-induced excitation of the external intercostals; yet, denervation of the external intercostals alters the inspiratory rib displacement only moderately (16). Therefore, to the extent that postinspiratory activity would also result from a spindle mechanism, it might be envisaged as an electrical characteristic of spindle-rich inspiratory muscles without any significant mechanical effect during normal, resting breathing.