Hyperoxia is a popular model of oxidative stress. However, hyperoxic gas mixtures are routinely used for chemical denervation of peripheral O2 receptors in in vivo studies of respiratory control. The underlying assumption whenever using hyperoxia is that there are no direct effects of molecular O2 and reactive O2 species (ROS) on brain stem function. In addition, control superfusates used routinely for in vitro studies of neurons in brain slices are, in fact, hyperoxic. Again, the assumption is that there are no direct effects of O2 and ROS on neuronal activity. Research contradicts this assumption by demonstrating that O2 has central effects on the brain stem respiratory centers and several effects on neurons in respiratory control areas; these need to be considered whenever hyperoxia is used. This mini-review summarizes the long-recognized, but seldom acknowledged, paradox of respiratory control known as hyperoxic hyperventilation. Several proposed mechanisms are discussed, including the recent hypothesis that hyperoxic hyperventilation is initiated by increased production of ROS during hyperoxia, which directly stimulates central CO2 chemoreceptors in the solitary complex. Hyperoxic hyperventilation may provide clues into the fundamental role of redox signaling and ROS in central control of breathing; moreover, oxidative stress may play a role in respiratory control dysfunction. The practical implications of brain stem O2 and ROS sensitivity are also considered relative to the present uses of hyperoxia in respiratory control research in humans, animals, and brain stem tissues. Recommendations for future research are also proposed.
- brain stem respiratory centers
- central chemoreceptors
- oxidative stress
- hyperoxic hyperventilation
oxygen is required to sustain life, but too much oxygen is toxic to the mammalian central nervous system (CNS) due to excessive production and accumulation of reactive oxygen species (ROS). Recall that “oxygen pressure in the mammalian CNS is maintained at a level which is sufficiently high to ensure undisturbed function of brain cells and sufficiently low to minimize generation of free radicals” (24). Regardless, high-dose O2 is routinely, but prudently, used in clinical and nonclinical settings to treat or prevent hypoxemia and tissue hypoxia, and controversially for other miscellaneous disorders (3, 9, 33, 64). Clinical symptoms of CNS O2 toxicity, presumably due to the effects of ROS, are manifested only when breathing high-dose O2 at significant hyperbaric pressure [i.e., >2 atmospheres absolute (ATA) O2]. However, human and animal studies indicate that exposure to hyperoxia at normobaric pressure (pure O2 at ∼1 ATA) also has subtle but damaging effects on neurological function in the immature CNS (2, 30, 31) and a modulating influence on neuronal excitability in the mature CNS (6, 7, 60).
One neural system that is particularly sensitive to an increase in inspired O2 is the respiratory control system. The ventilatory response to hyperoxia is a biphasic response. The first few breaths of hyperoxia typically decrease expired minute ventilation (V̇e) by inhibiting the peripheral chemoreceptors (21, 34, 68, 69). This initial effect of hyperoxia on breathing, however, is short lived, and continued breathing of the hyperoxic gas mixture for >1-2 min reveals a secondary increase in V̇e. The secondary response is called “hyperoxic hyperventilation,” and it occurs primarily through a dose-dependent increase in tidal volume (Vt), which may or may not be accompanied by an increase in respiratory frequency (fresp) (4, 14, 35, 36). In some animal studies, the secondary increase in V̇e is not observed unless the peripheral chemoreceptors are denervated (27, 41). Because hyperoxic hyperventilation is present in animals with peripheral chemoreceptor denervation (14, 27, 41, 69), the hyperventilation most likely is of central origin. In contrast, the initial hypoventilation or apnea is abolished by peripheral chemoreceptor denervation, which confirms that the early transient response is completely dependent on peripheral chemoreception (69).
Hyperoxic hyperventilation is a paradox in respiratory control. Unlike the hypoxic ventilatory response, which functions to maintain tissue oxygenation during hypoxemia (28, 29, 68), the role of hyperoxic hyperventilation in O2 homeostasis is unclear. The maximum level of inspired Po2 (PiO2) in our natural environment is only 160 Torr (0.21 O2 × 760 Torr at sea level = 160 Torr O2 = 0.21 ATA O2). Regardless, understanding the central effect of hyperoxia on respiratory-related neurons (21, 44, 57, 72) becomes very important considering the pervasive use of hyperoxia in respiratory control research (4, 13, 16, 27, 71), clinical medicine (3, 9, 57, 64), and civilian and military professions (18).
This paradox of ventilatory control, we believe, is significant in that it provides clues into the fundamentally important role of reduction-oxidation (redox) reactions, oxidative enzymes, and highly reactive end products, which include ROS (3, 22), in central control of breathing (see, e.g., Refs. 37, 38, 40). A similar role for redox signaling and ROS has been identified in O2 sensing by peripheral chemoreceptors (50). In addition, redox signaling and ROS are a general mechanism employed by many cell types to optimize matching between O2 demand and O2 supply in various organs (1). The fact that hyperoxic hyperventilation occurs at all suggests that brain stem neurons regulating breathing are sensitive to oxidative environments (44). Many neurons in respiratory control nuclei of the brain stem contain oxidative enzymes and O2-derived second messengers that are thought to participate in normal cell-signaling pathways (10, 40, 52, 63, 74). It is conceivable that respiratory-related neurons capable of redox signaling are stimulated and/or inhibited by hyperoxia (PiO2 >160 Torr or >0.21 ATA O2), with the net effect being stimulation of V̇e (i.e., hyperoxic hyperventilation). Mulkey et al. (44) recently proposed a similar hypothesis, explaining, in part, hyperoxic hyperventilation. These authors reported that hyperoxia, ROS, and cellular oxidation directly stimulated firing rate of putative central CO2 chemoreceptors in the solitary complex (SC) in rat brain stem slices. In this context, recall that hyperoxia is a commonly used experimental protocol in studies of oxidative stress at normobaric pressure (3, 6) and hyperbaric pressure (19, 20, 49, 65).
A goal of this mini-review, therefore, is to summarize the effects of hyperoxia on V̇e given its common usage, as cited above. A second goal is to summarize the cellular mechanisms that are proposed to explain the paradox of hyperoxic hyperventilation. In particular, we will focus on recent evidence supporting the hypothesis that central CO2 chemoreceptors are stimulated directly by ROS during hyperoxia (44). A final goal is to examine the practical implications of brain stem O2 and ROS sensitivity relative to our present understanding of common hyperoxic exposures and to suggest future research directions. The importance of the brain stem's sensitivity to redox signaling and oxidative stress, respectively, in normal and abnormal functioning of the respiratory control network remains to be determined, but accumulating evidence suggests that it is probably very important (13, 32, 37-39, 44, 46, 51, 53, 54, 70, 71). From this perspective, it is appropriate to recall the following passage (67): “Interest in studies of oxygen toxicity is not limited to those who contemplate the exotic environments of sea and space. Such studies may lead to clarification of oxygen effects in normal man at sea level and in patients with cardio-respiratory and other diseases.”
NORMOXIA VS. HYPEROXIA AND ROS PRODUCTION
Hyperoxia is defined as any level of Po2, inspired (in vivo) or superfusate (in vitro), that produces a neural tissue Po2 (PtiO2) greater than the range of PtiO2 values that occur in an animal breathing normobaric air (PiO2 of ∼160 Torr) (18, 43, 44). Depending on the region of the CNS, normoxic values for PtiO2 will range from a minimum of ∼3 Torr up to a maximum of ∼35 Torr (18, 43). Therefore, we will consider any value of PtiO2 >35 Torr (PiO2 of >160 Torr) as hyperoxic and capable of increasing ROS levels in the brain stem (6, 19, 20, 25, 65). The O2 continuum that humans routinely encounter in medical and professional settings is very broad, extending up to at least PiO2 = 2,280 Torr (equal to 3 ATA pure O2), which is called “hyperbaric oxygen” (HBO2) (18, 64). At ≤3 ATA O2, the direct effect of pressure per se on the brain stem is thought to be minimal (18, 45). Consequently, any effects of HBO2 on the brain stem are attributed to increased PtiO2 and ROS and not to pressure per se (17, 18, 44, 45).
During hyperoxia, as PtiO2 increases (18, 43), the body's antioxidant defenses are overwhelmed by increased production of ROS in the mitochondria, nucleus, cytosol, membranes, and extracellular fluid compartments (3, 12). ROS include various O2 free radicals and their reactive by-products, such as superoxide, hydrogen peroxide, hydroxyl radicals, nitric oxide, and peroxynitrite (19, 20, 23, 48, 49, 65); this list is by no means complete (3, 22). As reviewed elsewhere (3, 18, 22), ROS can react with various cellular targets, including lipid bilayers (lipid peroxidation), ion channels, and various enzymatic systems, resulting in changes in membrane excitability, synaptic transmission, gene induction, and cellular metabolism. How acute and chronic exposures to excess molecular O2 and ROS affect specific populations of brain stem neurons in the respiratory centers remains to be determined. Initial studies, however, indicate that not all neurons in respiratory-related areas of the brain stem are equally sensitive to hyperoxia and ROS (18, 44, 46). Moreover, previous research, which is summarized next, indicates that a broad range of hyperoxia and ROS stimulates ventilation, suggesting that the respiratory control system responds to a continuum of O2 (and ROS) that ranges from anoxia/hypoxia up through normoxia and hyperoxia.
EFFECTS OF HYPEROXIA ON RESPIRATION
Historically, the direct actions of hyperoxia and ROS on the brain stem at normobaric pressure have rarely been considered when interpreting respiratory control data collected under hyperoxic conditions. The popular interpretation when using hyperoxia under in vivo conditions is that any change in V̇e is related to “chemical denervation” or “physiological chemodenervation” of the peripheral chemoreceptors (see, e.g., Refs. 4, 8, 11, 28, 29, 71). Likewise, for in vitro studies (e.g., brain slices), the popular assumption is that the hyperoxic superfusates (25, 43), which are used routinely as the control O2 tension, have no effect on baseline activity or neuronal responsiveness to the stimulus being tested (see Ref. 18; however, see Refs. 25 and 60 for exceptions to this assumption). Conversely, when subjects are breathing HBO2, the cumulative effects of ROS are well recognized because of their role in CNS O2 toxicity (3, 19, 20, 64-66). Accumulating evidence supports the notion that the CNS responds to a continuum of O2 tension at both normobaric and hyperbaric pressures (6, 29, 34, 60, 73). If true, then we anticipate that the deleterious effects of HBO2 on the brain stem (14, 61, 66) are preceded by early neuronal events beginning at lower levels of PtiO2. We propose that hyperoxic hyperventilation is an example of the brain stem's high sensitivity to ROS, possibly an early physiological response in what eventually develops into impaired respiratory distress and failure during CNS O2 toxicity at large doses of HBO2 (14, 44, 61, 66). It follows then that the direct effects of O2 on the brain stem need to be considered when hyperoxia is used. Dripps and Comroe (21) first introduced the use of normobaric hyperoxia (100% O2, 8-min exposure) as a tool for physiological denervation of the carotid body chemoreceptors. They emphasized, however, the following caveat: “It must be remembered that a stimulant effect of oxygen which tends to increase respiration may be acting simultaneously to limit the extent of this immediate depression of minute ventilation.”
The “stimulant effect of oxygen which tends to increase respiration” (21) is well documented in human infants (16, 56, 58) and adults (4, 15, 21, 28, 35, 36, 55, 57) and various animal models (27, 41, 69). Because the dose of hyperoxia, and therefore ROS, is defined by the duration of the hyperoxic exposure and the level of hyperoxia, it is useful to examine studies that have used short and long exposures as well as different levels of hyperoxia at normobaric and hyperbaric pressures. At normobaric pressure, onset of O2 breathing usually evokes an initial transient hypoventilation (21, 69), which is not always observed (4, 27, 41, 55, 57). Within 1-2 min, V̇e begins to increase and is significantly elevated above the preexposure level within 5 min (28). Short exposures to isocapnic hyperoxia for only 5 min increase V̇e, on average, by ∼16% (28). By comparison, 5 min of isocapnic hypoxia (8-10% O2) increases V̇e, on average, by ∼37% (28).
Longer exposures to isocapnic hyperoxia reveal a larger and sustained hyperventilation that is dose dependent (4, 55, 57). Figure 1 shows the magnitude of the ventilatory response to graded levels of isocapnic hyperoxia breathed for 30 min [solid lines; values given in parentheses show the relative lack of change in end-tidal Pco2 (PetCO2) in Torr during isocapnic hyperoxia]. Switching from 21% to 30, 50 and 75% O2 increased V̇e, respectively, by 21, 61, and 115% (4). Figure 1 also shows that the magnitude of the hyperoxic hyperventilation is greatly reduced if PetCO2 is not regulated (i.e., poikilocapnic hyperoxia) (4). In this example, breathing 30 min of 75% poikilocapnic hyperoxia decreased PetCO2 from 41.3 Torr (air) to 37.7 Torr and increased V̇e by only 16% (dotted line in Fig. 1). In this same example (Fig. 1), V̇e increased by 115% during 75% isocapnic hyperoxia. Similar results were reported with 55 min and 8 h of isocapnic hyperoxia vs. poikilocapnic hyperoxia (55, 57) [for 8 h of hyperoxia, end-tidal Po2 was maintained at 300 Torr so as to avert pulmonary O2 toxicity (55)]. The present interpretation is that O2-induced hyperventilation results in hypocapnia, which combines with reduced activity by the peripheral chemoreceptors in high O2 to blunt the magnitude of the O2-induced hyperventilatory response (4, 57). In some cases, hyperventilation is completely masked during poikilocapnic hyperoxia compared with isocapnic hyperoxia (55). This may explain why the hyperoxic hyperventilation response has been missed by many investigators who have used poikilocapnic hyperoxia in their research.
In addition, the example in Fig. 1 illustrates that the ventilatory response to 30 min of isocapnic hyperoxia, at all levels of PiO2 tested, is not immediately reversible, suggesting that O2 evokes a long-lasting facilitation of V̇e. In this context, it is interesting to note that hyperoxia has another time-dependent stimulatory effect on respiration, which is dependent on the activity of neuronal nitric oxide synthase: hyperoxic potentiation of the hypoxic ventilatory response (29). Prebreathing 760 Torr O2 (1 ATA O2) for only 10 min increases the magnitude of the subsequent ventilatory response to isocapnic hypoxia, indicating that “hyperoxia may alter tonic outputs for central respiratory drives” (29).
Under hyperbaric conditions, hyperoxia has similar stimulatory effects on ventilation, at least initially. Prolonged exposure to high levels of HBO2 evokes irregular breathing and respiratory distress and, ultimately, respiratory failure in a dose-dependent fashion (61, 66). In 1921, Dautrebande and Haldane (15) reported that, for subjects who breathed 760 and 1,580 Torr (1.0 and 2.08 ATA) O2 for 5 min, PetCO2 was decreased by 1.5 and 3.5 Torr, respectively, which they attributed to the central effect of O2 causing graded hyperventilation (72). Lambertsen et al. (36) showed that breathing 2,660 Torr (3.5 ATA) poikilocapnic hyperoxia increased V̇e by 26%, primarily by increasing Vt, which then decreased PetCO2 by 6.7 Torr. In anesthetized rats, 30-60 min of 3,040, 4,560, 6,080 Torr (4.0, 6.0 and 8.0 ATA) O2 produced a dose-dependent increase in V̇e by increasing both Vt and fresp. Sectioning the glossopharyngeal nerves abolished the fresp response, but not the Vt response, to HBO2. Overall, a significant hyperventilation was still observed during O2 breathing at 4.0 and 6.0 ATA O2 but not at 8.0 ATA O2 because of a large reduction in fresp. Presumably, the inhibitory effects of 8.0 ATA O2 on fresp were due to CNS O2 toxicity (3, 61). Together, these findings and others (27, 41, 44) strongly suggest that hyperoxic hyperventilation is of central origin.
Indeed, prolonged exposure to HBO2 has deleterious effects on respiration. Simon and Torbati (61) found that continuous exposure to 2,280 and 3,800 Torr (3 and 5 ATA) O2 has profound effects on fresp (Vt was not measured) in awake rats. Rats who breathed 2,280 Torr (3 ATA) O2 had increased fresp above air control levels for ∼2 h, after which fresp declined to a minimum level (below air control) by 3 h. Next, fresp increased, in some cases, above the air control level, and clinical convulsions occurred along with shallow breathing, gasping, and restlessness. This period of respiratory distress was followed by respiratory failure and usually death after 6 h. Breathing 3,800 Torr (5 ATA) O2 induced the initial hyperventilation, but fresp became erratic and rapidly decreased after ∼15-20 min of HBO2 breathing, coincident with seizures and followed thereafter by unstable breathing. In the context of disordered breathing patterns caused by HBO2, it may be significant that normobaric hyperoxia, under certain conditions (e.g., sleep, periodic breathing), also destabilizes breathing, causing irregular breathing patterns and apnea (5, 13, 71).
In summary, these studies reveal several important features of O2 sensitivity of the respiratory control system as revealed by the ventilatory response to hyperoxia. 1) The brain stem areas controlling ventilation are extremely sensitive to increased PtiO2. Increasing O2 from 21% to only 30% while maintaining PetCO2 isocapnic caused a significant increase in V̇e. The increase in V̇e typically occurs within 1-2 min of exposure to hyperoxia. 2) Increasing PiO2 further from 0.21 to 0.50, 0.75, and 1.0-8.0 ATA O2 shows that the initial hyperventilatory response to O2 is dose dependent. 3) The ventilatory response to 30 min of isocapnic hyperoxia is not immediately reversible, suggesting O2 evokes a long-lasting facilitation of breathing (4, 29). 4) The hyperventilatory response shows only slight adaptation after 8 h when care is taken to avert pulmonary O2 toxicity at normobaric pressure (57). Conversely, prolonged exposures to HBO2 initially stimulates breathing but then leads to respiratory distress and, ultimately, respiratory failure. 5) O2-induced hyperventilation significantly reduces PetCO2 (4, 16, 35, 36, 57), which blunts or masks (55) the magnitude of the O2-induced hyperventilation. Thus hyperoxic hyperventilation is best measured during isocapnic hyperoxia (4, 55, 57).
PREVIOUSLY PROPOSED MECHANISMS OF HYPEROXIC HYPERVENTILATION
Seven potential mechanisms have been proposed over the years to explain hyperoxic hyperventilation. The most frequently cited hypotheses are that hyperoxia 1) decreases cerebral blood flow (CBF) and 2) decreases formation of reduced hemoglobin, thereby interfering with CO2 transport (the “Haldane effect”). The net effect is to increase Pco2 in the cerebral vasculature and neural tissue, which stimulates central CO2 chemoreceptors (16, 35, 36, 55). 3) Lactic acidosis resulting from HBO2-induced histotoxic hypoxia within the brain stem may also stimulate central chemoreceptors (14). These effects of CO2 and H+ are likely to be counterbalanced by the rapidly ensuing respiratory alkalosis that results from O2-induced hyperventilation (16). 4) In addition, it was proposed that O2 disinhibits an inhibitory input that is normally active in normoxia (41). 5) Long exposures to hyperoxia, resulting in pulmonary O2 toxicity, result in pulmonary congestion, irritation, and atelectasis, which may stimulate respiratory reflexes, leading to hyperventilation (cited in Refs. 16 and 35). Pulmonary O2 toxicity will be a factor with long exposures to hyperoxia, but it is not expected to contribute to the rapid hyperventilation that occurs after only 1-2 min of O2 breathing (28). 6) It has also been proposed that O2 directly stimulates respiratory neurons, which results in increased ventilation. No data were provided to support this hypothesis or which type of neuron may be involved (14, 41).
Recently, Mulkey et al. (44) provided the first evidence that neurons in the SC of the dorsocaudal medulla oblongata are directly stimulated by hyperoxia. The following section summarizes the primary findings in their study, which forms the basis for the hypothesis that 7) hyperoxic hyperventilation is initiated by increased production of ROS during hyperoxia, which directly stimulates central CO2 chemoreceptors in the brain stem (44).
O2 AND ROS DIRECTLY STIMULATE PUTATIVE CO2 CENTRAL CHEMORECEPTORS
The SC is one of several sites of CO2 chemoreception distributed throughout the brain stem (47, 59). Neurons in chemosensitive areas, such as the SC, share several features that suggest a common function in CO2 chemoreception (59). One of these features is the overwhelming tendency of certain neurons in chemosensitive areas to exhibit an increased firing rate and increased membrane input resistance (i.e., net decreased membrane conductance) during hypercapnic acidosis that is strongly dependent on intracellular pH (pHi) (26, 59).
In the study by Mulkey et al. (44), HBO2 was used to examine the effects of hyperoxia on SC neurons in the brain slice preparation. Normobaric hyperoxia was not used because the control level of O2 used in all brain slice studies is, by definition, already hyperoxic (0.95 O2 × 1 ATA = 0.95 ATA O2) (18, 25, 43). Consequently, HBO2, exposing brain slices to O2 at barometric pressure >1 ATA (17, 43, 44), is the only way to study the effects of further hyperoxia on neurons in the brain slice model, at least until a newer, lower (more physiological?) level of control O2 is established experimentally for the brain slice preparation (18, 25). The levels of HBO2 used (2-3 ATA O2) to test for O2 sensitivity produced a range of PtiO2 in the slice equivalent to the level of PtiO2 in the intact brain of a rat exhibiting signs of CNS oxygen toxicity (18, 43, 44). Thus the test levels of O2 used in these studies (17, 18, 43, 44) were physiologically relevant for evaluating O2 sensitivity of neurons.
The three main findings in the study of Mulkey et al. (44) were as follows. 1) Not all neurons in the SC are equally sensitive to hyperoxia. Sixty-two percent of the neurons tested showed no significant change in their excitability during ∼10 min of hyperoxia ranging from 2 to 3 ATA. Thirty-eight percent of the SC neurons tested, however, were stimulated by hyperoxia, showing increased membrane input resistance and increased firing rate. 2) The neurons stimulated by hyperoxia were the putative central CO2 chemoreceptors: 90% of the O2-sensitive neurons were also stimulated by hypercapnic acidosis, whereas 81% of the O2-insensitive neurons were CO2 insensitive. Figure 2A shows the integrated firing rate response of a SC neuron that was stimulated by normobaric hypercapnic acidosis (left) and hyperbaric hyperoxia (right) [the cell showed no significant response to pressure per se on compression with the inert gas, helium; not shown (45)]. When CO2-excited neurons were exposed to hyperoxia, there was usually a small increase in membrane input resistance, a small depolarization, and a significant increase in firing rate (44). 3) The ability of hyperoxia to increase firing rate and input resistance could be blocked by coexposure with an antioxidant and mimicked by exposure to chemical oxidants at control levels of Po2. Figure 2B shows the firing rate response of an O2-sensitive neuron that was blocked, reversibly, by coexposure with Trolox-C (a membrane-permeable analog of vitamin E). The present working model is that hyperoxia increases ROS, which inhibit a potassium channel(s), although the specific ionic targets have not been identified (17, 44, 46).
The above results indicate that putative central CO2 chemoreceptors will be the first neurons in the SC affected by breathing hyperoxia, suggesting that they are especially sensitive to O2 and ROS. This may explain why, in humans, there is a significant linear correlation (r = 0.83) between the magnitude of the ventilatory response to hypercapnia and the magnitude of the ventilatory response to 0.75 ATA isocapnic hyperoxia (4). The above findings also are noteworthy in that they provide the first evidence that stimulation of putative central CO2 chemoreceptors occurs in the absence of any secondary changes in Pco2 caused by decreased CBF and diminished reduction of hemoglobin (Haldane effect), which, until now, have been the favored mechanisms for explaining hyperoxic hyperventilation (4, 16, 35, 36, 56, 57).
In the intact animal, however, the cumulative effects of O2 and ROS and CO2/H+ (due to decreased CBF, Haldane effect, and so forth) on the brain stem are additive, especially during poikilocapnic hyperoxia. Figure 3 shows that the combined effect of CO2 and O2 on putative central CO2 chemoreceptors is to produce a large stimulation of firing rate (44). In these experiments, each neuron was stimulated by exposure to hypercapnia alone (Fig. 3, A-C), hyperoxia alone (Fig. 3, A and B), or chemical oxidant alone [Fig. 3C; N-chlorosuccinimide (NCS)]. When the same level of hypercapnia was combined with hyperoxia (Fig. 3, A and B) or NCS (Fig. 3C), the firing rate response of each cell was at least additive (Fig. 3B) and often greater (Fig. 3, A and C). Recent research indicates that chemical oxidants also have a large effect on pHi of SC neurons, causing a significant intracellular acidification and slowing of Na+/H+ exchange (46). The bases for these interactions between O2, ROS, CO2, and pHi require further investigation.
FUTURE DIRECTIONS FOR RESEARCH
There are at least three implications of brain stem O2 sensitivity that need to be considered in future studies given the present uses of hyperoxia in clinical and basic research. First, experiments that use O2 breathing have to take into account the central effects of increased O2 (4, 21). Future research should establish the temporal relationship between electrical signaling in neurons in central chemosensitive areas and electrical signaling by peripheral chemoreceptors during hyperoxia and hypoxia. Until that occurs, it has to be assumed that the initial hypoventilation [peripheral chemoreceptor inhibition by O2 and ROS (34, 68)] may be limited by the simultaneously occurring stimulation of V̇e [central chemoreceptor stimulation by O2 (44) and the secondary increase in CO2/H+ (4, 16, 35, 36)], as first proposed by Dripps and Comroe in 1947 (21). Thus caution should be exercised in attributing any change in V̇e during hyperoxia solely to “peripheral chemo-denervation.” For example, the central effects of O2 and ROS may be a factor in studies of periodic breathing (71), assessing peripheral chemoreceptor function in infants (8), characterizing the hypoxic ventilatory response (29), and studying interactions between the peripheral CO2 and central CO2 chemoreceptors (11). In particular, if O2 breathing lasts for more than 1-2 min, the increase in V̇e will affect results of ventilatory response tests (4). In addition, the ensuing arterial hypocapnia from hyperoxic hyperventilation will alter cerebral vascular tone and thus CBF and intracranial pressure. This latter issue becomes especially important when considering the effects of O2 therapy on CBF and intracranial pressure in cases of brain trauma (57). As previously stated (4, 57), it is premature to consider altering the present O2 therapy protocols to account for the effects of hyperoxic hyperventilation on CBF and acid-base balance until the appropriate clinical trials are conducted.
Second, because hyperoxia is a useful model of oxidative stress (3, 6, 18) and perturbs breathing, as discussed above, future research will want to determine how O2 and other oxidative conditions affect respiratory control. If redox signaling is key to normal respiratory control, then excessive amounts of oxidative stress may negatively impact normal cellular mechanisms of control. Thus it will be important in future studies to determine the effects of acute and chronic oxidative stress on respiratory rhythmogenesis and the various afferents that alter ventilation, for example, peripheral and central chemoreceptors (34, 42, 44). In addition, future studies will want to determine the effects of a broad range of O2 on neural activity and the mechanisms of hyperoxia-induced changes in neuronal plasticity, such as long-term facilitation (4, 29). Furthermore, it also will be important to determine the effects of oxidative stress over the course of development and maturation of the respiratory control system. In these contexts, it may be significant that normobaric hyperoxia increases the incidence of sleep-related apneic episodes in rats (13), introduces periodic breathing and destabilizes breathing further during periodic breathing in lambs (71), and destabilizes breathing in infants presenting with recurrent apnea and cyanosis (5). Likewise, other forms of chronic oxidative stress, caused by metabolic defects, may render the developing brain stem network vulnerable to impaired functioning under certain conditions (32, 54, 62). For example, high levels of iron (53, 70) and low levels of melatonin (39) result in chronic oxidative stress and occur in children that die from sudden infant death syndrome.
Finally, because of extensive use of in vitro tissue preparations of the CNS in respiratory control studies, and neuroscience in general, it is appropriate to consider the potential effects of a hyperoxic control solution on neuronal function. As discussed elsewhere (18), nearly all brain slice experiments are conducted under conditions of hyperoxia with 95% (0.95 ATA) O2 (43). The PtiO2 profile in a submerged, 300-μm-thick rat brain stem slice is equivalent to the PtiO2 in the cortex of a rat breathing 2.2-2.9 ATA O2 (18, 43). This means that the control PtiO2 level used in nearly all brain slice studies is effectively “HBO2” minus the physical pressure component (18). What effect this level of hyperoxia has on ROS production in various in vitro preparations requires further study (18, 25). However, the fact that hyperoxia has long-lasting effects on ventilation (4, 29), and selectively stimulates certain populations of SC neurons (44), is certainly a cause for concern. For example, recent pHi measurements in brain stem slices indicate that pHi of neurons in the SC, specifically within the nucleus tractus solitarius, decreases as PtiO2 decreases (mean ± SE): 95% O2 (7.44 ± 0.01), 60% O2 (-0.10 ± 0.01 ΔpH unit, where Δ indicates change), 20% O2 (-0.16 ± 0.01 ΔpH unit), and 0% (-0.16 ± 0.01 ΔpH unit) (Potter SJ, Putnam RW, and Dean JB, unpublished findings). This demonstrates that the use of a lower level of control O2 will affect “control” pHi in SC neurons, which is anticipated to significantly affect excitability of CO2-chemosensitive neurons and any other pHi-dependent processes that occur in other SC neurons.
Furthermore, levels of O2 in in vitro preparations of the CNS that are often referred to as hypoxia are in fact physiological (PtiO2 <35 Torr) (18, 43), and accounting for this fact may alter our interpretation of the responses of the brain to true hypoxia. Given the significant effects that elevated O2 levels have on ventilation, further studies of the role of hyperoxia on brain stem neurons are clearly of interest. Identifying the effects of the O2 continuum on basic cellular properties, which includes anoxia/hypoxia through normoxia and hyperoxia, we will gain insight into the O2 sensitivity of the brain stem and the role that redox signaling plays in respiratory control (1, 22, 37, 38, 44) as well as the possible role for oxidative stress in diseases of respiratory control (22, 32, 39, 53, 54, 62, 67, 70).
Funding for research presented from the authors' laboratories was provided by grants from the Office of Naval Research, Undersea Medicine Program (N000140110179 to J. B. Dean and R. A. Henderson and DURIP-ONR N000140210643 to J. B. Dean, R. W. Putnam, and R. A. Henderson) and the National Heart, Lung, and Blood Institute (R01 HL-56683 to R. W. Putnam and J. B. Dean).
The authors acknowledge Phyllis Douglas for excellent technical assistance in studies discussed in this mini-review.
- Copyright © 2004 the American Physiological Society