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Control of apnea in the hibernating ground squirrel Garland, Rhonda J. 1994

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CONTROL OF APNEA IN THE HIBERNATING GROUND SQUIRRELbyRHONDA J. GARLANDB.Sc. (Hons), Acadia University, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFNASTER OF SCIENCEMASTER OF ZOOLOGYinTHE FACULTY OF GRADUATE STUDIES(Department of Zoology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember, 1993© Rhonda J. Garland, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of O-iCThe University of British ColumbiaVancouver, CanadaDate____________DE-6 (2/88)11ABSTRACTDuring hibernation the breathing pattern of the golden-mantled ground squirrel consistsof short episodes of breathing separated by pauses ranging in length from less than a minute togreater than 30 minutes. A breathing pattern as such results in wide fluctuations in the partialpres sure of oxygen (P02) and carbon dioxide (P2) in the blood and lungs; the increase in P02and decrease in P2 during a breathing episode are reversed in the following apnea. Theseoscillations raise questions about the mechanisms involved in the initiation and termination ofbreathing episodes.Accompanying the profound reduction in ventilation is an alteration in the relativesensitivities to hypercapnia and hypoxia. While the ventilatory response to hypercapnia iselevated during hibernation compared to euthermia, the hypoxic ventilatory response (HYR)appears blunted, suggesting that changes in 02 have little or no role in the control of episodicbreathing. Despite an absolute reduction in the arterial oxygen partial pressure (Pao2) thresholdof the HVR, however, a strong correlation between the Pao2 values for the threshold of the HVRand the shoulder of the oxyhemoglobin equilibrium curve (OEC) persists in heterothermic rodentsas body temperature changes. It has been suggested that this may reflect either temperatureinduced changes in the response characteristics of arterial chemoreceptors or an ability to sensechanges in arterial oxygen content (Cao2).Thus, the first series of experiments in this thesis examined the extent to which changingCao2 independent of Pao2 with carbon monoxide hypoxia could contribute to the HVR inheterothermic (golden-mantled ground squirrels) and nonheterothermic (rats) rodents. The HVR111of isocapnic, anaesthetized rodents was assessed during both hypoxic hypoxia, which alters Pao2and Cao2 simultaneously, and carbon monoxide hypoxia, which alters Cao2 independent of Pao2.While both species exhibited ventilatory responses to hypoxic hypoxia and carbon monoxidehypoxia, the HVR of the squirrel was consistently higher than that of the rat. Reductions in Cao2independent of Pao2 could produce only 60% of the full HVR seen with hypoxic hypoxia in bothspecies. Simultaneous changes in Pao2 were necessary to produce the full response. While itseems likely that the results can be explained by the changes in the tissue P02 which would occurat receptor sites under various conditions, such an explanation is not totally supported by otherstudies.So, although the HVR in hibernating animals is blunted, the golden-mantled groundsquirrel can respond to changes in Cao2 independent of Pao2 and thus, changes in 02accompanying the ventilation-apnea cycle cannot be dismissed as playing a role in the controland genesis of episodic breathing. The second series of experiments in this thesis examined thepossible role of fluctuations in 02 and CO2 levels over the ventilation-apnea cycle in initiatingor terminating breathing episodes. Computerized tomography scans of two hibernating animalsindicated that the glottis was closed and that apneic oxygenation could not occur. Analysis ofend-tidal gas composition, indicative of arterial blood gas composition, revealed no clear cutthresholds in gas composition for initiating or terminating episodes of breathing. Over the courseof the breathing episode, however, 02 consumption fell exponentially while CO2 production fellin a linear fashion. The breathing episode terminated at the point where 02 consumptionasymptoted suggesting that the length of the episode was just sufficient to repay the 02 debtwhich accumulated during the preceding period of apnea. The oxidative costs associated withivbreathing episodes in these animals was calculated to be approximately 90% of the totalmetabolic rate during hibernation. This suggests that the metabolic rate is not constant duringhibernation but varies in a cyclic fashion associated with the breathing pattern. It further suggeststhat although blood gas levels play a key role in establishing the total level of ventilation, thecyclic variations in their composition , associated with periods of apnea and eupnea, do not bythemselves initiate or terminate breathing episodes.VTABLE OF CONTENTSAbstract jjTable of Contents vList of Tables viList of Figures viiChapter 1 General Introduction 1Chapter 2 The Ventilatory Response of Rodents to Changes in ArterialOxygen ContentIntroduction 6Materials and Methods 8Results 12Discussion 24Chapter 3 End-Tidal Gas Composition is Not Correlated with EpisodicBreathing in Hibernating Ground SquirrelsIntroduction 31Materials and Methods 33Results 37Discussion 49Chapter 4 General Conclusions 58Bibliography 63viLIST OF TABLESTABLE PAGETable 2.1 The effects of carbon monoxide hypoxia and hypoxic hypoxia 13on selected blood and respiratory variables.Table 3.1 Ventilatory variables for hibernating golden-mantled ground 40squirrels.vuLIST OF FIGURESFIGURE PAGEFigure 2.1 Relationship between Pao2 and minute ventilation, breathing 14frequency and tidal volume in rats and squirrels, expressedas % change from normoxic values, during graded hypoxichypoxia.Figure 2.2 Relationship between Cao2 and minute ventilation, breathing 17frequency and tidal volume in rats and squirrels, expressedas % change from normoxic values, during graded hypoxichypoxia.Figure 2.3 Relationship between Cao2 and minute ventilation, breathing 19frequency and tidal volume in rats and squirrels, expressedas % change from normoxic values, during graded carbonmonoxide hypoxia.Figure 2.4 Relationship between Cao2 and minute ventilation, expressed 22as a % change from normoxia, during graded hypoxic hypoxiaand carbon monoxide hypoxia.Figure 2.5 The effects of high and low extraction at the receptor level 26on the tissue Po2 of rats at different levels of carboxyhemoglobin (5A and 5B) and following sodium cyanatetreatment and anemia (5 C).Figure 3.1 Midsagittal computerized tomography scans of a hibernating 38golden-mantled ground squirrel.Figure 3.2 Traces of airflow and the percentage composition of CO2 42and 02 at the nares of a hibernating ground squirrel duringepisodic breathing.Figure 3.3 Partial pressures of end-tidal 02 and CO2 at the beginning 44and end of breathing episodes in hibernating golden-mantledground squirrels.Figure 3.4 The relationship between average O consumption/breath and 47average CO2 production/breath and position of the breath inthe breathing episode.Figure 3.5 Schematic of the relationship between average 02 55consumption/breath and position of the breath in thebreathing episode.vii’1CHAPTER ONEGENERAL INTRODUCTIONVentilation is regulated primarily to match pulmonary 02 and CO2 exchange to metabolictissue rates. While high metabolic rates generally require continuous ventilation to meet metabolicneeds, the need to breath continuously is often precluded at low metabolic rates. Thus, while thehigh metabolic rates of euthermic mammalian hibernators warrant a continuous pattern ofventilation, upon entrance into hibernation metabolism undergoes a profound reduction in thesespecies and continuous breathing is replaced by one of two basic patterns of intermittentbreathing. Single breaths separated by nonventilatory periods or apneas ranging from 30s - 6mmare exhibited by some species including the marmot, Columbian ground squirrel and 13-linedground squirrel (Endres and Taylor, 1930; Landau and Dowe, 1958; Malan et al., 1973; Milsomand McArthur, 1987). In other species, such as the hedgehog, dormouse, golden-mantled groundsquirrel and the little brown bat, intermittent breathing is manifest as a series of lung ventilations(episodes) separated by periods of apnea ranging from less than a minute to hours (Kristofferssonand Soivio, 1964; Hammel et al., 1968; Pajunen, 1970; Tähti and Soivio, 1975; Steffen andRiedesel, 1982; Milsom and McArthur, 1987; Thomas et al., 1990).Not surprisingly, intermittent ventilation results in wide fluctuations in lung and blood Poand, to a lesser extent, P2 and pH (Musacchia and Voilcert, 1971; Tähti and Soivio, 1975;Steffen and Riedesel, 1982). End-apneic arterial Po2 (Pao2)values as low as 10.5 Torr and arterialP2 (Paco2) values as high as 34.6 Torr have been reported for hibernating hedgehogs (Tähti2and Soivio, 1975). Since these values approximate the thresholds for the hypoxic and hypercapnicventilatory responses of hibernators (Endres and Taylor, 1930; Lyman, 1951; Biörck et al., 1956;Tähti, 1975; McArthur and Milsom, 1991), it is not unreasonable to suggest the end-apneic 02and CO2 influence the length of the apnea and in essence, act as thresholds for initiating abreathing episode. This has been postulated for lower vertebrates which also exhibit arrhythmicbreathing patterns (Burggren and Shelton, 1979; Shelton and Croghan, 1988; West et al., 1989).Since the intermittent breathing patterns exhibited by mammalian hibernators bear a strikingresemblance to those of lower vertebrates, with the exception that the interbreath interval occursat end-inspiration in lower vertebrates and at end-expiration in mammals, a similar mechanismfor initiating and terminating breathing may exist. Although no definitive blood gas thresholdsfor initiating or terminating breathing episodes have been identified, some studies do support theirexistence (Burggren and Shelton, 1979; Shelton and Croghan, 1988; West et a!., 1989). Studiesemploying unidirectional ventilation in lower vertebrates, however, have shown that episodicbreathing persists in the absence of blood gas oscillations (West et a!., 1987; Smatresk and Smits,1991). Thus, the exact role of the fluctuations in Pao2 and Paco2 in the control and genesis ofepisodic breathing in lower vertebrates is uncertain. Whether similar blood gas fluctuationsinitiate or terminate breathing episodes in hibernators is unknown.The levels of intermittent breathing seen in mammalian hibernators represent a 10 to 20fold decrease in overall ventilation compared to the levels of continuous breathing seen ineuthermia. Along with the dramatic changes in levels of ventilation and breathing pattern, therelative contribution of changes in Pao2 and PacojpH as stimuli to ventilation seem to bereversed during hibernation. While euthermic, the hypercapnic threshold of fossorial and semi-3fossorial animals is high at 3-7% CO2 and the sensitivity of these animals to changes in CO2 islow (Boggs et al., 1984). During hibernation, however, the threshold for the hypercapnicventilatory response (HCVR) may be as low as 1-3% CO2 (Endres and Taylor, 1930; Lyman,1951; Biörck et al., 1956; Tähti, 1975; McArthur and Milsom; 1991). In contrast, the stronghypoxic ventilatory response (HVR) seen in euthermic fossorial animals appears blunted duringhibernation. The typical hypoxic threshold of fossorial animals, which ranges from 8-16% 02(Boggs et al., 1984), plummets to as low as 3% during hibernation. In some species, respiratorydepression and death may occur before a ventilatory response to declining levels of inspired 02is initiated (McArthur and Milsom, 1991 a).This apparent reduction in the ventilatory sensitivity to severe hypoxia has led toconclusions that changes in arterial 02 have little role in the control of ventilation duringhibernation (Biörck et al., 1956; Tä.hti, 1975; Tihti et al., 1981). Hematological differencesbetween hibernating and euthermic animals, however, make direct comparisons of the Po2threshold of the HVR misleading. The drop in body temperature characteristic of hibernationinduces a severe left-shift in the oxyhemoglobin equilibrium curve (OEC). As a result, 02 bindingaffinity is elevated and thus, the P02 at which blood is 50% saturated (P50) during hibernationis reduced. The P50 falls from a euthermic value of 36 to 6.5 Torr during hibernation in 13-linedground squirrels (6°C; Musacchia and Voilcert, 1971), from 34 to 8.9 Torr in hedgehogs (5°C;Clausen and Ersiand, 1968), and from 18.8 to 5.8 Torr in golden-mantled ground squirrels (7°C;Magginnis et al., 1989). Consequently, the blood remains fully saturated at very low levels ofP02. McArthur and Milsom (1991a) superimposed the HVR of euthermic and hibernating golden-mantled ground squirrels on their respective OEC and observed that, despite a 13 Torr decrease4in the intraspecific P50 during hibernation, there remained a strong correlation between theinflexion point of the HVR and the shoulder of the OEC. In many species, the Pao2 at whichventilation begins to increase is correlated with the Pa02 of the inflexion point on the OEC wherearterial O content (Cao2) begins to significantly decrease. Consequently, animals with highoxyhemoglobin affinities have lower Pao2 thresholds for the HVR (Van Nice et al., 1980; Boggsand Birchard, 1983; Glass et al., 1983).Such a reduction in the Pao2 threshold of the HVR during hibernation means thatventilation would only increase when the Pao2 falls beyond the point that hemoglobin begins todesaturate. Two explanations have been put forward to account for the tight correlation betweenthe inflexion points of the OEC and the HVR in these species. It has been suggested that eitherthe HVR of these species may be more strongly correlated to changes in Cao2 than Pao2 or thattemperature induced changes in the OEC and HVR have been mediated by natural selection(Wood, 1984).Given this background information, the aim of the present study was two fold. In a firstseries of experiments the relative responses of heterothermic and nonheterothermic rodents tochanges in Cao2 independent of Pao2were examined to ascertain whether the correlation betweenthe inflexion points on the OEC and the HVR with changing body temperature was aconsequence of a unique ability of heterothermic species to sense changes in Cao2.This tests thehypothesis that changes in Cao2 are a better indicator of °2 availability than changes in Pao2 inanimals that undergo wide fluctuations in body temperature and would indicate whether theapparent blunting of the HVR of these species could be a consequence of a Cao2 mediatedresponse. In a second series of experiments the cyclic fluctuations in end tidal P02 and P25levels were examined over many ventilation-apnea cycles. This data was used to address thehypothesis that separate thresholds ofP02 orP2 for initiating or terminating breathing episodesin hibernators exist. This study also examined the hypothesis that 02 uptake during apnea throughan open glottis accounts for a large portion of the 02 requirements of the animal. Although theoccurrence of apneic oxygenation would not effect blood gas thresholds per Se, it wouldinfluence the time required to reach threshold and may account for the sizable variability in thelengths of the apneic periods often seen in episodic breathing patterns.6CHAPTER 2THE VENTILATORY RESPONSE OF RODENTS (RATS AND GROUND SQUIRRELS)TO CHANGES IN ARTERIAL OXYGEN CONTENTINTRODUCTIONExposure to reduced ambient 02 concentrations decreases both arterial oxygen content(Ca02) and partial pressure (Pao2). In many species the Pao2 at which ventilation begins toincrease is correlated with the Pa02 of the inflection point on the oxygen equilibrium curve(OEC) where Cao2 begins to decrease significantly. It is generally assumed that the stimulus forarterial chemoreceptors arises from the change in Pao2. Although the correlation between thethreshold of the hypoxic ventilatory response (HVR) and the inflection point on the OEC issuggestive of the presence of chemoreceptors responsive to changes in Cao2,most authors haveconcluded that this correlation is a consequence of natural selection acting on the Pao2 thresholdof the HVR in a manner which optimally protects arterial oxygen saturation (Van Nice et al.,1980). Birchard and Tenney (1986) directly investigated the correlation between Pao2,Cao2 andthe HVR by experimentally altering the intraspecific P02 at which blood is 50% saturated (P50)in rats by infusion of sodium cyanate. These authors found that, despite a left-shifted OECaccompanied by a 10 Torr decrease in the intraspecific P50, the Pao2 threshold of the HVR wasunchanged, supporting the idea that the HVR is mediated solely by changes in Pao2.In contrast to the findings of Birchard and Tenney (1986), however, the relationship7between the threshold of the HVR and the inflection point of the OEC is retained in species thatexperience natural fluctuations in P50. As body temperature falls in ectotherms (Glass et al.,1983) and hibernating endotherms (McArthur and Milsom, 1991a) the hypoxic threshold shiftsto increasingly lower values of Pao2, the values at which arterial blood begins to desaturate. Itis possible that temperature-induced changes in the response characteristics of arterialchemoreceptors could account for this variation in the hypoxic threshold. It is also possible thatthese species possess chemoreceptors capable of responding to changes in Cao2. It has beensuggested that for animals that undergo wide fluctuations in body temperature and exhibit a leftshifted OEC accompanied by an intraspecific decrease in P50, Cao2 would be a better indicatorof the oxygen status of the blood than would Pao2 (Wood, 1984).Based on these observations, the present study was undertaken to test the hypothesis thatheterothennic rodents do respond to changes in Cao alone. To this end, it examined thecomparative vendlatory responses to concurrent decreases in Pao2 and Cao2 as well as todecreases in Cao2 alone, of rodent species which can (golden-mantled ground squirrels) andcannot (rats) hibernate.8MATERIALS AND METHODSAdult Wistar rats (Rattus norvegicus; mean weight 247.8 g ± 6.6 g) and golden-mantledground squirrels (Spermophilus lateralis; mean weight 212.4 g ± 8.8 g) of either sex wereobtained from a commercial supplier. The animals were housed in a controlled-environmentchamber at an ambient temperature of 20°C ± 1°C under a 12L:12D photoperiod and providedwith food and water ad libitum.Surgical proceduresBoth species were initially induced into a light plane of anaesthesia with 3.5% halothanein air. Intraperitoneal injections of sodium pentobarbitol (rats: 6.5 mg/100 g; squirrels: 9.5mg/100 g) and atropine sulfate (0.35 mgIlOO g) were then administered to maintain anaesthesiaand minimize mucus secretion, respectively. Following exposure of the trachea by bluntdissection, a small incision was made between two cartilaginous rings and a 3 cm length ofpolyethylene tubing (PE-240) was inserted approximately 1 cm into the trachea and secured inplace. Both femoral arteries and one femoral vein were then exposed and cannulated withpolyethylene tubing (PE-50) filled with heparinized saline. Body temperature was monitored witha rectal thermistor and servo-controlled at 37°C with an infrared heat lamp.Experimental proceduresFollowing the surgery, the animals were placed supine in a 6 1 plexiglass chamber withports on either end to allow gas flow through the chamber. A small pneumotachograph was9attached to the tracheotomy tube and the differential pressure changes arising from air flowduring ventilation were monitored with a differential pressure transducer (Validyne modelDP1O3- 18). This signal was amplified and electronically integrated (Gould Universal Amplifierand Integrating Amplifier) to give tidal volume. The system was calibrated by injecting knownvolumes of air into the pneumotachograph. One arterial cannula was connected to a pressuretransducer to monitor arterial blood pressure. The other arterial cannula was connected in seriesto the venous cannula via 02, CO2 and pH microelectrodes. Continuous movement of arterialblood through the extracorporeal loop and back into the animal through the venous cannula wasensured by a peristaltic pump (Gilson) operating at a rate of 1 mi/mm. A constant plane ofanaesthesia, indicated by a weak limb-withdrawal reflex, was maintained throughout theexperimental procedure using supplements of sodium pentobarbitol (1.0-3.5 mg/30 mm).Analytical proceduresArterial P02,P2 and pH were continuously measured with miniature O, CO2 and pHelectrodes (Microelectrodes, Inc.) and displayed on a Radiometer PHM-73 pH/blood gas monitor.The P2 electrode was calibrated using water equilibrated with pre-analyzed gas mixtures(Medigas). The calibration of the P02 electrode utilized water equilibrated with pre-analyzed gasmixtures as well as 5mvI sodium bisulfate while precision buffers (Radiometer) were used tocalibrate the pH electrode. All calibrations were done at 37°C with all solutions passing throughthe electrodes at a rate of 1 mI/mm. Measurements of total Cao2 were made on 20 pl samplesof blood according to the method of Tucker (1967) using a Radiometer P02 electrode in a sealedchamber at 37°C. The gas mixtures administered to the animals were obtained by mixing air with1002, N2, CO2or CO with flow meters. Prior to reaching the experimental chamber, the gas mixturewas hydrated by bubbling it through a flask of water. The experimental gas mixtures were alsomonitored continuously using a Beckman OM-1 1 02 analyzer and LB-2 CO2 analyzer calibratedwith pre-analyzed gas mixtures. The differential pressure, tidal volume, Pao2Paco2and pHa andarterial blood pressure were all displayed continuously on a chart recorder (Gould).Experimental protocolThe animals were maintained under normoxic normocapnic conditions until blood gas andrespiratory variables stabilized. For each species, the animals were divided into two groups. Thefirst group was made progressively hypoxic by decreasing the fraction of inspired 02 (Fi02) inthe air through the addition of N2 (hypoxic hypoxia). In the second group hypoxia was inducedthrough the addition of carbon monoxide to the air (CO-hypoxia). In both groups the animalswere maintained isocapnic by the addition or removal of CO2 from the gas mixture. Animalsexposed to CO-hypoxia were maintained isoxic through alterations of the N2 concentration in thegas mixture. At each progressive level of graded hypoxia sufficient time was allowed for allvariables to reach new stable levels. At that time approximately 30 jil of blood was collected forthe determination of hematocrit and oxygen content. The level of hypoxia was increased until the02 saturation of arterial blood decreased to approximately 50%.Data analysisApproximately 30 s of breathing trace was recorded at high speed (5mm/sec) at each level11of hypoxia. Breathing frequency (fR) was determined for each 10-s interval, tidal volume (VT)was measured for fifteen consecutive breaths and minute ventilation (‘[E) was calculated as theproduct of the mean fR and the mean VT.Inter- and intraspecific comparisons between treatments were performed using two wayANOVAs and Tukey post hoc tests. Intraspecific comparisons within each treatment utilizedpaired t-tests. The fiducial limit of significance was set at P <0.05.12RESULTSNormoxiaThe mean values of blood gas and respiratory variables for rats and squirrels breathingair before exposure to either hypoxic hypoxia or CO-hypoxia are summarized in Table 2.1. Therewere no significant intraspecific differences between groups in any of the variables. Interspecificcomparisons revealed that fR and /E of rats were consistently higher than those of squirrels inboth groups.Hypoxic hypoxiaReducing the Fl02 in the experimental gas mixture resulted in a decrease in Pao2 from 63to 23 Torr and from 63 to 29 Torr in the squirrel and rat, respectively. Concurrent reductions inCao2 from 27.0 to 13.5 ml02J100 ml in the squirrel and 24.7 to 15.8 ml 02J100 ml in the rat(Table 2.1) confirmed that comparable levels of hypoxemia were produced in both species. Theeffects of changing both Pa02 and Ca02 on ventilation in both rodent species are shown in Fig.2.1. The relationships between 1E, fR and VT and Pao2 are all nonlinear. The mean rose fromthe nomioxic value of 35.3 to 253.7 ml/min100 g at the most severe level of hypoxia in thesquirrels and from 58.3 to 178.7 ml/min100 g in the rats, due to significant increases in fR andVT in both species (Table 2.1). The relative magnitude of the ventilatory response betweenspecies differed; ‘1E increased by 10-fold in the squirrels but only 6-fold in the rats (Fig. 2.1).This difference was a consequence of the lower resting ‘E of the squirrel since the absolutechanges in VE were not significantly different between species. When expressed as a13Table 2.1. The effects of carbon monoxide hypoxia and hypoxic hypoxia on selected blood andrespiratory variables. The values are expressed as means ± S.E.M. Hypoxic values were obtainedat the most severe level of hypoxia (50% drop in Cao2) in both groups. • indicates a valuesignificantly different from normoxia in same species with same treatment; ° significantlydifferent from normoxia value of squirrels with same treatment; + significantly different betweennormoxic and hypoxic values within a species between treatments (P < 0.05).HYPOXIC HYPOXIA CARBON MONOXIDEHYPOXIASQUIRREL RAT SQUIRREL RAT(N=6) (N=8) (N=6) (N=6)pHa normoxia 7.46 ± 0.02 7.49 ± 0.02 7.43 ± 0.02 7.46 ± 0.08hypoxia 7.44 ± 0.02 7.51 ± 0.03 7.41 ± 0.02 7.41 ± 0.02Paco2 normoxia 46.4 ± 3.5 41.4 ± 3.6 53.3 ± 4.2 43.5 ± 4.2(Torr) hypoxia 43.2 ± 4.5 .40.4 ± 3.5 51.5 ± 3.9 43.7 ± 4.0Pao2 normoxia 62.8 ± 4.5 63.2 ± 4.5 59.6 ± 8.2+ . 58.9 ± 4.6+(Ton) hypoxia 22.5 ± l.3 29.3 ± 24’ 62.6 ± 8.7 57.8 ± 4.9Cao2 normoxia 27.0 ± 0.6 24.7 ± 1.3 24.2 ± 2.2 27.7 ± 0.9(ml 02f100 ml) hypoxia 13.5 ± 08’ 15.8 ± 10’ 12.0 ± 1.0 14.6 ± 2.0’hematocrit normoxia 45.3 ± 0.9 47.6 ± 1.6 41.0 ± 2.9 46.0 ± 1.3(%) hypoxia 41.0 ± 2.2 45.4 ± 1.2 37.8 ± 2.3 40.5 ± 1.5’blood pressure normoxia 130.5 ± 10.2 153.8 ± 7.6 126.0 ± 8.0 160.8 ± 11.6(mm H20) hypoxia 134.2 ± 13.6 134.4 ± 10.5 127.0 ± 10.8 146.7 ± 9.5fR normoxia 42.0 ± 9.6 89.3 ± 6.5° 49.0 ± 9.4 95.0 ± 9.10(breaths/mm) hypoxia 115.5 ± 35.8’ 153.6 ± 13.9’ 100.5 ± 10.6’ 157.5 ± 9.1’VT normoxia 0.96 ± 0.15 0.65 ± 0.13 0.66 ± 0.13 0.56 ± 0.04(m]/lOOg) hypoxia 2.47 ± 0.41’ 1.15 ± 0.17’ 1.62 ± 0.44 0.94 ± 0.04’VE normoxia 35.3 ± 4.6 58.3 ± 4.9° 27.9 ± 3.4 54.2 ± 7.9°(zni/minlOOg) hypoxia 253.7 ± 57.2’ 178.7 ± 30.9 146.5 i 25.6’ 148.6± 13.3’14Figure 2.1. Relationship between Pa02 and minute ventilation, breathing frequency and tidalvolume, expressed as % change from normoxic values, during graded hypoxic hypoxia. (Rats=•; Squirrels= D)I....•.‘ciaui•000tjci0 0•F-00ciU0--150050100Pa(mmHg)02Pa0(mmHg)U Ua..0.U’ ‘UaI.11000- 800 600 400 200 0600400200 06004002000501000•a.i150JI16function of Cao2, the ventilatory response curves for hypoxic hypoxia were linear for both species(rat: ‘E = -6.38 Cao2 + 642; squirrel: ‘E = -11.9 Cao2 + 1273) over the experimental rangeof Cao2 (Fig. 2.2). In both species, pH, Paco2Hct and blood pressure were maintained relativelyconstant throughout the experimental series (Table 2.1).CO-hypoxiaDuring exposure to CO, the Pao2 did not deviate significantly from the normoxic levelin either species (Table 2.1). Similar drops in Cao2were produced in both species (squirrel: from24.2 to 12.0 ml 02/100 ml; rat: from 27.7 to 14.6 ml 02/100 ml). The effects of lowering Cao2,independent of Pao2, on the ventilation of squirrels and rats are presented in Fig. 2.3. Therelationships between fR, VT and and Cao2 were linear in both species (rat: ‘E = -3.55 Cao2+ 360; squirrel: ‘1E = -7.28 Cao2 + 784). The 12 ml 02/100 ml drop in Cao in squirrels wasaccompanied by a 5-fold increase in ‘E from 27.9 to 146.5 m1/min100 g achieved through asignificant increase in fR. In the rat, a 13 ml 02/100 ml fall in Cao2 was accompanied bysignificant increases in both fR and VT resulting in VE rising from 54.2 to 148.6 ml/minlOOg. Again, the absolute changes in ‘E were not significantly different between species but whenexpressed in relative terms, the squirrels appeared to undergo a proportionately greater increasein yE (Fig. 2.3). In both species, pH, Paco2 Hct and blood pressure were maintained relativelyconstant throughout the experimental series (Table 2.1).ComparisonsA summary of the ventilatory responses for both species to hypoxic hypoxia and CO17Figure 2.2. Relationship between Cao2 and minute ventilation, breathing frequency and tidalvolume, expressed as % change from normoxic values, during graded hypoxic hypoxia. (Rats=•; Squirrels= I:)Ca0(%control)0 0 0 0Ca0(%control)ciciIci1000 800600400200 0cici...cicicicici600400200 0600400200 0ci...ci..I.ci..•cici.•..cicici040ci cicici•ci•ci: ...80120040801200019Figure 2.3. Relationship between Cao2 and minute ventilation, breathing frequency and tidalvolume, expressed as % change from normoxic values, during graded carbon monoxide hypoxia.(Rats= •; Squirrels= D)1000600400800200°o600o,.I0600400°•_•J400•••.•••°200•040°10‘10Ca0(%control)Ca0(%control)C21hypoxia, expressed as a function of Cao2, is shown in Fig. 2.4. For clarity, this figure presentsthe least squares linear regression curves for the individual data points seen in Fig. 2.2 and Fig.2.3 for each treatment and species. While the slopes of the regression lines describing theventilatory responses to hypoxic hypoxia are significantly different between species, the slopesof the ventilatory responses to CO-hypoxia are not (Fig. 2.4). Comparisons between the slopesdescribing the two forms of hypoxia revealed no intraspecific differences. The effect of alteringPao2 and Cao2 simultaneously consistently exceeded the effect of altering Cao2 alone regardlessof the species. The inset in Fig. 2.4 expresses the VE during CO exposure as a ratio of the VEduring N2 exposure. In both species, changing Cao2 alone could only produce 60% of theventilatory response that occurred when both Pao2 and Cao2 were altered together.22Figure 2.4. Relationship between Cao2 and minute ventilation, expressed as % change fromnormoxia, during graded hypoxic hypoxia (JIB) and carbon monoxide hypoxia (COH). Theoriginal data points from figures 2 and 3 have been replaced with least squares linear regressioncurves for the rat (- - -) and the squirrel ( ). The inset depicts the ratio of the ventilatoryresponse to carbon monoxide hypoxia (COH ‘1E) to that of hypoxic hypoxia (HH ‘1E) from 40-90% of the control Cao2 range.r.t1.>HH0 C)1.0 0.8 0.6 0.41000 800600400200C CCOil0.2 OOL//III7/ 406080100Ca0(%control)COHni____ // 406080Ca2100(%control)24DISCUSSIONThe normoxic respiratory variables in Table 2.1 are similar to those found previously inrats (Cragg and Drysdale, 1983) and squirrels (Davies and Schadt, 1989) under pentobarbitolanaesthesia. Slight differences in values for fR and/or VT due to the depressant effects ofbarbiturate anaesthetic are apparent in both species when the values of the present report arecompared to those recorded for conscious animals (rats: Walker et al., 1985; Frappell et al.,1992; squirrels: Davies and Schadt, 1989; McArthur and Milsom, 1991a). The significantlyhigher resting ventilation of the rats compared to the squirrels is consistent with the relativehypoventilation of semifossorial species (Boggs et al., 1984).The ventilatory responses to hypoxic hypoxia presented in Fig. 2.1 are similar to thosereported in the rat (Cragg and Drysdale, 1983) and squirrel (McArthur and Milsom, 1991a). Sincethe animals in this study were maintained isocapnic, the ventilatory responses shown hererepresent true hypoxic ventilatory responses. The slight left-shift in the hypoxic ventilatoryresponse curve of the squirrel compared to the rat may be attributed to the higher Hb02 affinityof squirrel blood (P50=18.1 Torr; Magginnis et al., 1993) compared to that of rat blood (P50=35.5Torr; Birchard and Tenney, 1986) which would conform to reports that lower Pao2 thresholdsexist for the HVR of animals with lower P50s (Van Nice et al., 1980).During CO-hypoxia, both species showed a strong ventilatory response. Although we arenot aware of any previous accounts of the ventilatory responses of rodents to CO inhalation,similar observations have been reported for cats and goats (Santiago and Edelman, 1976;Chapman et al., 1982; Gautier and Bonora, 1983; Gautier et al., 1990). The average relative25increase in ventilation for both squirrels (52 1%) and rats (275%) during CO inhalation exceededthe values reported previously (216% increase in conscious goats: Santiago and Edelman, 1976;130% increase in anaesthetized cats: Gautier and Bonora, 1983; 130% increase in conscious cats:Gautier et al., 1990). Since the average change in Cao2 reported for these studies is comparableto that produced in the present study, the difference cannot be explained by the level of COadministered and it seems most likely that the difference is an effect of species.The relative ventilatory response of the squirrel was consistently greater than that of therat regardless of the form of hypoxic exposure. When the ventilatory responses to both forms ofhypoxia were expressed as a function of Cao2 (Fig. 2.4), it was clear that the ventilatory responseto hypoxic hypoxia exceeded that to CO-hypoxia in both species; while 60% of the fullventilatory response could be reached by changing Ca02 alone in both species (Fig. 2.4),concurrent changes in Pao2 were required to elicit the full response. This does not necessarilyimply that changes in Pao2 alone account for only 40% of the HVR. Since only Cao2 wasexamined independently, speculation remains as to whether the interaction between the twopotential stimuli is additive, multiplicative or redundant. Whatever the case, the data do suggestthat both species can respond to CO-induced changes in Cao2 alone.Despite the obvious distinction between hypoxic hypoxia and CO-hypoxia at the arteriallevel, it has been argued that the ventilatory responses to both are actually mediated throughchanges in tissue P02 (Mills and Edwards, 1968). Carbon monoxide alters the shape and positionof the OEC and has the potential to change tissue P02 at any given Pao2 (Haldane, 1912). Theextent to which tissue P02 is altered will depend not only on the level of carboxyhemoglobin(HbCO) in the blood but also on the degree of 02 extraction at the receptor level. In Fig. 2.5A26Figure 2.5. OEC for rat blood. Fig. 5A and 5B show OEC in the presence of different amountsof carboxyhemoglobin. The curves 1, 2 and 3 correspond to HbCO concentrations of 0, 20 and50%, respectively. In A, the effects of low (0) and high (•) extraction at the receptor level ontissue P02 are shown for points with the same Pao2 but different levels of Cao2. In B, the effectsof low and high extraction at the receptor level on tissue P02 are shown for points with the sameCao2 but different levels of Pa02. In C, OEC for control rats, sodium cyanate treated rats(redrawn from Birchard and Tenney, 1986) and rats with an anemia-induced 50% reduction inCao (calculated).27A2520151050B2510U50C25201510500 20 40 60 80 100Pa0 (mmHg)1231341528and 2.5B, OECs for rat blood containing 0-50% HbCO have been calculated using the methodsof Roughton and Darling (1944) and the standard curve of Birchard and Tenney (1986); Fig.2.5C contains the OEC for sodium cyanate treated rats (Birchard and Tenney, 1986) and acalculated OEC for the rat during anemia (50% reduction in hematocrit), two conditions whichalso alter Cao2 at constant Pao2. Assuming metabolism and tissue pH remain constant, the tissueP02 levels at both high and low levels of 02 extraction can be estimated for blood having thesame level of Pao2 but different levels of Ca2 (Fig. 2.5A) as well as for blood having the samelevel of Cao2 but different levels of Pao2 (Fig. 2.5B). The value for high extraction (25%) isbased on whole body arterio-venous Cao2 differences recorded in rats (Burlington and Milsom,unpublished data) while the low extraction value (5%) is based on reports of carotid body 02consumption (Purves, 1969). Based on the ventilatory responses recorded in the present study,the ventilation at any given combination of Pacj2 and Cao2can be estimated. From these estimatesinferences as to whether CO-induced changes in Cao2 can be acting solely through changes inP02 at the tissue level can be made.If extraction of 02 at the tissue level is either high or low, for the same Pao2, tissue Pao2would be lower during CO-hypoxia than during normoxia (HbCO=0%) (Fig. 2.5A). The higherventilation recorded during CO-hypoxia compared to normoxia (Fig. 2.2), therefore, is consistentwith changes in tissue P02 being the stimulus for the HVR. In contrast, the reduced HVR withCO-hypoxia compared to hypoxic hypoxia (Fig. 2.4; Table 2.1) could only be explained bydifferences in tissue P02 levels if extraction was low. For the same Cao2, high extraction wouldresult in a lower tissue P02 during CO-hypoxia than during hypoxic hypoxia (Fig. 2.5B). As aconsequence, ventilation should increase more with CO-hypoxia, which was not the case. Studies29that have examined the effects of experimentally-induced changes in the intraspecific P50 on theHVR or the effects of anemia on the HVR, however, are not totally consistent with tissue P02acting as the sole stimulus for the HVR with either high or low levels of tissue 02 extraction.The Pao2 threshold of the HVR remains unchanged in sodium cyanate treated rats exposed tohypoxic hypoxia (Birchard and Tenney, 1986) despite the fact that for any given Pa02, tissue P02would always have been lower in sodium cyanate treated rats compared to untreated animals(Fig. 2.5C). Furthermore, if tissue P02 were acting as the stimulus for the HVR, the ventilationof anemic animals with reduced Cao2 would always be expected to exceed that of non-anemicanimals since tissue Po2 will always be lower in anemic than non-anemic animals (Fig. 2.5C).The ventilatory responses of animals to anemia, however, have been somewhat equivocal. WhileCropp (1970) reported that the ventilatory response to 10% 02 was greater in anemic dogs thanit was in non-anemic dogs and a similar observation was made in goats during transient hypoxia(inhalation of several breaths of N2) no difference was found between anemic and non-anemicgoats during steady state hypoxia (12% 02 for 7 mm) (Santiago et a!., 1975). Thus, althoughmuch of the data is consistent with the hypothesis that the HVR is mediated solely throughchanges in tissue P02 if extraction at the receptor site is low, not all data fits this picture and itremains possible that some species can sense changes in Cao2per Se.The exact mechanism underlying the ventilatory response to CO-hypoxia remains obscure.It is generally accepted that the HVR is mediated by the carotid body chemoreceptors sensingchanges in Pao2. The question then arises, if the HVR in rodents is not due solely to changes intissue P02 can carotid bodies in ground squirrels and rats sense changes in Cao2 or is the HVRto changing Cao2mediated by some other receptor group? Both are possible. Many carotid body30denervated animals retain or regain an HVR (Bisgard et a!. , 1980; Smith and Mills, 1980;Maskrey et a!., 1981; Webb and Milsom, 1990), suggesting that a non-carotid bodychemoreceptor is capable of eliciting an HVR in these species. Furthermore, the HVR of carotidbody denervated animals resembles the hypoxic tachypnea characteristic of CO inhalation atconstant Pao2 (Santiago and Edelman, 1976; Chapman et a!., 1982; Gautier and Bonora, 1983;Gautier et al., 1990). That is, the HVR is reduced in magnitude and due primarily to changes inbreathing frequency. This is certainly true of the golden-mantled ground squirrel (Webb andMilsom, 1990). It follows, therefore, that some aspect of 02 delivery other than Pao2 may beresponsible for the HVR, acting at non-carotid body chemoreceptors, during CO-hypoxia in theground squirrels and rats. This does not rule out the possibility that the carotid bodies in thesespecies can sense changes in Cao2.In conclusion, it is clear that CO-induced changes in Cao2 independent of Pao2 are capableof eliciting a ventilatory response in both heterothermic and nonheterothermic rodent species.Although this study did not address mechanism per Se, it appears that either tissue P02 could beacting as the stimulus for the HVR during CO-hypoxia or that these animals can sense changesin 02 content per Se. Although it is easier to ascribe adaptive significance to this feature inheterothermic rodents, the data collected in the present study suggests it may be a commonfeature of all rodent species.31CHAPTER 3END-TIDAL GAS COMPOSITION IS NOT CORRELATED WITHEPISODIC BREATHING IN HIBERNATING GROUND SQUIRRELSINTRODUCTIONUpon entrance into hibernation, the breathing pattern of the golden-mantled groundsquirrel, Spermophilus lateralis, is manifest as episodes of lung ventilations separated byrelatively long nonventilatory periods or apneas (Twente and Twente, 1964; Steffen andRiedesel, 1982). A host of other mammals, including the hedgehog, dormouse and littlebrown bat, also exhibit this breathing pattern during hibernation (Kristoffersson and Soivio,1964; Hammel et al., 1968; Pajunen, 1970; Tähti and Soivio, 1975; Thomas et al., 1990).This intermittent ventilation results in wide oscillations in lung and blood P02 and Pc2(Musacchia and Volkert, 1971; Tähti and Soivio, 1975; Steffen and Riedesel, 1982); during abreathing episode, lung and blood P02 increase and Pc02 decreases while in the followingapnea, the reverse occurs. While Tähti (1975) originally suggested that the length of theapnea was most likely regulated by arterial others have postulated that the cyclicfluctuations in P02 and/or P2 acting via chemoreceptor feedback play a role in the controland genesis of episodic breathing (see Milsom, 1988; Shelton and Croghan, 1988, for reviewson arrhythmic breathing). The exact role played by the blood gas fluctuations remainsunknown and whether separate thresholds of P02 or P2 for initiating and terminatingepisodic breathing in hibernators exist remains unclear. The present study was designed to test32the hypothesis that such thresholds do exist and are instrumental in the genesis of breathingepisodes in the golden-mantled ground squirrel.Any relationship between metabolic rate, blood gas changes and the length of theapneic pauses will be modified if gas exchange occurs during apnea. An open glottis wouldallow oxygen to diffuse down the respiratory tract during a nonventilatory period, assisted bybulk flow of air, if the gas exchange ratio were less than one; a phenomenon called apneicoxygenation (Malan, 1982). In hibernating animals, with such low metabolic rates and longperiods of apnea, this could contribute a significant amount to the resting need for gasexchange. Based on observations of pressure fluctuations synchronous with the heart beats onplethysmograph recordings, Malan (1982) concluded that the glottis did remain open duringapnea in hibernating hedgehogs. Direct attempts to measure oxygen uptake in torpid batshave been equivocal. While some studies have reported apneic oxygenation possible onlythrough an open glottis (Szewczak and Jackson, 1992), some studies have not (Thomas et a!.,1990).Although such a phenomenon would not necessarily affect any blood gas thresholdsfor initiating or terminating breathing episodes, it would affect the rate at which these werereached and thus, play a role in the control of the breathing pattern. This would contribute tothe large variability in the length of apneic periods often seen with these breathing patterns.Given this, the present study also examined the presence of apneic oxygenation in thisspecies.33MATERIALS AND METHODSExperimental Animals. Golden-mantled ground squirrels (Spermophilus lateralis) of eithersex were obtained from a commercial supplier. The animals were housed in a controlled-environment chamber at an ambient temperature of 20 ± 1°C under a 12L: 12D photoperiodfrom June to October.Animals were induced into hibernation in early November by gradually reducing theambient temperature to 5 ± 1°C and changing the photoperiod to 2L:22D. Theseenvironmental conditions were maintained throughout the winter months, with food and waterprovided ad libitum throughout the entire year. During the period of induction intohibernation, which ranged from 2 to 4 weeks, the animals were not handled or disturbed. Theanimals periodically aroused from hibernation as has been reported previously for this, andother species (Pengelley and Fisher, 1961; Twente and Twente, 1978). To reduce theincidence of premature arousals during experiments, following this induction period theanimals were handled and fitted with a face mask (see below) 3-4 times each week forseveral weeks prior to experimentation. Animals were not used for experiments unless theyhad been in hibernation for at least 48 h.Computerized Tomography Scans. Silver disc, surface electrodes (Type E5SH, Grass) weretaped to small shaven spots on the midthoracic region of the back of two animalsapproximately 6 hours prior to the scanning procedure. Then, the animals were removed fromthe environment chamber and transported to the University Hospital in an ice chest packed34with ice. The animals were subsequently removed from the ice chest, placed in the scan unitand the electrodes were connected to an impedance converter and respiratory induced changesin impedance were monitored on a chart recorder. Each animal had a computerizedtomography (CT) scan (TCT 900S Series, Toshiba, Japan) made of the entire body during anonventilatory period. This procedure only took minutes and the animals remained in deephibernation throughout it as judged from the recorded breathing patterns and the animals’behavior.Measurements of Ventilation/End-Tidal Gases. A small face mask was made using the barrelof a 50 cc syringe molded with plasticine to fit the snout of the animal. The mask was placedover the snout of the animal and secured around the neck with a rubber cuff to create a tightseal. A small pneumotachograph was attached to the open end of the face mask. The totaldead space of the mask-pneumotachograph system was approximately 0.3 ml. The differentialpressure changes arising from air flow during ventilation were monitored with a differentialpressure transducer (Validyne model DP1O3-18). This signal was amplified and electronicallyintegrated (Gould Universal Amplifier and Integrating Amplifier) to give tidal volume. Thesystem was calibrated by injecting known volumes of air into the pneumotachograph. Airflow and tidal volume were monitored continuously on a chart recorder (Gould).The pneumotachograph was also equipped with a small outlet port 5 mm distal to theface mask (7 mm from the nares). A mass spectrometer (Centronic 200 MGA), connected tothe outlet port via a length of polyethylene tubing, continuously measured 02 and CO2concentrations. The sample rate of the mass spectrometer was adjusted (20-60 mI/mm) to35ensure the accuracy of end-tidal gas measurements. The output of the mass spectrometer wasmonitored continually and displayed on a chart recorder (Gould). The mass spectrometer wascalibrated using pre-analyzed gas mixtures (Medigas).Once instrumented, the animal was transferred to an open plexiglass chamber (14 X23 X 12 cm) housed in a 250 1 refrigerator maintained at 5 ± 1°C with a continuous supplyof fresh air. The animal was left undisturbed until the episodic breathing pattern of deephibernation was well established. Once deep hibernation was assured, the breathing pattern ofthe animal was monitored closely and the speed of the chart recorder increased to 5 mm/secjust prior to or at the onset of each ventilatory period. Breathing traces for each animal wererecorded for 4-8 hrs.Data Analysis. Breathing traces were analyzed for overall breathing frequency (flj, thenumber of breaths in each episode, the duration of the episode or ventilatory period (TvP), theduration of the nonventilatory period between episodes (TNvP) and the length of the pausebetween breaths in an episode (TP). Tidal volume (VT), end-tidal O and CO2 composition(% of expired gas) and the duration of inspiration (TI) and expiration (TE) for each individualbreath were also recorded. The change in the 02 and CO2 concentrations of the expired gaswere measured at 0.2 s intervals for each breath and plotted against corresponding incrementsof expiratory volume. The area under these curves was measured to give breath-by-breathvalues for oxygen consumption (c02) and carbon dioxide production (‘2). For each of 5animals, 4 breathing episodes ranging in size from 8-27 breaths were analyzed breath bybreath for and ‘co2. Ventilatory variables were averaged to give mean values for eachsquirrel; these were then averaged to give a grand mean.Statistical analysis of the Vo2 and Vco2 profiles was performed using one-wayANOVAs. The level of significance was set at P< 0.05.3637RESULTSComputerized Tomography Scans. Figure 3.1A depicts a golden-mantled ground squirrel in ahibernating position. Panel 3.1B shows a midsagittal CT scan of a hibernating ground squirrelin this same position. On the left is a colored gradation scale ranging from white to black. Inthe picture, white represents the most dense tissue and black the least. Thus, the sinuscavities, trachea and lungs contain air and are clearly recognized as the least dense areas. Thescan was taken during the nonventilatory period marked on the breathing trace in the lowerpanel (3.1D) of the figure. The CT scan in panel 3.1C is an enlargement of the head andthorax shown in the panel 3.1B with pertinent structures labelled. Immediately behind thetongue of the animal is the epiglottis which can be seen to occlude the airway. CT scans ofboth animals yielded similar images in both mid- and parasagittal sections.Measurements of Ventilation/End-Tidal Gases. Episodic breathing patterns were observed inall hibernating ground squirrels. Table 3.1 lists the ventilatory variables recorded duringhibernation. Breathing episodes consisted of 1-53 breaths (mean: 14.2) and were separated bynonventilatory periods ranging from 26 seconds to 23.8 minutes (mean: 6.1 mm.). Ventilatoryperiods were classified as distinct episodes if the duration of the following nonventilatoryperiod exceeded the duration of the ventilatory period. Breathing episodes were alwaysinitiated with an inspiratory movement and terminated at end-expiration. The time requiredfor expiration exceeded the time required for inspiration in all animals with the pausebetween breaths within an episode ranging from 0 to 47.7 sec (mean: 1.95 see).38Figure 3.1. Midsagittal computerized tomography scans of a hibernating golden-mantledground squirrel. Panel 1A depicts a golden-mantled ground squirrel in the hibernatingposition. Panel lB is a whole body CT scan of the animal. Panel 1C is an enlargement of thehead and thorax in panel lB with pertinent structures labelled. The lower panel (1D) is atrace from an impedance converter showing a breathing episode (downward deflectionsrepresent respiratory movements) and a portion of the following apnea (large upwarddeflections during apnea represent mechanical interference from the scanner, small upwarddeflections are cardiogenic oscillations). The arrow marks the time during the apnea when theCT scan was taken.39©A2 mm ‘©lungtrachea—iepiglo ft/s —tonguenasopharynx —40Table 3.1. Ventilatory variables for hibernating golden-mantled ground squirrels.VariableVT (ml) 2.09 ± 0.03TI (see) 0.99 ± 0.01TE (see) 1.25 ± 0.01TP (see) 1.95 ± 0.27breaths/episode 14.2 ± 1.9TvP (see) 47.7 ± 7.8TNvP (see) 368.6 ± 70.0fR (breaths/mm) 2.6 ± 0.71VE (mi/mm) 5.53 ± 1.56V02 (mI/mm) 0.135 ± 0.0481co2* (mI/mm) 0.095 ± 0.03 1Values are means ± S.E.M.N=7 except * where N=5.41Traces of pneumotachograph air flow and the percentage composition of CO2 and 02at the nares of a hibernating ground squirrel for three breathing episodes are shown in theupper panel of Fig. 3.2; in the lower panel, the time scale has been expanded for a singleepisode. While the animal was not breathing or while inspiring, the gas measured was theinspired gas containing 21% 02 and 0% CO2. The % 02 and CO2 in the end-tidal gas weremeasured for every breath and values were converted to partial pressures (PET02 and PET2,respectively). End-tidal 02 was lowest (range: 69.5-120.6 Torr) and CO2 highest (range: 19.6-42 Torr) at the beginning of the episode (Fig. 3.3). In 36 of 60 episodes analyzed, the lowestPETo2 was seen on the first breath of the episode (22/60 on the second and 2/60 on the third).The highest PET2corresponded to the first breath in only 15 of 60 episodes. Most often thehighest PETCO2 was characteristic of the second (21/60) or third (16/60) breath in the episode.As the breathing episode progressed, PET02 increased (range: 92.1-124.9 Torr) and PETo2decreased (range: 14.1-38.5 Torr) (Fig. 3.3).The range of PETO2 and PETCO2 values recorded at the beginning (lowest PET2 andhighest PETCO2)and end (last breath) of episodes was wide (Fig. 3.3) and no clear cutthresholds for turning breathing on or off were apparent. One animal did show a tightclustering of PET2 and two animals showed a tight clustering of PET2values at the onsetof breathing episodes (Fig. 3.3). Also, there was no correlation between PETOJPETCO2at thebeginning of the episode and the length of either the preceding apnea or the subsequentbreathing episode. As well, no correlation was found between PETOJPETCO2at the end of thebreathing episode and the length of the following apnea.Average Vo2 values for individual animals ranged from 0.0400- 0.288 mI/mm (mean42Figure 3.2. Traces of airflow and the percentage composition of CO2 (%C02)and 02 (%02) atthe nares of a hibernating golden-mantled ground squirrel during (upper panel) three breathingepisodes and (lower panel) for a single breathing episode recorded with an expanded timescale. Airflow is in ml/niin.+6001Ih1iI11111LiIIOILl—i-Flow0j-200%C011111ItiaJL2j21%0215ImmFlow%C02LJAN\MrAc5secFigure 3.3. Partial pressures of end-tidal O (upper panel) and CO2 (lower panel) at thebeginning (0) and end () of all breathing episodes monitored in 5 hibernating goldenmantled ground squirrels.44PET0(mniHg)PET0(mmllg)•t’•)CCCCC0CC00CoCCCIII—m—iicn000©IIII000cm3cuo0IcoGUIICI.I_I.—0nocoB0ocxaUI0..I..—_II0IL1I460.135 mi/mm) and from 0.025-0.190 mi/mm (mean 0.095 mi/mm), giving a meanrespiratory quotient of 0.70 (Table 3.1). In Fig. 3.4, the O2 and ‘Jo2 of each breath havebeen standardized with respect to where the breaths occurred in an episode.“2 per breathincreased in the second 10% of the episode and then underwent a linear decline throughoutthe remainder of the episode. There were no significant differences between ‘co2 values forbreaths at the beginning and end of the episode. In contrast, the fall in‘O2 per breath overthe course of the episode was exponential, appearing to asymptote in the last 20% of theepisode. The decrease in the V02 values became significant at the 70% mark.Figure 3.4. The relationship between average 02 consumption/breath (upper panel) andaverage CO2 production/breath (lower panel) and position of the breath in the breathingepisode. Thus, the first (10%) histogram in the upper panel represents the average Oconsumption/breath of all breaths occurring in the first 10% of a breathing episode.47480.10z 0.080p0.0610.04* *0 0.02 fl fl000 — - - - -‘-- - - - - I I10 20 30 40 50 60 70 80 90 1000.100.08W00.06Oc 0.02 [‘1F’]0.00 — -‘- - —i- . — - - - -1__ - I I I I10 20 30 40 50 -60 70 80 90 100TEMPORAL DISTRIBUTION WITHIN EPISODE (%)49DISCUSSIONCritique of Methods: CT Scan. We had initially hoped to address the question of apneicoxygenation by measuring gas exchange during apnea directly. The methods used formeasuring the extremely low levels of seen in hibernating animals (both closed andopen-circuit systems) yield measurements close to the margins of error of the methodsthemselves. Measuring 1O2 due to possible apneic oxygenation is even more problematic. Inthe present study both open and closed-circuit systems were used in an attempt tocontinuously measure 02 uptake and CO2 production of hibernating ground squirrels over theventilation-apnea cycle. Gases were analyzed by mass spectrometry and gas chromatography.Even so, measurements of 02 uptake during apnea were equivocal, and none exceeded therange of error for the equipment. As a result, quantitative measurements were abandoned.A computerized tomography (CT) scan yields a 3-dimensional computerizedreconstruction of the degree to which different tissues absorb transmitted X-rays at the timeof the scan. From the stored information, static images can be constructed of different planesthrough the tissue. By performing a series of parasagittal scans at 0.5 mm increments throughthe tissue, a 3-dimensional image of the internal structures can be envisaged, including theglottis and its relationship to the larynx. From this conclusions can be drawn vis-a-vis theextent to which apneic oxygenation is possible.Critique of Methods: End-Tidal Gases. Ideally, exploring the existence of P02 or P2thresholds which trigger a ventilatory event should involve monitoring changes in these50variables at the receptor site. This requires the knowledge of the receptor sites involved andmeasurements of tissue gas tensions which are highly invasive. Arterial blood gases aregenerally considered a suitable substitution in such studies. Although arterial blood gases areonly one of the main determinants of tissue gas tensions, they generally reflect tissue P02 andP2 levels closely. Similarly, alveolar gas composition closely reflects the arterial gas levels.One fundamental requirement of the present study was to avoid arousing thehibernating animals. It was, therefore, necessary to utilize the least invasive technique tomonitor P02 and P2 levels over the ventilation-apnea cycle. End-tidal gas measurements canbe obtained noninvasively and are considered a reasonable approximation of arterial blood gaslevels (Steffen and Riedesel, 1982). PETcj2 values typically exceed arterial P02 values. Theextent to which the end-tidal P02 values exceed true arterial levels (the alveolar-arterial (A-a)P02 difference) is dependent on the degree of diffusion limitation and pulmonary ventilation-perfusion mismatch (Dejours, 1981). The size of the A-a difference should remain constant aslong as these factors remain constant. The diffusing capacity depends on the area of thealveolar membrane available for gas transport and the thickness of the diffusion pathway,while the ventilation-perfusion relationship is influenced by posture, shunting and alveolarrecruitment. Since there is no reason to believe that any of these factors would change fromone ventilatory period to the next, the present study assumes that the A-a difference remainsconstant. Therefore, although there may be absolute differences between alveolar and arterialgas levels, this should not effect inferences concerning the existence of ventilatory thresholds.51Apneic Oxygenation. Apneic oxygenation, the movement of 02 from the atmosphere to thelungs during apnea, is dependent on an open glottis. Such movement would arise fromdiffusion and bulk convection if the respiratory quotient were less than one. Malan (1982)presumed that pressure fluctuations in body plethysmographic recordings which coincidedwith heart beats during apnea were adiabatic fluctuations resulting from air flow through anopen glottis and concluded that apneic oxygenation must occur. While positive pulsescorrelated with the heart beat do appear in the plethysmogram if the airway is patent, Mills(1969) demonstrated that the amplitude of the wave would actually increase if the glottis wasclosed due to mechanical considerations. Thus, the assumptions that the glottis must be openfor cardiogenic oscillations to show up as pressure fluctuations on the plethysmograrn are notvalid and direct measurements of gas exchange are required to demonstrate apneicoxygenation.Further indirect arguments supporting the occurrence of apneic oxygenation werebased on calculations for oxygen consumption in the hibernating hedgehog (Malan, 1982).Unless the glottis remained open during apnea, this study concluded that ongoing metabolismcould not be supported. These calculations assumed a constant metabolic rate over theventilation-apnea cycle implying that the oxidative cost of breathing was negligible. This isclearly not true and will be discussed further below.Direct measurements of apneic oxygenation have been equivocal. Thomas et al. (1990)reported measurable 02 uptake during periods of apnea in Myotis lucfugus, but the valueswere only a fraction of the uptake expected were the glottis to have been open. On the otherhand, Szewczak and Jackson (1992) reported 02 uptake values in torpid Eptesicus fuscus52during apnea which were consistent with the glottis remaining open.In the present study, the scans of the two animals, taken at random during apneicperiods, both showed glottal closure. This would suggest that apneic oxygenation does notoccur in this species.“On/Off’ Thresholds for Intermittent Breathing. The breathing patterns and ventilatoryvariables presented in Table 1 are similar to those reported previously for hibernatingSpermophilus lateralis (Steffen and Riedesel, 1982; McArthur and Milsom, 199 ib). A facemask and pneumotachograph have been employed for measuring ventilatory variables in paststudies and do not appear to disturb the animal’s episodic breathing pattern.The mean end-tidal gas pressures agree well with those reported for the same speciesby Steffen and Riedesel (1982), although the ranges reported for both PETO2 and PETCü2 werewider in the present study. Nonetheless, the end-tidal gases reported here do appear to be areasonable approximation of arterial blood gases. Although the only blood gas valuedocumented for hibernating S. lateralis is an arterial Pco2 value of 28 Torr (Twente andTwente, 1964), Musacchia and Volkert (1971) have reported arterial Po values ranging from35- 120 Torr and concomitant arterial Paco2 values ranging from 29-37 Torr in the 13-linedground squirrel during hibernation. Tähti and Soivio (1975) collected blood gas values withthe knowledge of ventilatory state, finding arterial P02 values ranging from 10.5 Torr at theend of an apneic period to 120.3 Torr immediately following ventilation. In the same study,arterial P2 values peaked at 34.6 Torr at end apnea and fell to 26.5 Torr after a breathingepisode. Given this agreement between studies, the wide range of PET02 and PETCO2 values53recorded at the beginning and end of breathing episodes in the present study does not supportthe existence of thresholds in P02 or Po2 for turning breathing on or off. The tight clusteringof values in animals 4 and 5 (Fig. 3.3), however, suggests that under certain circumstanceson/off thresholds may appear evident.Figure 3.4 provides an interesting comparison of the“02 and “co2 profiles over theventilatory period. The was high at the beginning of the episode and underwent anexponential decline until reaching an asymptote in the last 20% of the episode. In contrast,after reaching a peak value 20% into the breathing episode, the ‘co2 underwent a slight butsteady decline throughout the remainder of the breathing episode. This difference in theand ‘co2 profiles during the breathing episode may be attributed to the slow mobilization ofCO2 stores. Dejours (1981) states that most of the CO2 accumulated during apnea is notreleased into the lungs but remains within the blood and tissues because of their highcapacitance for CO2. Consequently, lower end-tidal CO2 values and, therefore, Vco2 valuesmay be expected in the first breath of an episode. As the breathing episode progresses, CO2stores are mobilized from the blood and tissues to the lung, end-tidal CO2 values peak andthen progressively decline.The “co2 profile provides no indication as to what terminates the breathing episodeand despite the fact that no thresholds in PETO2 were found for terminating the breathingepisode, the episode appeared to end when Vo2 had reached an asymptote. If this asymptoteis assumed to represent steady-state, it appears that the animal terminates the breathingepisode once the oxygen uptake matches ongoing metabolism, ie. the 02 debt from theprevious apnea has been repaid. How this is accomplished in the absence of any distinct54threshold in PET2 is unclear.Oxidative Cost of Breathing. The Vo2, “co2 and RQ values in the present study fall withinthe range of values previously reported for this and other species (Kayser, 1961; Hamrnel etal, 1968; Steffen and Riedesel; 1982; McArthur and Milsom, 1991b). Based on theassumption that the asymptote in the V02 profile represents the amount of 02 required to meetongoing metabolism, the data can also be used to calculate the oxidative cost of breathing.Thus, the shaded area in Fig. 3.5 represents the amount of the 02 consumption required tosupport the resting metabolic rate during the ventilatory period and the unshaded arearepresents the amount of the O consumption required to repay the02-debt accumulatedduring the apnea. Dividing the shaded area by the length of the ventilatory period and theunshaded area by the length of the preceding apnea will give values for the V02 duringbreathing and the‘O2 during apnea, respectively. Assuming the only difference betweenperiods of apnea and eupnea is the act of breathing, the oxidative cost of breathing will be theV02 during breathing less the ‘O2 during apnea. This oxidative cost can be expressed as afraction of the total V02.The values obtained by calculating the oxidative cost of breathing in this manner reachup to 90%. This seems excessively high. However, this discrepancy may be resolved if thecost of other events commonly associated with a breathing episode are hidden within thecalculated value. Milsom (1992) has reported increases in electroencephalographic (EEG) andelectromyographic (EMG) activity just prior to and during breathing episodes in hibernatingS. lateralis suggesting there is increased central neural activity and resting body tone duringFigure 3.5. The relationship between average 02 consumption/breath and position of thebreath in the breathing episode. Sum of shaded areas equals V02 required for metabolicprocesses during the breathing episode. Sum of non-shaded areas equals02-debt frompreceding apnea. See text for details.55liz0.100.080.06 0.04Iz 02 I-I--I--I-•1I-I-00.02•0.00102030405060708090100TEMPORALDISTRIBUTIONWITHINEPISODE(%)057breathing episodes. A tachycardia preceding and during the breathing episode has also beenreported for S. lateralis (Steffen and Riedesel, 1982; Milsom, 1992). It is possible, therefore,that the oxidative cost of breathing calculated in the present study is actually the cost of acomplex set of events associated with a breathing episode. In view of the fact that theoxidative cost of breathing (and associated events) could reach such levels and that apneicoxygenation is not possible with a closed glottis, strategies to reduce the cost of breathingtake on great importance.In conclusion, the specific factors involved in the initiation and termination ofbreathing episodes remain obscure. No clear cut thresholds in end-tidal gas composition couldbe identified in this study for turning breathing on and off. Although changes in blood gascomposition appear to influence the breathing pattern, the control and genesis of episodicbreathing could not be ascribed to this single variable. Furthermore, apneic oxygenation didnot occur in this species and the oxidative cost of breathing, with its associated events, wereestimated to account for most of the resting metabolic rate of these animals duringhibernation.58GENERAL CONCLUSIONSMammalian hibernation is characterized by profound reductions in body temperatureand metabolism and the conversion of continuous breathing to intermittent breathing. In thegolden-mantled ground squirrel intermittent breathing is manifest as a series of breathingepisodes separated by variable periods of apnea. Such a breathing pattern allows significantfluctuations in lung and blood P02 and Pc2 levels; during a breathing episode, lung andblood P02 increase and P2 decrease while in the following apnea, the reverse occurs(Musacchia and Volkert, 1971; Tthti and Soivio, 1975; Steffen and Riedesel, 1982). Previousstudies on arrhythmic breathing in lower vertebrates have suggested that these cyclicfluctuations in P02 and/or P2 acting via chemoreceptor feedback play a role in the controland genesis of episodic breathing. These studies have hypothesized that specific blood gasthresholds for P02 and P2 exist and that, in response to changing 02 and CO2 stores,chemoreceptor drive gradually diminishes over the ventilatory period and gradually increasesover the nonventilatory period to these thresholds where breathing is terminated or initiated,respectively (Shelton and Croghan, 1988). The data in support of this hypothesis remainequivocal, however (Milsom, 1990). Given this, the main goal of the present study was to testthe hypothesis that blood gas fluctuations play a role in the control of episodic breathing inhibernating mammals or, more specifically, that there are separate thresholds in P02 and/orP2 which turn breathing on and off.Before addressing this hypothesis, however, the ability of these animals to respond tochanges in 02 and CO2 was examined. A reduced hypoxic ventilatory response and elevated59hypercapnic venifiatory response are seen in animals during hibernation compared toeuthermia and this has led many authors to conclude that changes in arterial 02 play little rolein the control of the episodic ventilation during hibernation (Biörck et al., 1956; Tthti, 1975;Tähti et al., 1981). Indeed, the Pao2 threshold of the hypoxic ventilatory response is lowerduring hibernation than euthermia. On closer examination, however, this reduction actuallyequates to a left-shift in the Pao2 threshold of the hypoxic ventilatory response in conjunctionwith the temperature-induced left-shift in the oxyhemoglobin equilibrium curve. As a result,the correlation between the inflexion points on the hypoxic ventilatory response curve andoxyhemoglobin equilibrium curve is retained during hibernation (McArthur and Milsom,1991a). It appears, therefore, that regardless of temperature these animals do not elicit anhypoxic ventilatory response until arterial blood begins to desaturate. This suggests either thatthere is a temperature-induced change in the response characteristics of arterialchemoreceptors or that these species are capable of responding to changes in Cao2.In the present study the hypoxic ventilatory response to concomitant changes in Cao2and Pao2 (hypoxic hypoxia) as well as to changes in Cao2 independent of Pao2 (CO-hypoxia)were examined in both heterothermic and nonheterothermic rodent species. While bothspecies exhibited ventilatory responses to hypoxic hypoxia and CO-hypoxia, the hypoxicventilatory response of the squirrel was consistently stronger than that of the rat. Furthermore,the ventilatory response to hypoxic hypoxia exceeded that to CO-hypoxia in both species;reductions in Cao2 alone could produce only 60% of the ventilatory response seen duringhypoxic hypoxia and simultaneous changes in Pa02 were required to elicit the full response.Despite the relative reduction in the hypoxic ventilatory response induced by CO-hypoxia, it60is clear that both species could respond to changes in Cao2 alone.Such a phenomenon may account for the persistent correlation between the inflexionpoints of the hypoxic ventilatory response curve and the oxyhemoglobin equilibrium curveduring hibernation. Since the carotid body chemoreceptors are the only chemoreceptor groupknown to elicit a ventilatory response to changes in arterial O delivery in mammals, andthese receptors have been shown to respond only to changes in Pao2, it has been generallyaccepted that the hypoxic ventilatory response is mediated solely through changes in Pao2.Thus, although many species increase ventilation at the Pao2 at which arterial blood begins todesaturate, this is generally assumed to be a consequence of natural selection acting to protectarterial 02 saturation and not a response to changes in Cao2per se (Van Nice et al., 1980).Many carotid body denervated animals, however, either retain or regain an HVR (Bisgard etal., 1980; Smith and Mills, 1980; Maskrey et al., 1981; Webb and Milsom, 1990), suggestingthat some other chemoreceptor is capable of eliciting a ventilatory response to a change insome aspect of 02 delivery. Curiously, the hypoxic ventilatory response of golden-mantledground squirrels to CO-hypoxia at constant Pao2 and to hypoxic hypoxia after carotid bodydenervation are both manifest primarily as an increase in breathing frequency and bothproduce 60% of the full ventilatory response induced by hypoxic hypoxia in the intact animal(Webb and Milsom, 1990). These observations indicate that changes in Cao2 may be sensed ata non-carotid body chemoreceptor and that the left-shifted hypoxic ventilatory response seenin hibernating animals is due to stimulation of this chemoreceptor group. It is also possiblethat the carotid bodies of these species are also sensitive to changes in Cao2.Whatever the mechanism, having shown that the golden-mantled ground squirrel was61capable of responding to changes in both Cao2 and Pa02, it was concluded that changes in Ocould not be ruled out as contributing to the control of episodic breathing. The present studythen examined the existence of specific thresholds in P02 and P2 for the initiation and/ortermination of breathing episodes. No clear thresholds in P02 or P2 were found for turningbreathing on or off. Since these animals are capable of responding to changes in Cao2independent of Pa02, it was conceivable that the onset and termination of breathing episodeswas correlated to Cao2 thresholds. When the end-tidal P02 values of the present study, whichare assumed to be close approximations of Pao2, were expressed as Cao2 values, however, nothresholds were seen. In fact, the range of P02 recorded both at the onset and termination ofthe breathing episodes all represented arterial°2 saturations of 100%. Thus, it appears thatarterial blood does not undergo any appreciable desaturation during apnea; venous blood mustbe totally recharged from lung 02 stores.Furthermore, the length of the apnea was independent of the length of the previous orfollowing breathing episode and the length of each breathing episode was independent of thelength of the previous or following apnea. Since the glottis remained closed in this speciesduring apnea, the variability in the lengths of the nonventilatory periods could not beattributed to apneic oxygenation. Thus, not only was there no indication of clear thresholdsfor initiating or terminating the breathing episode, there was also no clear correlation betweenthe length of the breathing episode and the magnitude of the blood gas changes preceding orfollowing it. The breathing episode did appear, however, to end when the 02 consumptionprofile asymptoted, suggesting that breathing ceased when body 02 stores were replenishedand thus, the animal was responding to some aspect of oxygen delivery. It remains unclear,62however, what aspect of oxygen delivery triggers the tennination of the ventilatory periods ifthis is the case.These data conform with a growing body of evidence from studies in lowervertebrates which suggest that while changes in chemoreceptor drive establish the overalllevel of ventilation, cyclic blood gas fluctuations are not necessary for the genesis of anepisodic breathing pattern (West et a!., 1987; Smatresk and Smits, 1991). This stresses thedifference between the control of total ventilation and the control of breathing pattern. 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