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Department of Physiology and Pediatrics, Medical College of Wisconsin, Zablocki Veterans Affairs Medical Center, and Department of Physical Therapy, Marquette University, Milwaukee, Wisconsin 53226
Submitted 4 September 2003 ; accepted in final form 2 June 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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30–90 min after bilateral injection. Thereafter, breathing frequency declined, central apneas resulted in arterial hypoxemia (arterial PO2 40 Torr) and hypercapnia (arterial PCO2 60 Torr), and, 11 ± 3 min after the peak tachypnea, respiratory failure was followed by cardiac arrest in three airway-intact goats. However, after the peak tachypnea in four tracheostomized goats, mechanical ventilation was initiated to maintain arterial blood gases at control levels, during which there was no phasic diaphragm or abdominal muscle activity. When briefly removed from the ventilator (90 s), these goats became hypoxemic and hypercapnic. During this time, minimal, passive inspiratory flow resulted from phasic abdominal muscle activity. We estimate that 70% of the neurons within the pre-BötzC area were lesioned in these goats. We conclude that, in the awake state, the pre-BötzC is critical for generating a diaphragm, eupneic respiratory rhythm, and that, in the absence of the pre-BötzC, spontaneous breathing reflects the activity of an expiratory rhythm generator. respiratory rhythm generator; terminal apnea; inspiratory and preinspiratory neurons
Recently, it has been proposed that, within the medulla, there are two respiratory rhythm generators, which are mutually inhibitory and each capable of generating a rhythm under different conditions: one is an inspiratory generator composed of pre-BötzC inspiratory neurons, and the second is an expiratory generator composed of preinspiratory (pre-I) neurons ventrolateral to the rostral Bötzinger complex (4, 8, 14, 17). Supposedly, the pre-BötzC rhythm generator is normally dominant, and it inhibits the rostral pre-I neurons (14), which initiate rhythmic abdominal muscle contraction when pre-BötzC inspiratory neuronal activity is depressed (4, 8).
We recently found in awake goats that 29% bilateral reduction of pre-BötzC area NK1R-expressing neurons resulted in transient periods of coactivation of inspiratory and expiratory muscles (26). One possible explanation for this effect is that the pre-BötzC lesion reduced the inhibition of the rostral pre-I neurons, resulting in simultaneous activation of both rhythm generators.
The present study was designed to gain insight into the role of and/or the relationship between these two rhythm generators in an awake animal. If the pre-BötzC is normally dominant and it elicits an inspiratory muscle rhythm characteristic of eupnea, then large (or total) pre-BötzC area lesions would eliminate phasic diaphragm activity and uncover the activity of the hypothesized expiratory rhythm generator. The primary objective of the present study was to test this hypothesis.
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MATERIALS AND METHODS |
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Experimental design. Because the objective was large (or total) destruction of pre-BötzC area neurons, the small saporin conjugated to substance P (SP-SAP)-induced pre-BötzC area lesions described previously in seven goats (hereafter referred to as group I) (26) were simply expanded with injection of a neurotoxin specific for glutamatergic neurons. These goats were instrumented with electrodes for recording respiratory muscle activity and with microtubules chronically implanted into the pre-BötzC area. Accordingly, for the purposes of this study, ibotenic acid (IA; 10 µl of 50 mM) was injected bilaterally into the pre-BötzC area of awake goats. These goats either died (n = 3) or were euthanized (n = 4) within 6 h of the neurotoxin injection.
Four additional goats were also instrumented with respiratory muscle electrodes and chronically implanted microtubules. In three of these goats (hereafter referred to as group II), one microtubule was implanted into the pre-BötzC on one side with the contralateral microtubule slightly rostral or medial to the pre-BötzC area. These three goats were implanted before the goats of group I; thus the "misplacement" of one microtubule in each goat reflects our search for the proper coordinates for implantation into the pre-BötzC area. In the fourth (control) goat, both microtubules were implanted into nuclei distal to the pre-BötzC. These four goats were studied for 5 h after bilateral IA injection. Eupneic breathing, arterial PCO2 (PaCO2), and CO2 sensitivity were assessed on several days following IA injection (see Ref. 26 for protocol). These goats were killed 7–14 days after IA injection for histological purposes.
Surgical procedures. Animals were anesthetized initially with xylaject-ketaset (1:24) intravenously, and after intubation anesthesia was maintained with 1.5% halothane in 100% O2. Detailed surgical procedures (under sterile conditions) and medication protocol have been described elsewhere (26). Briefly, in an initial surgery, the carotid arteries were relocated to just below the skin, and electrodes were inserted into the diaphragm and three upper airway muscles (thyroarytnoid, posterior cricoidarytnoid, and inferior pharyngeal constrictor). After at least 3 wk, microtubules were chronically implanted bilaterally into the medulla, and in two goats abdominal electromyogram electrodes were implanted. Because of evidence of airway obstruction after IA injection, a tracheostomy was created 3–4 days before the IA injection study (n = 4).
Medication after surgery and during entire period. Following the initial surgery, goats were medicated with ceftiofur sodium (6 mg/kg im) every day to minimize infection. Following craniostomy surgery, dexamethasone was administered to minimize brain swelling (internal jugular, 3 times a day for 7 days, starting dose of 4.0 ml and decreasing by 0.5 ml per day). Antibiotics [ceftiofur sodium (every day) and chloremphenicol (20 mg/kg iv, 3 times a day) for the first 3 days and gentamyacin (6 mg/kg, every day) thereafter] were administered for the duration of the studies.
Procedures and protocols.
To monitor inspiratory flow (
I), a pneumotachograph was attached to the inspired port of a breathing valve. The valve was attached to either a muzzle mask (airway intact) or endotracheal tube (tracheostomized). The pneumotachgraph was connected to a recorder, which was connected to a computer. Diaphragm and (if instrumented) upper airway and abdominal muscle activities were recorded. Arterial blood was sampled for arterial blood-gas determination, and arterial blood pressure (ABP) was measured from a chronically indwelling arterial catheter. Rectal temperature was recorded after each blood sampling.
For all IA studies, breathing, respiratory muscle activities, ABP, and heart rate (HR) were monitored continuously while the subjects breathed room air, and arterial blood was withdrawn at least every 30 min. After a 15-min control period, IA (10 µl, 50 mM) was injected into one microtubule, and 1 h later an identical injection was made into the contralateral microtubule. About 30–90 min later (explained in the RESULTS section), we began to mechanically ventilate the tracheostomized goats to maintain PaCO2 at or near control levels. Mechanical ventilation continued for up to 4 h, but periodically the ventilator was turned off to determine whether the goats were capable of spontaneous breathing.
Brain perfusion, fixation, and histology. On termination of the goat, the brain stem was removed, postperfusion fixed and cryoprotected, frozen, and sectioned (25 µm) for hematoxylin and eosin (H&E) staining (26). Living and dead neurons were counted at 200-µm intervals in a 1.2 x 1.8-mm area just ventral to nucleus ambiguus and medial to spinal trigeminal nucleus, beginning 2.0 mm rostral to the obex, and extending to 4.0 mm rostral to the obex both in a group of unoperated control goats and in the lesioned goats. In addition, we identified dead neurons throughout the area surrounding the microtubule implantation site and the rostral ventral medulla. The appearance of dead neurons delineates the area of the lesion, and the reduction in living neurons in the lesioned goats compared with the unoperated goats provides a quantitative estimate of the lesion extent.
Data analysis.
Pulmonary ventilation (I), tidal volume, breathing frequency (f), ABP, and HR were analyzed on a breath-by-breath basis in 1-min bins. Linear regression analysis was used to calculate ventilatory CO2 sensitivity [change in (
)I/PaCO2; l·min–1·Torr–1], and ANOVA was used to determine statistical significance between mean data values with Bonferroni post hoc test to establish differences when P < 0.05 (by ANOVA).
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RESULTS |
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Injection of IA into the pre-BötzC area of awake goats resulted in a significant (P < 0.001) tachypneic hyperpnea (Table 1, Figs. 1 and 2). In six of seven goats, these increases were evident within 5 min after unilateral injection, and, in all goats, the increases were significantly (P < 0.001) accentuated by IA injection into the contralateral pre-BötzC area. A peak tachypneic hyperpnea was reached 30–90 min after the contralateral injection. The injection of IA also increased phasic and/or tonic diaphragm, abdominal (Figs. 1B and 2B), and upper airway muscle activities (data not shown). Diaphragm and abdominal muscle activities during even the most pronounced tachypnea were in opposite phase with each other (Fig. 2B). Arterial blood gases and pH were not altered by the unilateral IA injection (P > 0.10, Table 1), but at the peak hyperpnea, arterial PO2 (PaO2) and arterial pH were reduced (P < 0.001), and PaCO2 was increased (P < 0.001) above control (Table 1). HR was not changed throughout the period up to and including the peak tachypnea (P > 0.10), but mean ABP was elevated as tachypnea reached a peak (P < 0.001, Table 1). The peak tachypneic hyperpnea was sustained for <5 min, after which breathing decreased and transient apneas were observed (Fig. 1, C–E). In three airway-intact goats, apneic periods increased in duration and eventually ended with small (<25 ml/breath), brief inspirations (Fig. 1, C–E). Within minutes, breathing and respiratory muscle activity ceased and cardiac failure followed (11 ± 2 min after the peak tachypnea, Fig. 1E). Based on restlessness, bulging eyes, and erect ears, the goats appeared mildly distressed during the maximal tachypnea, but the signs of distress decreased as breathing decreased, and there were no signs of distress during the minutes before death.
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I preceding death, suggesting airway obstruction (Fig. 1E). Therefore, subsequent goats were tracheostomized (days before IA injection), and mechanical ventilation was initiated when apneic periods and severe hypoxemia and hypercapnia became apparent (30–90 min after contralateral injection). While ventilated, no phasic diaphragm and abdominal muscle activities were evident, but tonic activities were variable and were accentuated during occasional movement of the entire body. The average PaCO2 and PaO2 during this period were 42.0 ± 1.0 and 70.8 ± 10.2 Torr, respectively. To determine whether the goats were capable of spontaneous breathing and phasic respiratory muscle activity, goats were taken off the ventilator for brief periods (90 s) (Fig. 2, C and D). After 15–30 s, spontaneous, highly irregular I was apparent. Tonic diaphragm and abdominal muscle activities increased with time off the ventilator. In one goat, phasic diaphragm and even greater phasic abdominal muscle activity (Fig. 2C) was present. All phasic muscle activities were synchronous and in opposite phase of I, thus indicating a passive I (35–190 ml/breath). In the other three tracheostomized goats, there was minimal phasic diaphragm activity, which was not related to the minimal I (Fig. 2D). The average PaCO2 and PaO2 after 60–90 s of spontaneous breathing were 56.9 ± 4.6 and 39.7 ± 9.1 Torr, respectively. Thus with no indication of recovery, the goats were euthanized with intravenous injections of buthanasia 5 h after the initial IA injection. Studies on group II goats. Unilateral IA injection into the pre-BötzC area of group II goats (n = 3) increased f for up to 5 h (Fig. 3). These goats were mildly hypercapnic (PaCO2 = 45–55 Torr) and hypoxemic (PaO2 = 30–60 Torr) during these studies (Table 2, Fig. 3). However, the morning following IA injection and for 7–14 days thereafter, f, eupneic arterial blood gases, and CO2 sensitivity did not differ from values obtained before IA injections (P > 0.10).
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Histology. In the group I goats, dead or dying neurons were found bilaterally only within an area 1.5 mm in the rostral/caudal direction from the center of the injection tube tip, no more than 0.5 mm lateral or medial from the edge of the implant tract, and ventrally 1.5–2.0 mm from the end of the microtubule (Fig. 4). In the group II goats, dead neurons were found unilaterally in a pattern similar to group I goats (Fig. 4). In the goat with microtubules implanted into distal nuclei, no dead or dying neurons were found in the pre-BötzC area.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Sites of injection and extent of lesions. In anesthetized cats and rats, injection of the glutamate receptor agonist DL-homocysteic acid into the pre-BötzC area increases tonic and/or phasic phrenic nerve output, whereas injections into other distal or proximal nuclei do not increase respiratory rhythm (1, 15, 22). Because IA is a glutamate receptor agonist, the IA-induced tachypnea and subsequent progression of tachypnea following contralateral IA injection into the pre-BötzC area in all group I goats (n = 7) provide strong physiological evidence that injections were made into (at least part of) the pre-BötzC areas in those goats. In the group II goats, IA induced a tachypnea only when injected into the pre-BötzC area. IA injection did not result in tachypnea when injections were made into areas outside the pre-BötzC (group II contralateral injections and control goat).
We routinely utilize H&E staining to identify dead and dying neurons, which defines the area and extent of the lesion. However, at least 6 h are required after neuronal death for the neuronal changes to occur that permit the stain to distinguish living from dead neurons (3). Therefore, in the goats that died or were euthanized <6 h after IA injections (group I), the H&E stain would not identify the neuronal death caused by IA. However, in the group II goats that were killed 7–14 days after IA injections (n = 3), the neurons killed by IA were identified. Some of the neurons destroyed by IA may have already been phagocytosed; thus quantitation of the dead vs. living neurons may underestimate the extent of the lesion. Accordingly, we counted living neurons in the pre-BötzC area of three groups of goats: unoperated, unilateral IA lesioned goats (group II), and bilateral IA plus SP-SAP goats (group I). We are uncertain of the exact boundaries of the pre-BötzC in goats (26), but a reasonable estimate is 2.6–3.6 mm rostral to the obex, which is the area of cell counts listed below. In group II goats, the pre-BötzC-intact side had the same number of living neurons as the unoperated goats. On the pre-BötzC lesioned side, the number of living neurons was only 44% of the living neurons in the unlesioned side, indicating that 56% of that pre-BötzC was destroyed. In group I goats, the total number of bilaterally counted living neurons in the pre-BötzC area was 83% of the total bilateral living neurons in the unoperated goats (Fig. 5). This decrease likely only reflects neuronal death caused by SP-SAP. If we assume that the subsequent IA injection caused the same degree of neuronal death as in goats that had only an IA injection (56% decrease), then the estimated living neurons in the SP-SAP plus IA-injected goats would be only 30% of living neurons in the unoperated goats. In other words, we estimate that 70% of all neurons within the pre-BötzC area were destroyed in group I goats.
Because dead neurons were found outside the described pre-BötzC area, we cannot be certain that it was only pre-BötzC neuronal death that eliminated diaphragm activity. Others have shown that injections of glutamate receptor agonist in areas adjacent to the pre-BötzC have no or minimal effect on breathing (1, 15, 22); thus destruction of neurons in these areas would not be the likely causes of the elimination or reduction of diaphragm activity observed in our group I goats.
Effects of the neurotoxin injection. IA is a glutamate receptor agonist that is neurotoxic, supposedly by irreversibly binding to glutamate receptors (preferentially N-methyl-D-aspartate) (2), therefore inducing continual cellular excitation/stimulation, resulting in eventual metabolic exhaustion and death. If the binding is to receptors on respiratory neurons, respiratory motor output in an anesthetized animal often will initially increase but subsequently decrease (16). This temporal pattern is usually observed within minutes after injection of a neurotoxin. To our knowledge, the only previous study that followed the temporal pattern of breathing after injection of a neurotoxin in the awake state was after microinjections on the ventrolateral medullary (VLM) surface (5). These previous ejections may have affected parafacial pre-I (17) neurons to mediate the observed tachypnea. However, IA ejection on the VLM surface did not cause any apneic periods, conceivably because, even if VLM neurons were destroyed, the pre-BötzC area remained intact and capable of rhythm generation. In the present study, the prolonged tachypnea after IA injection suggests that there was a prolonged net excitatory effect of the injection that killed the neurons and eliminated phasic diaphragm activity.
Gray et al. (6) demonstrated that >80% bilateral destruction of NK1R-expressing neurons was necessary to affect breathing and blood gases in awake rats. We previously demonstrated that 29% bilateral destruction of NK1R-expressing pre-BötzC area neurons in awake goats had minimal effect on arterial blood gases but caused transient alterations in breathing rhythm and/or pattern (26), and we reported here that 70% destruction of the pre-BötzC eliminates eupneic breathing. Thus our data support the conclusions of Gray et al. that the physiological effects of pre-BötzC area lesions are related to the extent of cell death (6) and that the pre-BötzC is critically important to the generation of an inspiratory and eupneic respiratory rhythm.
Abdominal muscle activity following IA injection.
Janczewski et al. (8) and Mellen et al. (14) have recently demonstrated expression of rhythmic expiratory muscle respiratory-related activity following a µ-opioid-induced depression of pre-BötzC inspiratory neurons. These data provide evidence of a second rhythm generator (expiratory), which is only expressed with depression of the inspiratory rhythm generator. This expiratory rhythm generator is rostral to the pre-BötzC area ventrolateral to the rostral Bötzinger complex and/or in the parafacial region (4, 8, 14, 17). In goats during the tachypneic period following IA injections, rhythmic abdominal muscle activity was clearly present, and it was characteristically 180° out of phase with diaphragm activity. However, 4+ h after IA injection, while the goats were mechanically ventilated, tonic abdominal muscle activity was present, and only when the animal was taken off the ventilator did the activity become rhythmic. This rhythmic abdominal muscle activity was synchronous with diaphragm activity, and all muscle activity was in opposite phase of I, indicating that the inspiration was passive. Thus these data provide further evidence of an expiratory rhythm generator that is expressed when there is a disruption of the more dominant inspiratory rhythm generator.
Gasping.
Spirometry flow recordings following pontile lesions in decerebrate cats are characterized by rapid onset of I and either short inspiratory time or in an irregular flow pattern (12, 13, 24, 25). Although a similar pattern of I was recorded just before terminal apnea in three airway-intact goats following pre-BötzC lesioning (Fig. 1E), this does not provide conclusive evidence of gasping. Additionally, brief cessation of mechanical ventilation after large pre-BötzC area lesions resulted in spontaneous I and respiratory muscle activity that was primarily passive due to expiratory muscle contraction and was clearly not gasplike. These findings may indicate that the expiratory rhythm generator is dominant over the gasping mechanism, that the hypoxemia was not sufficient to induce gasping, and/or that the gasping center was also eliminated. The latter alternative is consistent with the postulate of Lieske et al. (11) that the pre-BötzC is involved in the generation of gasps among other respiratory patterns.
Summary and conclusion. The present findings demonstrate that bilateral stimulation of the pre-BötzC area with IA resulted in a prolonged tachypneic hyperpnea, which eventually subsided, and eupneic respiratory rhythm was eliminated, resulting in respiratory failure in airway-intact goats. However, when terminal apnea was prevented by mechanical ventilation, spontaneous breathing was evident during brief cessation of mechanical ventilation. This breathing was primarily a result of contraction of abdominal expiratory muscles. Thus we conclude that 1) large bilateral pre-BötzC lesions eliminate eupneic respiratory rhythm and diaphragm activity, and 2) after large pre-BötzC area lesions, spontaneous breathing reflects primarily the activity of an expiratory rhythm generator.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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