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Am J Physiol Regul Integr Comp Physiol 281: R528-R538, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 2, R528-R538, August 2001

Brain stem PO2 and pH of the working heart-brain stem preparation during vascular perfusion with aqueous medium

R. J. A. Wilson1, J. E. Remmers1, and J. F. R. Paton2

1 Department of Medical Physiology and Biophysics, Heritage Medical Research Building, University of Calgary, Calgary, Alberta, Canada; and 2 Department of Physiology, University of Bristol, Bristol BS8 1TD, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The rat working heart-brain stem preparation (WHBP) is an in situ preparation having many of the advantages associated with in vitro preparations while retaining cardiovascular response functionality and an eupnoeic respiratory motor pattern. The preparation is perfused arterially with an aqueous medium having a much lower oxygen-carrying capacity than blood. To evaluate the efficacy of the artificial perfusion in providing adequate gas exchange within the brain stem, we used polarographic PO2 and pH microelectrodes to determine the tissue PO2 and pH of the medulla oblongata at various depths. When the perfusate was equilibrated with 5% CO2 and 95% O2, average tissue PO2 was 294 Torr and no hypoxic areas were encountered. Tissue pH was remarkably uniform throughout the tissue, and on average was only 0.04 ± 0.02 pH units more acidic than that of the perfusate. Increasing the PCO2 of the perfusate increased tissue PO2 and decreased arterial resistance. Decreasing perfusate PCO2 (while keeping pH constant) decreased tissue PO2 and reduced the respiratory activity. These results suggest that arterial PCO2, independent of arterial pH, is an essential variable in determining both respiratory drive and cerebrovascular perfusion. We conclude that the medulla of the WHBP is oxygenated and within a physiological pH, which accounts for the eupneic pattern of respiratory motor activity it generates. Furthermore, this preparation may be a useful model for exploring mechanisms of central chemoreception as well as the dynamics of the cerebral vasculature responses following changes in blood gases.

cardiovascular; respiration; breathing; eupnea


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

SUPERFUSED ISOLATED MAMMALIAN brain stem en bloc and slice preparations have propelled respiratory neuroscience in recent years (for reviews, see Refs. 2, 33, 17). Without afferent inputs and blood perfusion, they have a simplicity and mechanical stability lacking in their in vivo counterparts. Such preparations have led to both the identification of the preBotzinger Complex as an important site for respiratory rhythmogenesis (e.g., Refs. 13, 39) and significant advances in determining the cellular properties and modulation of many classes of rhythmically active neurons (e.g., Refs. 5, 16, 26). Whereas these preparations have served well in studies of a discrete site of rhythmogenesis, their usefulness in investigating how the complete respiratory circuit generates eupnoeic breathing (i.e., "eupnogenesis") has been questioned (11, 40, 41).

One of the major drawbacks of rat and mouse superfused en bloc brain stem preparations is that they have anoxic and very acidic cores (pH 6.5) (6, 22). Their use is limited to neonatal preparations, and they must be maintained under conditions of severe hypothermia (27°C). Under these conditions, peripheral circuitry within 450-700 µm of the ventral surface is oxygenated (e.g., Refs. 6, 22), but the properties and function of deeper circuits are likely to be compromised. Furthermore, the cervical spinal bursts produced by superfused brain stem preparations differ from those produced by other neonatal preparations lacking anoxic cores (11, 45). For example, they have a rapid-onset decrementing shape (a characteristic of gasping) that is insensitive to strychnine and bicuculline (5, 25). Slice preparations are well oxygenated throughout (e.g., Ref. 1), but the preBotzinger slice is rhythmogenic only if from neonates and in the presence of elevated K+ (39). Thus the behavioral significance of the mechanism of rhymogenesis in these preparations is unclear (e.g., Refs. 11, 31, 41). Finally, whereas the simplicity deafferentation affords can have considerable advantages in determining central neuronal mechanisms, deafferentation also precludes the use of these preparations for studying the interactions between respiratory rhythmogenesis and autonomic reflexes as well as kinesiological aspects of ventilatory control such as those regulating the upper airway.

To understand the neuronal control of eupnoeic respiration in mammals, we have turned to a working heart-brain stem preparation (WHBP) (27-29). This preparation is perfused artificially with an aqueous medium through the descending aorta. Despite the low O2- and CO2-carrying capacity of this medium, the preparation is highly reflexive and allows studies of autonomic reflexes both in neonates and adults. Importantly, the brain stem of the WHBP generates robust respiratory bursts, having a pattern comparable with that of eupnea in vivo but with improved mechanical stability for intracellular analysis.

Although the phrenic nerve motor pattern generated by the WHBP (or in vivo) is exquisitely sensitive to PO2 levels and was used originally as an index of the adequacy of central oxygenation (27), tissue PO2 has not been measured. With the growing interest in the preparation (e.g., 7, 9, 11, 30, 32, 42), we have performed PO2 and pH depth profile measures through the medulla. We show that arterial perfusion with an aqueous medium can provide adequate brain stem oxygenation and a normal tissue pH. Furthermore, we demonstrate that cerebral vascular responses are intact and that the respiratory activity is reduced during hypocapnic perfusion at normal physiological pH. We suggest that this preparation offers unique advantages for studying eupnogenesis per se, its reflex control, and cerebrovascular responses in mammals.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

WHBP. Wistar rats (120-150 g, 4-6 wk old) were kept according to standards set by the home office guidelines. Eight rats were used for tissue PO2 measurements, and five rats were used for tissue pH measurements. WHBPs were prepared as described previously (27). Briefly, rats were anesthetized deeply in halothane until respiration was depressed and animals failed to respond to noxious paw pinch. Animals were then transected below the diaphragm, and the head, neck, and thorax were immersed in ice-cold artificial cerebrospinal fluid [ACSF; consisting (in mM) of 125 NaCl, 5 KCl, 1.25 MgSO4, 24 NaHCO3, 1.25 KH2PO4, 2.5 CaCl2, and 10 D-glucose] equilibrated with 95% O2 and 5% CO2 (pH of ~7.4). Preparations were decerebrated at the precollicular level, and all intracranial tissue rostral to this transection was aspirated. The dorsal roof of the cranium, cerebellum, and choroid plexus was removed, exposing the fourth ventricle. The thorax was opened by removing the ventral portion of ribs T8-T12, and the right phrenic nerve was isolated. Preparations were subsequently transferred to a recording chamber where the descending aorta was cannulated with a double-lumen catheter. Preparations were artificially perfused retrogradely through the catheter with ACSF plus 1.25% ficoll (Sigma) at 31°C and equilibrated with one of two tanks of premixed gas containing either 4.99 or 5.99% CO2 in O2. (Note that experiments involving PO2 measurements used the former gas mixture, whereas those involving tissue pH measurements used the latter.) Modified ACSF equilibrated with these gases had pH's of ~7.40 and 7.35, respectively. The modified ACSF, which was passed through a bubble trap and a 24-µm filter before reaching the preparation, was driven by a peristaltic pump (Watson-Marlow 502S). The aortic pressure at the tip of the cannula was monitored using a Statham pressure transducer (Gould). Perfusate leaked from numerous cut vessels in the preparation and was collected and recirculated following reoxygenation (total volume of perfusate ~200 ml). Rhythmic contractions of respiratory muscles recommenced a few minutes after the initiation of perfusion. These movements were arrested by paralyzing the preparation with 0.3 µg/ml vecuronium bromide. Recording of the central respiratory rhythm was obtained from a phrenic nerve (PRN) using a suction electrode (tip diameters ~100 µm). To obtain this rhythm, preparations require "tuning," which was achieved by adjusting the rate of arterial perfusion by adjusting pump speed. Tuning was successful in 95% of preparations. Typical flow rate used was 30 ml/min. Preparations remained viable, as judged by the ability to produce a eupneic motor pattern during control conditions, for the duration of the experiment (2-3 h).

pH and PO2 electrodes. A macro combination pH electrode (Corning) was placed in a well of perfusate produced by the walls of the empty cranium. This electrode was calibrated against commercial pH buffers (Sigma). Tissue pH was measured using pH microelectrodes with beveled tips of 2-10 µm (#823, Diamond General) and a pH amplifier (model 2000, A-M Systems). The reference of the macro combination electrode doubled as the reference for the pH microelectrodes. pH microelectrodes were calibrated after each experiment in the well produced by the walls of the empty cranium. To calibrate, we manipulated the pH of the perfusate in the well by changing the fractional concentrations of CO2 in the equilibrating gas mixture between ~5 and 8%. This allowed us to calibrate the pH microelectrodes in the cranium against macroelectrode measurements. Tissue PO2 was monitored using Clark-style microelectrodes with guard cathodes (tip diameter 25-30 µm; model 737GC, Diamond General) and a polarographic amplifier (model 1900, A-M Systems). The PO2 electrodes were calibrated after each depth profile in tonometers equilibrated with either 95% O2 and 5% CO2 or 100% N2. Previously, we found these electrodes have a mild flow sensitivity (the PO2 reading was 1.9% greater with flow than without), although this difference was not significant statistically (paired t-test: P = 0.09) (46).

pH and PO2 tissue depth profiles. Electrodes were attached to a motorized drive (Nanostepper, Scientific Precision Instruments) and positioned over either 1) the dorsal-lateral surface of the medulla corresponding to 1 mm rostral to calamus scriptorius and ~2.0 mm lateral to midline [so that tracks would intersect the ventral respiratory group (VRG)] or 2) the dorsal medial surface around calamus scriptorius and 0.5 mm lateral to midline so that tracts would pass through the nucleus of the solitary tract (NTS). Each profile began by lowering the electrode to the surface of the tissue, which was determined visually as a slight dimpling at the surface when viewed at ×60 magnification using a dissection microscope (Zeiss). Electrodes were then lowered through the tissue at a rate of one 7-µm step every 1/2 s, with pauses of 20 s every 49 µm to make measurements (20 s was about twice the time for readings to equilibrate).

Perfusion manipulation. To determine whether cerebral vascular responses were intact, we equilibrated the perfusate with a number of different premixed gas mixtures. The fractional concentration of gases in these mixtures was determined using CO2 (901.Mk2, PK Morgan) and O2 (type OA 250, Taylor ServoMex) gas analyzers. These gas mixtures included 1) hypercapnic-hyperoxia (8.48% CO2 in O2), 2) hypercapnic-hypoxia (8.75% CO2, 5% O2 in N2), 3) normocapnic-hypoxia (5.42% CO2, 5% O2 in N2), and 4) anocapnic-hypoxia (8% O2 in N2). In some experiments, a HEPES-buffered perfusate (HEPES perfusate) was used. This consisted of modified ASCF in which the sodium bicarbonate was exchanged for 25 mM HEPES (pH adjusted to 7.35 with NaOH). HEPES perfusate was equilibrated with 100% O2 unless otherwise stated.

Data acquisition and analysis. Data were digitized (Cambridge Electronic Design 401) and archived as computer files using Spike2 software (Cambridge Electronic Design). Nerve recordings (digitized at 2.5 kHz), arterial pressure (digitized at 20 Hz), tissue PO2 or pH readings (digitized at 100 Hz), and digital pulses signaling stepwise displacements of the electrodes were recorded concurrently within the same file. Files were analyzed in either Spike2 software or exported to Axograph (Axon Instruments). Burst duration (i.e., Ti) and frequencies were determined before smoothing. To determine when neuronal activity peaked within phrenic bursts, extracellular nerve recordings were full-wave rectified and digitally filtered (3-Hz cutoff). An automated event detection routine was used to identify phrenic bursts. The occurrence of the peak was expressed as a percentage of the width of the smoothed burst at one-half amplitude. The neuronal equivalent to minute ventilation was calculated by integrating the smoothed bursts over a 60-s period. For depth profiles, values of pH and PO2 at the end of pause periods were used (allowing the readings 20 s to settle). In most preparations, the effects of altering perfusate on tissue PO2 or pH were determined at a depth of 2.2 mm. Measurements were made immediately before changing perfusate properties for the "before" and "during" conditions and once readings had reached steady state for the "after" condition. Note, therefore, that the measurements of PO2, pH, and arterial pressure provide information as to the direction of effect, not the absolute magnitude at steady state. Data are expressed as means ± SE. "Average tissue" pH and PO2 were calculated from depth profile data by determined animal average values from dorsal lateral tracts. These values (from all animals) were then averaged. Paired t-tests were used for statistical comparisons of dual conditions. Repeated-measures ANOVA was used for analysis of multiple conditions. If multiple-condition data failed a normality test, Friedman repeated-measures ANOVA on Ranks was used. After the ANOVA, the Bonferroni t-test was used to test for post hoc significant difference between one condition and remaining conditions. The Student-Newman-Keuls (SNK) method was used for all other post hoc testing.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Under control conditions (perfused with ACSF equilibrated with 95% O2 and 5% CO2, pH 7.4, 31°C), 12 of the 13 preparations used in this study produced rhythmic augmenting phrenic discharges similar to those reported previously for this preparation (27, 28). Bursts in these preparations occurred at a rate of 13.9 ± 2.0 bursts per minute (n = 12) and had a duration (i.e., Ti) of 0.98 ± 0.10 s with the peak occurring at 70 ± 2% of the duty cycle (e.g., Fig. 1A). The other preparation, which had an inadvertent unilateral lesion in the rostral pons, produced long-duration (>4 s) phrenic bursts that peaked within the first 1.5 s.


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  		Fig. 1.   Phrenic discharges and PO2 depth profiles through the medulla in a working heart-brain stem preparation (WHBP). A: burst triggered average of phrenic discharges produced by 1 of the WHBPs used in this study during control conditions (perfused with modified perfusate equilibrated with 4.99% CO2 in O2, pH ~7.4 at 31°C). Solid line shows average of 20 bursts, dashed lines depict confidence limits (2 × SE). B: average of PO2 depth profiles from microelectrodes placed initially at the level of calamus scriptorius and 0.5 mm lateral to the midline so to tract through the nucleus of the solitary tract (NTS). C: average of PO2 depth profiles from the dorsal-lateral surface of the medulla, 1 mm rostral to calamus scriptorius, and ~2 mm lateral to the midline aimed at the ventral respiratory group (VRG). Depth profiles were performed during control conditions. Points in B and C are means ± SE from 3 and 8 preparations, respectively.

Tissue PO2. When the WHBP was perfused with modified ACSF equilibrated with 95% O2 and 5% CO2 to give a PO2 of 720 ± 1 Torr, the PO2 in the perfusate above the ventral surface of the brain stem was 561 ± 58 and the average tissue PO2 was 293.7 ± 45 Torr (n = 8). The lowest tissue PO2 encountered was 29 Torr (recorded in 1 preparation immediately below the dorsal-lateral surface). The PO2 of the preparation that had nonaugmenting long-duration bursts was one of the best oxygenated; the lowest PO2 recorded in this preparation was 164 Torr at a depth of 300 µm.

Tissue PO2 varied systematically with depth. In the region of the NTS (Fig. 1B), tissue PO2 increased from 300 ± 63 Torr just below the surface to 457 ± 65 Torr at a depth of 1 mm (n = 3). For depth profiles from the dorsal-lateral surface 1 mm rostral to calamus scriptorius and aimed at the VRG, tissue PO2 also varied with depth (ANOVA: P < 0.001, n = 8; Fig. 1C). Within the first 700 µm of the surface, average tissue PO2 increased, reaching a maximum of 349 ± 53 Torr. With greater depth, tissue PO2 decreased, reaching a minimum of 215 ± 43 Torr at 1,300 µm (the PO2 at 1,300 µm was significantly less than that at 500-800 µm; Bonferroni t-test, P = 0.01-0.03). In five of eight preparations, a second peak was observed at 1,800 µm. However, overall tissue PO2 at the second peak was not significantly different to that at 1,300 µm.

Effects of hypercarbia on tissue PO2 and arterial pressure. Increasing the level of hypercarbia from 4.99 to 8.48% (Fig. 2) caused a significant increase in tissue PO2 (SNK: q = 9.32, P < 0.001, n = 7), despite the slight decrease (i.e., 3.49%) in the fractional concentration of oxygen in the gas mix used to equilibrate the perfusate. This treatment produced a statistically significant fall in arterial pressure (SNK: q = 5.59, P < 0.01, n = 6).


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  		Fig. 2.   Tissue PO2 and arterial pressure in response to perfusion with hypercapnic-hyperoxic perfusates. A: brief exposure to hypercapnic-hyperoxic perfusate caused increases in tissue PO2 (measured 2.2 mm below the dorsolateral surface of the medulla, 1 mm rostral to calamus scriptorius; top trace) and a fall in arterial pressure (measured in the descending aorta; bottom trace). Tissue PO2 and arterial pressure data summarized in B and C, respectively. During the "before" and "after" condition, preparations were perfused with bicarbonate-based perfusate equilibrated with 5% CO2 in O2. *Significant difference (P < 0.05) from control. No significant difference from control. Bars show means ± SE.

The influence of hypercapnic perfusate on tissue PO2 (Fig. 2) suggests that the WHBP retains vascular responses to changes in arterial PCO2 and/or pH. To determine whether arterial PCO2 triggered these responses independently of arterial pH, we performed the experiment illustrated in Fig. 3. When the control perfusate (equilibrated with 5% CO2 and 95% O2) was exchanged for a hypocapnic HEPES perfusate of the same pH (equilibrated with 100% O2), tissue PO2 approached zero within 60 s (SNK: q = 5.69, P < 0.01, n = 4; Figs. 3 and 4A). Whereas the most likely explanation for this finding is that vasoconstriction was triggered by the reduction in CO2, we found no evidence for an increase in arterial pressure. On the contrary, in all four preparations, arterial pressure decreased, although this trend was not significant (SNK: q = 3.17, P = 0.07, n = 4; Figs. 3 and 4B). Changing from normocapnic bicarbonate perfusate to hypocapnic HEPES perfusate and then back again had a significant effect on the neuronal equivalent of minute ventilation (ANOVA: F = 6.06, P > 0.05; Fig. 4C). Changing to the hypocapnic HEPES reduced burst frequency in three of four preparations (to 50 ± 10% of control) and reduced burst amplitude in all four preparations (to 72 ± 7% of control). Burst duration, on the other hand, was reduced only slightly (to 94 ± 11% of control).


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  		Fig. 3.   Arterial PCO2 was essential for maintaining tissue PO2 in the WHBP. Replacing a bicarbonate-based perfusate (equilibrated with 5% CO2 in O2) with 25 mM HEPES perfusate (equilibrated with 100% O2) of the same pH caused a rapid and reversible reduction in tissue PO2 (top trace), a depressor response (middle trace), and a reduction in the frequency of respiratory bursts (bottom trace). Recordings were made 2.2 mm below the dorsal-lateral surface of the medulla. ACSF, artificial cerebrospinal fluid.



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  		Fig. 4.   Summary data demonstrating dependence of PO2 (A, D), arterial pressure (B, E), and respiratory activity (C, F) on perfusate PCO2. A-C: effect of switching from a bicarbonate-based perfusate equilibrated with 5% CO2 in O2 to a HEPES perfusate equilibrated with 100% O2 (n = 4). D-F: effect of switching perfusate from HEPES perfusate, equilibrated with 5% CO2 in O2, to HEPES perfusate equilibrated with 100% O2 (n = 3). Measurements made 2.2 mm below the dorsal-lateral surface of the medulla. Bars show means ± SE. *Significant difference (P < 0.05) from control unless indicated otherwise. nsd, No significant difference from control. The neuronal equivalent of minute ventilation was determined by integrating smoothed phenic bursts over 60 s.

Interestingly, when the HEPES perfusate was terminated and returned to the control bicarbonate perfusate, the tissue PO2 increased and overshot the control level by 214 ± 78 Torr (SNK: q = 4.45, P < 0.05, n = 4; Figs. 3 and 4A). Whereas arterial pressure increased on returning to bicarbonate-based perfusate (SNK: q = 5.73, P < 0.02, n = 4; Fig. 4B), the increase was not significantly greater than control levels (SNK: q = 2.56, P = 0.12, n = 4). The drop in tissue PO2 may have been caused by the addition of HEPES to the perfusate or the removal of CO2 and bicarbonate. However, we found that switching a HEPES perfusate equilibrated with 5% CO2 and 95% O2 to a perfusate of the same composition but equilibrated with 100% O2 also caused a precipitous fall in tissue PO2 (SNK: q = 4.58, P < 0.05, n = 3; Fig. 4D). In this case, the fall in PO2 was accompanied by an increase in arterial pressure (SNK: q = 4.58, P < 0.05, n = 3; Fig. 4E). During perfusion with the acapnic HEPES perfusate, the neuronal equivalent of minute ventilation was reduced significantly (Fig. 4F), both compared with control (SNK: q = 5.7, P < 0.05) and when 5% CO2/95% O2-equilibrated HEPES perfusate was reinstated (SNK: q = 5.5, P < 0.05). Acapnic HEPES perfusate reduced burst amplitude in all three preparations (to 35 ± 19% of control) but had a mixed effect on burst frequency, reducing it in two preparations but increasing it in the third. No significant overshoot in PO2 (as compared with the initial normocapnic HEPES condition) occurred when 5% CO2/95% O2-equilibrated HEPES perfusate was reinstated (SNK: q = 3.27, P > 0.05, n = 3; Fig. 4D).

When hypoxic-hypercapnic perfusate was used, the tissue PO2 fell significantly (SNK: q = 8.55, P < 0.001, n = 6; Figs. 5A and 6B) as did the arterial pressure (SNK: q = 6.3, P < 0.001, n = 6; Figs. 5A and 6C). Interestingly, after this hypoxic challenge, the PO2 rebounded rapidly and overshot control levels by 159 ± 43 Torr (SNK: q = 6.56, P < 0.001, n = 6; hatched area in Fig. 5A). Global cerebral vascular resistance as monitored by measuring arterial pressure in the descending aorta recovered more slowly than that of the tissue PO2 and without an overshoot (SNK: q = 2.55, P = 0.09, n = 6; Fig. 5C).


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  		Fig. 5.   Effect of hypercapnic-hypoxic perfusate on the medulla tissue PO2 and arterial pressure of the WHBP. A: example of medulla PO2 (depth: 2.2 mm; top trace) and arterial pressure (bottom trace) when normocapnic-hyperoxic (5% CO2 in O2) bicarbonate-based perfusate was switched to a hypercapnic-hypoxic (8.75% CO2 and 5% O2 in N2) perfusate as indicated by the solid bar. Note that after perfusion with hypercapnic-hypoxic perfusate, tissue PO2 overshot control levels (hatched area). B: summary PO2 data. C: summary arterial pressure data. Bars in B and C show means ± SE from 6 preparations. *Significant difference (P < 0.05) from control.



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  		Fig. 6.   pH depth profiles of the medulla in the WHBP. pH depth profiles were made from the dorsal-lateral surface of the medulla, 1 mm rostral to calamus scritptorius, and ~2 mm lateral to the midline aimed at the VRG. pH microelectrodes were advanced through the tissue in a rapid series of 7-µm steps with a 20-s pause every 49 µm to allow stabilization for measurements. Preparations were perfused with bicarbonate-based perfusate equilibrated with 5.99% CO2 in O2 (pH 7.34). Data points and error bars represent means ± SE for 5 animals to a depth of 1.6 mm and for 4 animals thereafter.

Tissue pH. pH depth profiles were determined for tracts from the dorsal-lateral surface of the medulla, 1 mm rostral to calamus scriptorius and 2 mm lateral to the midline, aimed at the VRG (Fig. 6). When the perfusate in the reservoir was equilibrated with 5.99% CO2 in O2, average tissue pH (7.30 ± 0.02) was slightly more acidic than that of the perfusate (7.34 ± 0.03; paired t-test: t = 4.27, P < 0.05, n = 5). In addition, the tissue was only slightly more acidic at the surface (pH 7.27 ± 0.06) than at a depth of 2.2 mm (pH 7.31 ± 0.07; SNK: q = 7.31, P < 0.01). These data suggest that tissue pH is determined, in large part, by that of the perfusate. Evidence that the electrodes were sensitive to tissue pH is illustrated in Fig. 7. When the perfusate was made hypercapnic by increasing the fractional concentration of CO2 in the equilibrating gas from 5.9 to 8.48% (in O2), the pH of the perfusate dropped by 0.16 pH units and tissue pH dropped by 0.13 ± 0.02 pH units (SNK: q = 11.85, P < 0.001, n = 5; Figs. 7 and 8A). Arterial pressure also fell (SNK: q = 4.55, P < 0.05, n = 5; Fig. 8B). In contrast, when control perfusate was changed to a perfusate that was both hypocapnic (0% CO2) and hypoxic (8% O2 in N2), i.e., an alkaline and hypoxic perfusate that might be expected to produce metabolic acidosis, tissue pH (SNK: q = 26.7, P < 0.001, n = 3; Figs. 7 and 8C) and arterial pressure increased (SNK: q = 7.16, P < 0.02, n = 3; Figs. 7 and 8D).


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  		Fig. 7.   Sensitivity of tissue pH in the medulla of the WHBP. Example of tissue pH and arterial pressure measurements during perfusion with bicarbonate-based hypercapnic-hyperoxia (8.48% CO2 in O2) and hypocapnic-hypoxic (8% O2 in N2) perfusates as indicated by the solid bars. Preparations were perfused with bicarbonate-based perfusate equilibrated with 5.99% CO2 in O2 before and after exposure to the test perfusates.



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  		Fig. 8.   Summary data demonstrating sensitivity of medulla tissue pH (A, C, E) and arterial pressure (B, D, F) in the WHBP. A and B: effect of switching to a hypercapnic-hyperoxic perfusate (equilibrated with 8.48% CO2 in O2). C and D: effect of switching to a hypocapnic-hypoxic perfusate (equilibrated with 8% O2 in N2). E and F: effect of switching to a normocapnic-hypoxia perfusate (equilibrated with 5.42% CO2/5% O2 in N2). Data bars show means ± SE. Note that the hypocapnic perfusate caused a rapid rise in tissue pH (C), and normocapnic perfusate produced no pH change (E) despite tissue hypoxia in both cases. During the before and after conditions, preparations were perfused with bicarbonate-based perfusate equilibrated with 5.99% CO2 in O2. Bars show means ± SE. *Significant difference (P < 0.05) from control.

Normocapnic-hypoxia had no significant effect on tissue pH (SNK: q = 2.31, P = 0.15, n = 4), although in all four of the preparations tested, tissue pH decreased slightly (Fig. 8E). Similarly, whereas this perfusate increased arterial pressure in three of four preparations (Fig. 8F), this trend was not significant (ANOVA F = 2.17, P = 0.19, n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

In this study, we have determined the disposition of the medulla of the rat WHBP preparation in terms of PO2 and pH. Our results prove the efficacy of the perfusion technique by demonstrating that the adult brain stem is remarkably well oxygenated and has a normal physiological pH. Under control conditions, for example, when the perfusate was equilibrated with 5-6% CO2 in O2, the brain stem was hyperoxic (averaged tissue PO2 >150 Torr) and tissue pH was uniform with depth. Tissue pH was similar to that set within the perfusion reservoir (i.e., when the carbogen contained 5.99% CO2, the pH in the perfusion reservoir was 7.34 ± 0.03 and the average tissue pH was 7.30 ± 0.01). Thus, because the composition of the artificial perfusate can be precisely controlled, it makes the WHBP a suitable model for studying the dynamics and modulation of cerebral vascular responses as well as central respiratory chemosensitivity.

Comparison of PO2 depth profiles with other brain stem preparations. PO2 depth profiles have been measured in both superfused and other arterially perfused brain stem preparations (e.g., Refs. 19, 22, 36, 44, 46). Superfused brain stem preparations rely on diffusion of gases between the tissue and the stirred perfusate flowing above their surface. The considerable diffusion distances involved give rise to steep PO2 and pH gradients. In superfused brain stem preparations of rat and mouse, diffusion limitations are significant. In the neonatal rat superfused en bloc brain stem preparation, for example, oxygen is present only within 450-700 µm of the surface, leaving a large anoxic core that either overlaps with, or is adjacent to, the VRG (e.g., Refs. 6, 22). These preparations experience a combination of both metabolic and respiratory acidosis, with tissue pH falling to as low as 6.2 (22). The higher metabolic rate (e.g., Ref. 10) and large size of mature mammalian brain stems exacerbate these problems, precluding the use of superfusion for their maintenance in vitro.

Intra-arterial perfusion reduces the problems associated with diffusion distance limitations and can allow the in vitro study of both neonatal and mature mammalian brain stems (e.g., Ref. 19). Schäfer et al. (36) demonstrated adequate tissue oxygenation at 26-27°C in an isolated adult guinea pig brain stem preparation that was both superfused and internally perfused through the basilar artery with an aqueous solution. With the solution equilibrated with 5% CO2 in O2, tissue PO2 fell from 423 Torr at the ventral lateral medulla surface to 219 Torr at a depth of 2 mm. This "source and sink" PO2 depth profile indicates that diffusion of oxygen from the surface of the medulla, where the PO2 is highest, may have contributed to tissue oxygenation. However, when internal perfusion was terminated, the tissue PO2 gradient became much steeper and an anoxic core was found, extending to within 500 µm of the ventral medullary surface. Tissue pH of this preparation was not reported.

In the WHBP, we found no significant difference between PO2 measurements made immediately below the dorsal-lateral surface of the medulla and those at 2 mm deep. In fact, for the tracts aimed at both the VRG and through the NTS, the trend immediately below the surface was toward an increase in PO2 (Fig. 1). This trend is consistent with a preparation in which the major source of tissue O2 is supplied by internal perfusion rather than from diffusion from the tissue surface. Unlike the aortic-perfused guinea pig preparation (8, 34), the WHBP of the rat and mouse produces robust eupnoeic discharges (27, 28) without the need for fluorocarbon to increase the oxygen content of the perfusate. The combination of a low perfusate viscosity and high perfusion rate together with slight hypothermia (to decrease metabolic demand) may ensure that the brain stem of the WHBP is hyperoxic.

Despite the robust motor pattern produced by the WHBP, we cannot exclude the possibility that hyperoxia may have detrimental effects on neuronal tissue (4, 38). In future studies, hyperoxia could be avoided by perfusing at slightly higher temperature (21) or with a lower PO2.

Regional variations in PO2. We observed a trend for an increase in tissue PO2, with depth within the first 700 µm from the surface, followed by a significant but local decrease in tissue PO2 at a depth corresponding to the core of the parvocellular reticular zone (~1.3 mm below the surface). These regional differences probably signify regions of different metabolic rate and/or vascular perfusion (e.g., Refs. 14, 36).

Uniform tissue pH, despite regional variations in PO2. We found both a significant arterial-to-tissue drop in PO2 and large regional variation in PO2. However, tissue pH differed only slightly from that of the perfusate (i.e., by 0.04 pH units) and was remarkably uniform with depth (Fig. 6). This was unexpected, because in superfused preparations, a fall in tissue PO2 with depth goes hand in hand with a fall in pH produced by both respiratory and metabolic acidosis. Assuming a respiratory quotient of one, we calculate that the arterial-to-tissue PO2 differences would be accompanied by a drop of 0.14-0.30 pH units (see APPENDIX). Furthermore, given the interstitium PO2 differences we observed and assuming that interstitial bicarbonate concentration is uniform with depth, our calculations suggested regional differences in interstitium pH of 0.16 pH units. However, these pH differences were not observed, suggesting a state of disequilibrium exists between PCO2 and pH at the point of measurement [i.e., pH is higher for a given PCO2 than predicted by the Henderson-Hasselbalch (HH) equation].

Disequilibrium between CO2 and pH probably occurs within the capillaries of the WHBP, given the rapid transit of the perfusate and the slow hydrolysis of CO2. In effect, the CO2 entering the capillaries is likely to be expelled before being fully hydrolyzed. However, rather than being confined to the capillaries, it is more conceivable that the tip of the electrode resided, for the most part, in the interstitial compartment. Under normal circumstances, CO2 flux through this compartment occurs through diffusion, a relatively slow process, incompatible with disequilibrium. Only if the blood-brain barrier at the site of measurement is compromised allowing bulk fluid flow and the convective expulsion of CO2 from the interstitium would disequilibrium be anticipated.

Although the integrity of the WHBP's blood-brain barrier has yet to be examined, our data do suggest a very well-perfused preparation in that normocapnic hypoxic perfusate, which would be expected to lead to metabolic acidosis, had no effect on tissue pH (Fig. 8E). Furthermore, when acapnic hypoxic perfusate was used, tissue pH increased despite the increase in arterial resistance (Fig. 8C).

CO2-dependent vasoconstriction and vasodilatation. We found that increasing perfusate PCO2 increased tissue PO2 (Fig. 2B), whereas decreasing perfusate PCO2 had the opposite effect (Fig. 4, A and D). Given the effects of hypercapnia on cerebral blood flow described in vivo (e.g., Refs. 3, 23, 24), the most probable explanation for the changes in tissue PO2 is that the level of PCO2 in the perfusate is a determinant of cerebral blood flow in the WHBP. This explanation is consistent with the observations that hypercapnic bicarbonate-based perfusate decreased (Fig. 2C), whereas hypocapnic bicarbonate perfusates increased arterial pressure (Figs. 7 and 8D). Changing from normocapnic to hypocapnic HEPES perfusate had similar effects (Fig. 4E).

However, such an explanation is not entirely consistent with the results obtained when bicarbonate-based normocapnic perfusate was exchanged for HEPES perfusate equilibrated with 100% O2. As with the other hypocapnic perfusates, tissue PO2 fell (Fig. 4A), but in this case, there was no change in arterial pressure (Fig. 4B). The reason for this paradoxical result is a matter of speculation. One possibility is that arterial pressure recorded in the descending aorta may be an inadequate measure of changes in local cerebral blood flow. For example, global vascular resistance may have been unchanged by this manipulation, despite local vasoconstriction leading to a fall in tissue PO2 at the recording site because of the vasodilatation of other blood vessels. We note that functional magnetic resonance imagery studies have found qualitative differences in response to hypercapnia of cerebral blood flow between different brain regions (15).

In conclusion, the primary result of this study is that the brain stem of the WHBP is neither hypoxic nor acidic when a normocapnic-hyperoxic perfusate is used. Thus our data complement previous work using this preparation that has demonstrated its utility in studying central neuronal mechanisms underlying eupnea as well as central mechanisms regulating cardiovascular and upper-airway motor outflows. We also provide evidence suggesting that vascular responses are intact. Thus we propose this preparation may be suitable for studying the cerebrovasculature and its intimate relationship with central chemoreception (20).

Perspectives

This study demonstrates the WHBP provides an alternative to en bloc preparations for the study of mammalian brain stem function; the major advantages of the WHBP being that it retains cardiovascular responses, produces a eupneic motor pattern, and has a core that is neither acidic nor anoxic. For most questions, these advantages should outweigh the simplicity afforded by the lack of cerebral vascular influences when using en bloc preparations.

This study also demonstrates that removing CO2/bicarbonate from the perfusate without changing arterial pH reduced respiratory activity (e.g., Figs. 3 and 4). This result illustrates the importance of arterial PCO2 as a necessary chemostimulant for eupnogenesis, regardless of either the site of action (e.g., intracellular or extracellular) or the nature of the chemostimulant (e.g., PCO2 or pH) at the receptor level. Removing CO2/bicarbonate from the perfusate also reduced tissue PO2, demonstrating the importance of PCO2 in maintaining blood flow. Given that manipulations that alter tissue PO2 may have both direct and indirect effects on the activity of respiratory neurons (e.g., Refs. 12, 18, 43), we speculate that the cerebral vascularture may be an important component of the CO2/H+-sensing mechanism in vivo. The WHBP provides a good model to test this possibility.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Assuming 1) aerobic metabolism and 2) that for a given location within the brain stem, the rate of flux of O2 molecules into the tissue equals the rate of flux of CO2 out of the tissue, then according to Fick's law
P<SC>co</SC><SUB>2<SUB>Tissue</SUB></SUB><IT>−</IT>P<SC>co</SC><SUB>2<SUB>Arterial</SUB></SUB><IT>=</IT><FR><NU>D<SC>o</SC><SUB>2</SUB><IT>·</IT>(P<SC>o</SC><SUB>2<SUB>Arterial</SUB></SUB><IT>−</IT>P<SC>o</SC><SUB>2<SUB>Tissue</SUB></SUB>)</NU><DE>D<SC>co</SC><SUB>2</SUB></DE></FR>
where
D<SUB>gas</SUB><IT>=</IT><FR><NU>solubility</NU><DE><RAD><RCD>mw</RCD></RAD></DE></FR>
The solubility of CO2 at the WHBP operating temperature (31°C) is 0.0345 ml · 100 ml-1 · Torr-1 (37), whereas that of O2 is some 20 times lower. Thus Dgas (the diffusion constant) is about 17 times greater for CO2 than O2. Given that arterial PO2 was at least 550 Torr and tissue PO2 ranged between 200 and 350 Torr, the PCO2 difference between the perfusate in the lumen of the arterial and the interstitium would be expected to range between 11.5 and 20.5 Torr. If the interstitial bicarbonate concentration is equal to that of the perfusate (i.e., 25 mM) and if hydrolysis of CO2 reaches equilibrium, then according to the HH equation, the arterial-interstitium PCO2 differences would be accompanied by a drop of 0.14-0.30 pH units. Similarly, if the extracellular bicarbonate concentration was uniform throughout the tissue, the differences in interstitium PCO2 at different depths would be expected to produce regional differences in interstitium pH of 0.16 pH units. However, this was not the case.


    ACKNOWLEDGEMENTS

R. J. A. Wilson was supported by the Parker B. Francis Foundation for Pulmonary Research and the Wellcome Trust. J. E. Remmers was supported by the Canadian Institute for Health Research. J. F. R. Paton is supported by the British Heart Foundation (BS/93003).


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. A. Wilson, Dept. of Medical Physiology, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1 (E-mail: wilsonr{at}ucalgary.ca).

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.

Received 11 January 2001; accepted in final form 19 April 2001.


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DISCUSSION
APPENDIX
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Am J Physiol Regul Integr Comp Physiol 281(2):R528-R538
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