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1Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; and 2Department of Medicine, People's Hospital, Beijing University, Beijing 100044, China
Submitted 19 February 2003 ; accepted in final form 13 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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short-term potentiation; nitric oxide synthase; ventilation; respiratory control; genetics
E). Rather, they and
other authors (1,
8,
38) have suggested a link
between PB and other posthypoxic ventilatory behavior. Indeed, the absence of
STP in the C57BL/6J (B6) mouse, a strain in which PB occurs, may remove a
stabilizing mechanism, thereby increasing the propensity for PB. Similarly,
the presence of PHFD in B6 mice might account for its tendency to exhibit PB.
The present study further examines this relationship by using pharmacological
manipulation of posthypoxic ventilatory behavior. Nitric oxide (NO) is a messenger molecule involved in the regulation of respiration, with three NO synthase (NOS) genes having distinct localized functions (24, 25). Specifically, neuronal NOS (nNOS)-generated NO is responsible, in part, for modulating gain of hypoxic responsiveness in the mouse (22). Subramanian et al. (32) showed significant strain differences in rats in regard to metabolism and f, including posthypoxic ventilatory response, under NOS blockade by using a nonspecific NOS inhibitor. Furthermore, knockout mice deficient in the nNOS isoform and mice given 7-nitroindazole (7-NI; a specific nNOS inhibitor) exhibit attenuated STP (20). Given this background, the hypothesis was that a pharmacological approach directed at reducing nNOS would produce PHFD and PB in A/J mice, a strain in which PB is usually not seen. A secondary intent was to determine whether nNOS plays a role in the expression of PB in the B6 strain.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Experimental protocols. Animals were tested over 4 days in a time period restricted to 12:00 PM to 3:00 PM. Measurements were made when animals were awake, as assessed by behavioral observation. On days 1 and 3, the mice were put into the chambers at the normal start time but did not undergo any testing. After testing hours, they were returned to the Animal Resource Center.
On days 2 and 4, the animals received one of two
experimental protocols: a 7-NI protocol and a vehicle protocol. Except for the
injection received, these protocols are identical. Half of the animals in each
strain received 7-NI on day 2 and the vehicle on day 4
(protocol A); the other half received the reverse protocol
(protocol B; Fig. 1).
Animals were first weighed and then placed in the test apparatus at 10:00 AM
and given 60 min to acclimate. At 11:00 AM, the mice received an
intraperitoneal injection of either peanut oil alone (vehicle) or 7-NI
dissolved in peanut oil at a dose of 60 mg/kg
(17). We allowed 1 h for the
drug to take effect before baseline breathing was recorded. Mice were then
given a 5-min exposure to hyperoxia (100% O2). The mice next
received a 5-min challenge of poikilocapnic hypoxia (8% O2-balance
N2), after which the gas was flushed out of the chamber for 
10
s and the chamber was reoxygenated with 100% O2. Most of the mice
then received a 5-min hypercapnic challenge (95% O2-5%
CO2) and a 5-min isocapnic hypoxic challenge (10% O2-3%
CO2). Finally, mice were given another poikilocapnic hypoxia
challenge but after flushing were reoxygenated with room air. Four B6 mice did
not receive any hypercapnic or isocapnic hypoxic challenges, but instead they
faced repeated challenges of poikilocapnic hypoxia followed by either room air
or 100% O2. Steady-state respiratory variables were measured in the
fifth minute of all tests, and there was a 10-min interval between each
challenge. Hypoxic ventilatory response (HVR) was defined as the percent
change in E from air to the end of
the fifth minute of poikilocapnic hypoxic challenge. Animals were visually
observed for sleeping behaviors during all measurements. Body temperature was
measured with a thermocouple inserted rectally at a depth of 3.5 cm and
was assessed immediately after the last hypoxic challenge.
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Measurements of ventilatory behavior. We used whole body plethysmography by the open-circuit method (29), modified for the unanesthetized and unrestrained mouse for measurement of ventilatory behavior (13). Animals were placed in a round Lucite chamber (600-ml volume) containing an inlet port for the administration of test gases (see Experimental protocols). An outlet port was connected to a vacuum sufficient to create a bias flow of 300 ml/min through the chamber, as measured by a flow rotameter. Because respiration immediately after hypoxia was a focus of the experiment, test gases were flushed out of the chamber at a flow rate of 15 l/min for 10 s, and then the flow rate through the chamber was returned to baseline. The chamber was connected to one side of a pressure transducer (Validyne DP45, Validyne Engineering, CA) with a sensitivity of ±2 cmH2O, referenced to a chamber of equal volume. As the animal breathed, swings in chamber pressure were recorded and then processed to a voltage signal. Comparison of this voltage signal to calibration volumes permitted an estimation of values that would represent VT. For each animal, the calibration volumes before and after each testing period and the voltage signals were recorded on a strip-chart recorder (Linerecorder WR3320, Graphtec, Irvine, CA) and stored in a computer with respiratory acquisition software (LabView programming by I.C.E, Cleveland, OH). The fractional content of O2 and CO2 was measured by sampling the gas exiting the chamber (Beckman OM-11 and LB-2 analyzers, respectively, Beckman Instruments, Pittsburgh, PA). With the chamber empty, a calibration volume of 0.25 ml of air was repeatedly introduced into the chamber before and on completion of recording. Other calibration volumes above and below this volume were also routinely performed as a quality control for the linearity of the voltage signal. A thermometer-hydrometer probe was inside the chamber to monitor chamber temperature, chamber humidity, and barometric pressure. Two setups were available, each using identical transducers and monitors. Animals were studied in tandem.
There were no differences in chamber temperature (24.10 ± 0.03°C), chamber humidity (28.3 ± 0.2%), or barometric pressure (742.6 ± 0.2 Torr) that differed significantly with day of testing or by strain.
PB was defined as cyclic fluctuations in VT and f interrupted by
periods of apnea or near apnea
(14). Apnea was defined as an
end-expiratory pause of 
2 average breath durations. We only scored events
as PB if at least three cyclic fluctuations of VT and f were
observed. We used three parameters to quantify PB: 1) the cycle
length (Tc; in s) or the time between successive points of minimum ventilation
in the periodic pattern, 2) the apnea length (Ta; in s), if
applicable, and 3) the strength of the oscillation (M; unitless), a
measure of how much the ventilation changes as the pattern goes from its point
of maximum ventilation
(Emax) to its point of
minimum ventilation
(Emin)defined by Waggener
et al. (35). For nonapneic
oscillations, M was defined as the ratio of
Emax -
Emin over
Emax +
Emin, and for apneic
oscillations, this same index was calculated as the ratio of Tc over Tc - Ta
(14,
35).
Data analysis. Ventilatory parameters were measured continuously
throughout the testing period, and scored by computer by using a
respiratory-based software program (Lab View programming by I.C.E.). The
following variables were analyzed: inspiratory VT (in µl), f (in
breaths/min), and E (in ml/min;
VT x f). Sighs or sniffs were excluded in the analysis. Sighs
were signals that exceeded by 150% the average VT for the past 4 s.
Sniffs are identified as rapid, very shallow oscillations in voltage, which,
on visual examination, are temporally related to exploratory behavior when the
animal appears to be awake. Tc and M were measured with the computer program.
During O2 reoygenation,
E, VT, and f were
determined when the concentration of the inspired O2 was between 40
and 50% (30 s after reoxygenation).
Significant differences between strains and drug treatments were detected by using ANOVA and Tukey's post hoc test. Differences with P < 0.05 were considered significant. All results are expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Ventilatory behavior during resting air breathing with 7-NI. A significant decrease in resting f was exhibited by B6 mice when 7-NI was administered. In A/J mice, the decrease in resting f did not reach significance. The magnitude of this decrease in f was significantly greater in B6 mice (P = 0.025). There were no significant within-treatment differences between strains for any resting breathing variables.
There was no difference in CO2 production
(CO2) or O2
consumption (O2) between
strains or between treatments. The decline in respiratory quotient (RQ)
between treatments for both strains was not significant
(Table 1). Body temperature
decreased significantly in B6 but not in A/J mice after 7-NI administration.
E/CO2
did not vary significantly between vehicle and 7-NI treatments for either
strain (Table 1).
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Respiration during steady-state isocapnic hypoxia, poikilocapnic
hypoxia, hypercapnia, and hyperoxia while under the influence of 7-NI.
For each strain, f was decreased after 7-NI administration for all
steady-state challenges, and this reached significance in all cases except for
A/J mice during hyperoxia and isocapnic hypoxia
(Fig. 2). This decrease in f
resulted in a significantly decreased
E for both strains during
hypercapnia and poikilocapnic hypoxia. With isocapnic hypoxia, however, only
B6 mice also demonstrated a significantly decreased
E due to 7-NI. Although both strains
exhibited decline in f during hyperoxia, this was offset by a nonsignificant
increase in VT such that there was no difference in
E between treatments for either
strain.
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Generally during steady-state challenges after 7-NI administration, f,
VT, and E changed
similarly for A/J and B6 mice. However, B6 mice showed a statistically greater
decline (measured in percent change from vehicle) than did A/J mice for f
during the fifth minute of hyperoxia and hypercapnia challenges
(Fig. 2). Because of similar
VT values, this did not result in a strain difference in
E during either challenge.
Ventilatory response to hypoxia with vehicle was similar for both strains.
With 7-NI, HVR in A/J mice decreased from 120 ± 41 to 20.4 ±
12%, and HVR in B6 mice decreased from 104 ± 24 to 16 ± 11%.
Thus both strains showed a similar significant decline in HVR with 7-NI.
PHFD and STP under the influence of 7-NI. Neither A/J nor B6 mice
with vehicle showed PHFD (Fig.
3). Furthermore, under vehicle, neither strain demonstrated either
STP or a drop in posthypoxic VT or
E. However, with 7-NI, there
occurred a large decrease in E for
both strains after reoxygenation. This was accomplished in A/J mice by a
significant decrease in both the f and VT components of ventilation
(Fig. 3). B6 mice also had a
significantly decreased f, but the decline in posthypoxic VT
compared with resting breathing was not statistically significant. In summary,
with vehicle, neither strain showed STP or PHFD; with 7-NI, both strains lack
STP and demonstrate PHFD.
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Effects of 7-NI on PB. 7-NI dramatically affected the occurrence and severity of PB in B6 animals, while having little effect in producing PB in A/J mice (Figs. 4 and 5, A and B). Half of the B6 animals tested experienced PB during resting air breathing with 7-NI, whereas none of the A/J animals showed PB during this treatment (Fig. 4). B6 animals with vehicle showed periodic breathing upon reoxygenation with either air or 100% O2, and all B6 mice showed apneic PB after 7-NI administration (Fig. 5A). The major effect of 7-NI in these animals was to increase Tc and Ta of PB, while leaving M similar (Fig. 6). Apneas reached up to 11 s in length and were often followed by very few breaths (Fig. 5A).
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In contrast, A/J mice did not show a tendency toward apneic PB with 7-NI administration (Fig. 5B). With vehicle, one of six A/J animals showed nonapneic periodic breathing with reoxygenation to 100% O2 (Tc = 11.58 s, M = 0.34). After 7-NI administration, this one animal showed no PB, but three other A/J mice showed nonapneic PB (Tc = 5.5 ± 0.82 s, M = 0.42 ± 0.12). Whereas no A/J mouse had PB under vehicle and posthypoxic reoxygenation to air, one had PB under 7-NI (Tc = 9.031, M = 0.76).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Several respiratory variables measured in mice differ from the results of earlier studies and are attributed to the injection of the vehicle. Notably, B6 mice did not exhibit statistically significant PHFD with vehicle, and A/J mice did not exhibit STP with vehicle, contrasting Han et al. (13) (Fig. 3). Also in contrast to Han et al., there were no significant strain differences in resting breathing or metabolic variables after administration of vehicle (Table 1). The vehicle injection could affect various components of the control system, including cortical inputs that modulate the integrative aspects of brain stem ventilatory pathways (15). Although in the present study we did not specifically test mice that did not receive any injection, the vehicle (peanut oil) affected ventilatory characteristics. The mechanism of this vehicle effect is beyond the scope of this study, but use of the vehicle was a necessary control to study the effect of the drug.
Several authors have related PB to the absence of STP. Ahmed et al. (1) showed a negative relationship between STP and Cheynes-Stokes breathing, a PB disorder. Younes (38) considers the neural afterdischarge that results in STP of breathing to be the most important factor that stabilizes ventilation and prevents PB. More recently, Dempsey et al. (8) and Powell et al. (28) noted STP as an important factor in promotion of a regular breathing pattern. Han et al. (14) suggested that strain differences in STP could be causing the strain differences seen in PB. The present study provides the opportunity to test the hypothesis that strain-related differences in the mouse are related to posthypoxic ventilatory behavior. Neither PHFD nor STP was observed after vehicle in either strain, but the B6 mice still exhibited PB. Furthermore, after 7-NI administration, both strains exhibited PHFD and apneic PB was only observed in B6 mice. Therefore, some mechanism or system interaction explains strain differences in PB in the mouse, even after vehicle.
Clinical studies correlate hypoxic sensitivities with the occurrence of PB (2, 23, 36), and models suggest that breathing instability can occur when there is increased gain (4, 19). nNOS-derived NO has previously been found to inhibit carotid body sensitivity to hypoxia in mice (22, 29), and Ogawa et al. (27) proposed that NO affects ventilatory behavior in Sprague-Dawley rats by serving to integrate chemoreceptor input in brain stem neurons. Kline et al. (22) reported respiratory oscillations in mutant mice deficient in nNOS, and attributed the effect to increased controller gain, but could not exclude an effect by the central controller (nucleus tractus solitarii). However, in contrast to the findings of Kline et al. (22) in genetically engineered mice, both the A/J and B6 strains exhibited a similar decline in HVR when given 7-NI. This drop in HVR is consistent with an overall decrease in responsiveness to hypoxia occurring with NOS inhibition. One report (12) found that specific nNOS blockade in rats had no effect on respiratory responses, although both the drug and the species are different from the present study. Therefore, it is again important to note that only B6 mice experience greater PB with 7-NI, including a tendency toward PB during resting air breathing and increases in Ta and Tc after hypoxia. We speculate that tonic nNOS function acting at a central respiratory controller plays an important role in maintenance of HVR in both A/J and B6 mice. The observation that A/J mice did not experience PB under the effect of 7-NI reinforces a concept that the strain-related PB is not due to differences in steady-state chemoresponsiveness (14).
The decrease in resting ventilation due to 7-NI was insignificant for both
strains, although the f component was significantly depressed in B6 mice. This
finding stands in contrast to the increased f and
E of nNOS knockout mice
(22) and the previously
reported increased f for 7-NI-treated mice
(20). Hence tonic nNOS
modulation of resting breathing among different mouse strains may differ
qualitatively and/or quantitatively, a finding also reported in rats
(32). Indeed, B6 mice
exhibited a decrease in resting f due to 7-NI that is significantly greater
that the decrease seen in A/J mice.
Inhibitors of NOS can significantly affect body temperature in mice and rats, and such effects can differ by strain (5, 11, 26, 32). The present study describes a decrease in temperature that is significant for only the B6 mice, the strain that experiences greater instability of breathing. Assuming that a decrease in temperature would lead to a damping of the ventilatory control system, making it more stable and less prone to PB, the effects of 7-NI on temperature cannot explain the strain difference in PB observed.
Previous studies with NOS blockade have yielded mixed results regarding the
role of NO in the maintenance of metabolism
(3,
7,
19,
30,
32), and its influence may
depend on the state of hypoxia or normoxia
(11). In the present study,
the effect of 7-NI on body metabolism was to decrease
CO2,
O2, and RQ, but none of
these reached statistical significance. Furthermore, any changes appear
similar between strains, indicating that strain differences in PB cannot be
easily attributed to changes in body metabolism.
Limitations. Animals were without food and water during the testing period, possibly leading to changes in metabolism. However, because half the animals received 7-NI first, and half received vehicle first, concern about such effects are reduced, because each animal acted as its own control.
Only one dose of 7-NI (60 mg/kg) was administered. This dose should provide maximal NOS blockade in rats (17); however, the dose for maximal NOS inhibition in mice has not been determined. The weight of the animals in each strain was different, and dose x weight effects could have contributed to the strain differences in PB seen. Different mouse strains could also respond differently to the same dose of 7-NI via direct biochemical mechanisms, contributing to the strain differences observed. Kalisch et al. (17) also showed that NOS activity was lowest 0.5 h after 7-NI injection and slowly returned to preinjection levels over several hours. If it is assumed that this pattern holds for mice, the effects of 7-NI could change slightly over the testing period. Both strains of mice, however, were given the same protocol with the same postinjection timing, and it is therefore unlikely that this could cause the strain differences observed. Although the B6 mice were significantly younger than the A/J mice tested in the present study, observations of two older B6 mice with and without 7-NI have shown similar effects of 7-NI on PB (unpublished results). The strain x age effects cannot, therefore, be easily attributed to the age difference between strains.
Although a state of wakefulness was noted visually, we did not attempt to objectively record state. Changes in alertness can affect respiratory drive and integration of breathing (4, 8), and therefore strain-related differences in vigilance might have affected our results.
Whole body plethysmography of unrestrained animals presents limitations to the measurement of VT. Accuracy of VT is dependent on many environmental and chamber variables, and even under ideal testing circumstances calibrations may be imperfect at different frequencies (32). Although most environmental factors were held constant, falling body temperatures under 7-NI could introduce error into VT measurements. The conclusions drawn from this study, however, can be made without absolute accuracy of VT estimates.
The precise location of the differences in PB is not identified in the present study. Structural components might include features of carotid body or nucleus tractus solitarii organization and connections; however, functional differences are also possible. Notably, A/J mice have previously been found to have carotid bodies with decreased size and function compared with the DBA/2J strain (37). Ventilatory response to hypoxia (percent change from baseline) is similar for the B6 and A/J strains with either vehicle or 7-NI. Whether differences occurring in carotid body morphology or in the central connections responsible for the ventilatory response to 7-NI explain these strain differences in PB is unknown.
In conclusion, 7-NI, an nNOS inhibitor, further dissociates the A/J and B6 mouse strains in regard to periodic breathing. PHFD is induced in both strains when 7-NI is administered. Yet the A/J strain does not experience PB after a hypoxic episode. In contrast, the B6 strain continues to exhibit PB after hypoxia, and in fact it experiences greater instability of breathing and incidence of PB with administration of 7-NI. It is unlikely that strain-related differences in STP or PHFD are primary determinants of strain-related PB. NO seems to play an important role in Ta and Tc in the B6 mouse. These results should further our understanding of PB in humans, which occurs in the context of newborn breathing, breathing at altitude, and breathing disorders related to sleep apnea and congestive heart failure (1, 10, 18, 38).
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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