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Oxygen supply and respiratory-like activity in the isolated perfused brainstem of the adult guinea pig
246
Brain Research, 618 (1993) 246-250 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19072
Oxygen supply and respiratory-like activity in the isolated perfused brainstem of the adult guinea pig Thorsten Sch~ifer
b, Marie-Pierre
Morin-Surun a and Monique Denavit-Saubi6
a
" Biologie Fonctionnelle du Neurone, Institut Alfred Fessard, CNRS, Gif-sur-Yvette (France) and b Department of Applied Physiology, Ruhr-Unicersity, Bochum (Germany) (Accepted 2 March 1993)
Key words: In vitro; Respiration; Hypoglossal nerve; Brain tissue oxygen; Hypoxia; Ischemia
In isolated brainstem preparations of mature guinea pigs the respiratory network remains functional only if perfused internally via the basilar artery with Krebs solution equilibrated with 95% O 2 / 5 % CO 2. In order to determine the oxygen availability in this preparation, we measured tissue partial oxygen pressures at the level of respiratory-related neurons using oxygen-sensitive microelectrodes. To estimate oxygen consumption we studied the effects of ischemia and cyanide-induced blockade of oxidative metabolism in relation to the respiratory-like rhythmic activity recorded from the hypoglossal nerve. The pO 2 profiles obtained from 9 stepwise measurements from the ventral to the dorsal surface decreased from 423+_32 (SE) mmHg on the ventral surface to 219±64 mmHg at 1900 ~m and stabilized near this value up to a depth of 5000/zm. In the superfused preparation without internal perfusion pO 2 was 0 mmHg within the first 500/Lm. An interruption of perfusion resulted in a rapid (less than 2 rain) decrease of tissue PO2 to 0 mmHg. During the ischemic period, respiratory-like neural activity exhibited first an increase in frequency and tonic discharge, followed by a marked decrease in both parameters. Cyanide added to the perfusate caused an immediate increase of tissue p O 2 and the drop of tissue p O 2 associated with ischemia was abolished. We conclude that there is a considerable oxygen consumption but no hypoxic or anoxic core in the isolated perfused brainstem at the level of the respiratory-related neurons.
INTRODUCTION
T h i s s t u d y was p e r f o r m e d to e x a m i n e t h e c o n d i t i o n s o f o x y g e n s u p p l y in t h e i n t e r n a l l y p e r f u s e d in v i t r o e n
I n v i t r o p r e p a r a t i o n s o f n e u r o n a l tissue h a v e b e e n
b l o c b r a i n s t e m o f t h e m a t u r e g u i n e a pig a n d to d e t e r -
w i d e l y u s e d for n e u r o p h y s i o l o g i c a l a n d p h a r m a c o l o g i -
mine whether tissue hypoxia might be a contributing
cal studies. T h e
f a c t o r to t h e s l o w e r r e s p i r a t o r y - l i k e f r e q u e n c y o b s e r v e d
o x y g e n t e n s i o n i n s i d e t h e slices o r
tissue blocks, h o w e v e r , s i g n i f i c a n t l y a f f e c t s t h e p e r f o r -
in this p r e p a r a t i o n
m a n c e o f s i n g l e n e u r o n s a n d t h e n e u r o n a l c i r c u i t r y 6.
P r e l i m i n a r y r e s u l t s o f this w o r k h a v e b e e n p u b l i s h e d in a b s t r a c t f o r m tS.
Therefore,
knowledge
of the
tissue
oxygen
profile
compared
to in v i v o c o n d i t i o n s .
w i t h i n t h e s e i s o l a t e d p r e p a r a t i o n s is e s s e n t i a l in o r d e r to i n t e r p r e t t h e r e s u l t s c o r r e c t l y .
MATERIALS AND METHODS
The perfused isolated brainstem preparation of the a d u l t g u i n e a pig has b e e n s h o w n to b e a u s e f u l t o o l to s t u d y r h y t h m i c n e u r o n a l d i s c h a r g e t°. P r o v i d e d t h a t this in v i t r o p r e p a r a t i o n
is p e r f u s e d w i t h a n o x y g e n a t e d
K r e b s s o l u t i o n via t h e b a s i l a r a r t e r y , s p o n t a n e o u s respiratory-like
activity c a n b e
recorded
from neurons
w i t h i n t h e r e s p i r a t o r y a r e a s , as w e l l as f r o m t h e r o o t l e t s o f t h e h y p o g l o s s a l n e r v e s for u p to 8 h 11. A s w e will d e m o n s t r a t e , t h e l a c k o f i n t e r n a l p e r f u s i o n r e s u l t s in a loss o f r e s p i r a t o r y - l i k e activity in t h e h y p o g l o s s a l n e r v e .
Ten adult male guinea pigs (body weight 180-350g), were obtained from an authorized supplier. The surgical procedure was performed as described elsewhere 1°. In short, the animals were anesthetized with sodium pentobarbital (30-40 mg/kg i.p.), and decapitated. The brainstem and cerebellum were rapidly removed, mounted in a recording chamber with the ventral surface upwards, and continuously superfused with Krebs solution equilibrated with 5% CO2-95% 0 2 at 26_+ I°C at a flow rate of 2.0-2.5 ml/min. The rostral end of the basilar artery was cannulated and retrogradely perfused with Krebs solution equilibrated with the same gas mixture. The flow rate of the internal perfusion was 1.5-1.8 ml/min. The superfusion and perfusion medium consisted of (in mM): NaCl 124,
Correspondence: M.-P. Morin-Surun, Institut Alfred Fessard, C.N.R.S., Biologie Fonctionelle du Neurone, 1 av. de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France. Fax: (33X1)69070538.
247 followed by a decrease as the electrode approached the cerebellum, which is not perfused by the retrograde basilar perfusion. In the superfused preparation without internal perfusion tissue p O 2 was zero 500 /~m below the VLMS (Fig. 1B), and no respiratory-like activity could be recorded from the hypoglossal nerve rootlets. The interruption of perfusion caused an immediate and rapid drop of tissue p O 2 reaching zero within 2 min (Fig. 2). Under these 'ischemic' conditions, respiratory-like nerve activity first showed an increase in tonic discharge and a concomitant acceleration of the burst frequency, followed by a slowing of the burst frequency and a decrease in tonic activity below preischemic values. Changes in the respiratory burst amplitude were less pronounced. After resuming the perfusion, partial oxygen pressures quickly recovered, and respiratory-like activity returned to baseline values (Fig. 2). If ischemia was induced in the later portion of the experiment (e.g. 4-6 h after decapitation), the loss of neural activity was irreversible. The time course of 'ischemia'-induced changes in tissue p O 2 were similar in different experiments (n = 9): after 30 s of ischemia, p O 2 dropped to 50.2 + 7.9 (S.E.)%, after 60 s to 22.4 + 6.6%, and after 120 s to 1.7 + 3.2% of the initial value. Blockade of oxidative metabolism with the addition of 10 mM sodium cyanide to the internal perfusate caused a rapid increase of tissue p O 2 (Fig. 3A) from 100 to 360 mmHg and an irreversible arrest of respiratory-like hypoglossal nerve activity. The interruption of perfusion during cyanide administration no longer caused a drop of tissue p O 2 (Fig. 3B). The postischemic increase in p O 2 in the presence of cyanide might indicate that the blockade of oxidative
KCI 5, MgSO 4 2.2, K H 2 P O 4 1.2, N a H C O 3 26, CaCI 2 2, glucose 10, H E P E S 20, without addition of an oxygen carrier. Respiratory-like neural activity was recorded from the hypoglossal roots using suction electrodes, full wave rectified and integrated using a passive R C integrating circuit with a time constant of 20 ms. Tissue p O 2 m e a s u r e m e n t s were performed in steps of 100 ~ m from the ventral to the dorsal medullary surface at the level of the ventral respiratory group ( V R G ) at 1.5-2.5 m m rostral to the obex and 1.5-2.5 m m lateral to the midline 13 using an electrically driven micromanipulator. Two types of oxygen-sensitive microelectrodes (Clark type, tip 20/~m, and combined needle electrodes, cathode 25 tzm, D i a m o n d Electro-Tech Inc., A n n Arbor, MI, U.S.A.) with time constants < 3 s for a 90% response and a stirring sensitivity less than 1% relative to unstirred m e d i u m , connected to an Oxygen Meter (Model 781, Strathkelvin Instruments, Glasgow, U.K.), were used. Electrodes were allowed to stabilize for at least 4 h prior to use and calibrated in Krebs solutions equilibrated with 100% N 2 and air at the same temperature as the bathing solution before and after the experiments to control for probable drifts. A profile of tissue oxygen was constructed with 9 traces obtained from 9 animals. In each trace p O 2 m e a s u r e m e n t s were made at 100 /xm intervals from the surface to a depth of 6 m m below the ventrolateral medullary surface (VLMS). To study the effect of 'ischemia' the electrode was then placed at depths between 1 and 3 m m and the internal perfusion was stopped for 2 min in these 9 experiments. O n e trace was obtained from a superfused preparation without internal perfusion of the arterial system. In one experiment oxidative metabolism was finally blocked by the addition of 10 m M sodium cyanide (NaCN) to the perfusate. All signals were displayed on an oscilloscope and recorded on digital tape and a thermal recorder.
RESULTS External superfusion and internal perfusion of the isolated brainstem preparation with oxygenated Krebs solution provide high tissue partial oxygen pressures as demonstrated in Fig. 1A. A m e a n p O 2 of 522_+ 33 (SE) mmHg in the bath above the ventrolateral medulla decreased to 423 _+ 32 mmHg on the surface, dropped further to 219 + 64 mmHg at 1900 /~m below the VLMS and remained stable to a depth of 5000 tzm,
i 500
i
~
DRG i A
B
~~
soo
3oo •
%300 ~' 200
200
100
0 -1000
0
I000
2000
3000
Depth below VLMS [~m]
4000
5000
6000
0 -1000
0
1000
~.000
Depth below V L M S # z m ]
Fig. 1. A: tissue p O 2 profile (means and standard errors) obtained from 9 traces beginning on the ventrolateral medullary surface (VLMS) to 6 m m below VLMS in 9 brainstem preparations at the level of the ventral respiratory group. The value at - 5 0 0 /xm indicates bath p O 2. The shaded bars show the sites of the ventral ( V R G ) and dorsal ( D R G ) respiratory groups. B: tissue p O 2 profile in a superfused preparation without internal perfusion.
248 Stop of Perfusion ~
A
mmHg
metabolism was not complete at the onset of ischemia, which caused a further reduction of oxygen consumption in this experiment.
150(~-J~I rain
0j
DISCUSSION
100
75 50
~ 25 ~
0
~, -25 -50 -75 -100 -1
0
1
2
3
4
5
6
Time [0.5 rain Intervals] Fig. 2. A: original recording of tissue p O 2 obtained 2.2 m m below the ventrolateral medullary surface. With onset of 'ischemia', p O 2 rapidly drops to 0 m m H g accompanied by a dramatic change in respiratory-like activity. Interruption of perfusion for 2 min is indicated by the horizontal bar. N.XII: hypoglossal nerve activity, int. N.XlI.: full wave integrated hypoglossal nerve activity (time constant 20 ms). B: relative changes in respiratory-like minute activity (mean burst amplitudes of integrated N.XI! activity x respiratory frequency in bins of 0.5 min) before, during (hatched area) and after ischemia (same experiment as A).
A
Our data show that oxygen supply is sufficient to maintain oxidative metabolism in the internally perfused brainstem preparation without the use of an oxygen carrier, and that the slower respiratory rhythm is not due to tissue hypoxia at the level of respiratoryrelated neurons. Polarographic oxygenxxxxx microelectrodes 2 have been widely used to determine the oxygen supply to different tissues. Direct measurements of tissue oxygen in cortical slices of guinea pigs 5, maintained in a solution bubbled with 95% 0 2 and 5% CO 2, revealed an anoxic core in the middle layers of the slice when the thickness exceeded 430 izm. Similar results were found in brain slices from adult rats. However, in slices from neonatal tissue, p O 2 values were significantly higher and an anoxic layer was only found if the tissue thickness exceeded 1500 izm 8. The isolated en bloc preparation of the brainstem and spinal cord from neonatal rata ~7, superfused with a bathing solution equilibrated with 95% O z and 5% CO 2, was shown to have an anoxic core beginning at a depth of about 700 /xm below the ventral surface 4. In addition, tissue p H decreases at a rate of 0.1 p H units/100 Ixm, reaching values of 6.9 in the center of the preparation ~2. Nevertheless, in this neonatal in vitro preparation the essential part of the respiratory rhythm generating network is reputedly under aerobic conditions 4.
B 5°°]
400-
s,o.
350-
4°°1
300-
/
I
250200-
,oo
150-
Control
100-
,oo
50
tNaCN lOmM r
0 -2
-,
'o )
2
Time [mini
j
'4
F
0
i
-2
-1
0
1
2
3
4
Time [min]
Fig. 3. A: blockade of oxidative metabolism with the addition of 10 m M NaCN to the internal perfusate causes a rapid increase of tissue p O 2 at 2.5 m m below VLMS. Time course of one experiment. B: during NaCN application (upper trace) internal perfusion arrest (indicated by the shaded area) no longer causes a drop in tissue p O 2 as compared to control conditions (lower trace). The post-ischemic increase in pO 2 might reflect an incomplete blockade of oxidative metabolism at the onset of ischemia in this experiment.
249 The present study provides data to characterize the oxygen supply in the isolated perfused brainstem preparation of adult guinea pigs. The oxygen profile of the non-perfused tissue bloc is very similar to the profile of the neonatal rat brainstem-spinal cord preparation 4 with an anoxic core beginning at a depth of 500 tzm. In the perfused preparation no anoxic region could be detected. A p O 2 gradient of 99 mmHg was found in the bath when approaching the brain surface. This gradient could be the combined result of the oxygen consumption of the tissue and an unstirred liquid layer around the preparation which would restrict the delivery of oxygenated Krebs solution. Furthermore a small stirring artefact resulting from the flow above the preparation might have led to a slight overestimation of the bath pO~. However, the stirring sensitivity of the electrodes was negligible. The p O 2 gradient arising between the surface and 2000 /zm below can be attributed to oxygen consumption and not to incomplete perfusion or diffusion limitations, because cyanide blockade of oxidative metabolism immediately increased tissue p O 2 levels in this area. The small but consistent p O 2 increase at about 2000 t~m from the VLMS might reflect an improved oxygenation near the perforant artery that extends from the VLMS to the dorsomedial medulla. Ischemia produced a rapid drop of p O 2 to 0 mmHg with similar time courses at different recording depths. The concomitant biphasic response of the respiratorylike hypoglossal nerve activity was similar to the effects of hypoxia on respiratory related neurons and phrenic nerve activity in vivo TM, where hypoxia as well as ischemia induces an initial augmentation of respiratory activity followed by a secondary depression. The same observations have recently been reported from the perfused brainstem preparation ef adult rats, whereas the neonatal brainstem-spinal cord preparation showed a much higher hypoxia tolerance ~. During arterial occlusions in air-breathing cats 9 cortical tissue p O 2 values fall to below 5 mmHg within less than 10 s. Tissue p O 2 values in this preparation exceed those reported in vivo. For example, the average tissue oxygen tension in the cerebral cortex of rats breathing 21% 0 2 is about 31.8 mmHg 7. This hyperoxia is probably the result of bubbling the perfusate with 95% 0 2 and decreasing the oxygen consumption of the tissue by maintaining the preparation at lower temperatures than in vivo. In anesthetized rats and cats elevations of partial oxygen pressures above normal values elicited discrete deviations in neocortical DC potentials 16. These effects, however, are at least in part attributed to an associated increase of the carbon dioxide pres-
sure resulting from higher concentrations of oxygenated hemoglobin in the venous blood (Bohr effect). The elevation of tissue pO 2 in hippocampal slices from 150 to 300 mmHg led to depolarization and an increase in the membrane input resistance 3. Similar effects in our en bloc preparation cannot be ruled out, but oxygen pressures for the most part were below this critical value. Furthermore, the discharge frequency of respiratory-related neurons in vitro is comparable to that found in vivo u. In conclusion no hypoxic or anoxic core was revealed in the isolated brainstem preparation of adult guinea pigs when perfused with an oxygenated Krebs solution via the basilar artery. Average pO 2 values exceeded in vivo values without use of special oxygen carrier molecules added to the perfusate. As proof of considerable oxygen consumption, the arrest of perfusion led to an immediate drop in tissue pO 2 to 0 mmHg paralleled with a transient increase of tonic hypoglossal nerve activity and an increased respiratory-like burst frequency. This was followed by a decrease in tonic activity and a marked reduction of the respiratory-like burst frequency. Acknowledgements.
The authors wish to thank Dr. P. Lacombe for his helpful advice in oxygen measurements. T. Sch~ifer was supported by an European Neuroscience Programme Short-Term Fellowship of the European Science Foundation. This work was supported by CNRS.
REFERENCES 1 Ballanyi, K., Kuwana, S., V61ker, A., Morawietz, G. and Richter, D.W., Developmental changes in the hypoxia tolerance of the in vitro respiratory network of rats, Neurosci. Lett., 148 (1992) 141-144. 2 Baumg~irtl, H., Systematic investigations of needle electrode properties in polarographic measurements of local tissue pO 2. In A.M. Ehrly, J. Hauss and R. Huch (Eds,), Clinical Oxygen Pressure Measurement, Springer, Berlin, 1987, pp. 17-42. 3 Bingmann, D., Kolde, G. and Speckmann, E.J., Effects of elevated PO2-values in the superfusate on neuronal activity in hippocampal slices. In M.R. Klee, H.D. Lux and E.J. Speckmann (Eds.), Physiology and Pharmacology of Epileptogenic Phenomena, Raven, New York, 1982, pp. 97-104. 4 Brockhaus, J., Ballanyi, K., Smith, J.C. and Richter, D.W., Microenvironment of respiratory neurones in the isolated brainstem of neonatal rats, Pfliigers Arch., 418 Suppl. (1991) R15. 5 Fujii, T., Baumg~irtl, H. and Liibbers, D.W., Limiting section thickness of guinea pig olfactory cortical slices studied from tissue pO 2 values and electrical activities, Pfiiigers Arch., 393 (1982) 83-87. 6 Haddad, G.G. and Donnelly, D.F., 0 2 deprivation induces a major depolarisation in brain stem neurons in the adult but not in the neonatal rat, J. Physiol., 429 (1990)411-428. 7 Hagendorff, A., Zimmer, K. and Grote, J., A new model for long-term investigations of cerebral oxygen supply in rats, Adv. Exp. Med. Biol., 277 (1990) 145-150. 8 Jiang, C., Agulian, S. and Haddad, G.G., 0 2 tension in adult and neonatal brain slices under several experimental conditions, Brain Res., 568 (1991) 159-164. 9 Leniger-Follert, E., Direct determination of local oxygen con-
250
10
11
12
13
sumption of the brain cortex in vivo, Adu. Exp. Med. Biol., 94 (1977) 325-330. Morin-Surun, M.P. and Denavit-Saubi6, M., Rhythmic discharges in the perfused isolated brainstem preparation of adult guinea pig, Neurosci. Lett., 101 (1989) 57-61. Morin-Surun, M.P., Boudinot, E., Sarraseca, H., Fortin, G. and Denavit-Saubi~, M., Respiratory network remains functional in a mature guinea pig brainstem isolated in vitro, Exp. Brain Res., 90 (1992) 375-383. Okada, Y., Mfickenhoff, K. and Scheid, P., Tissue pH in the isolated brainstem of the neonatal rat, International Conference, Modulation of respiratory pattern: peripheral and central mechanisms, Lexington, Kentucky, 1991, p.67. Richerson, G.B. and Getting, P.A., Medullary respiratory neurons in the guinea pig: localization and firing patterns, Brain Res., 591 (1992) 79-87.
14 Richter, D.W., Bischoff, A., Anders, K., Bellingham, M. and Windhorst, U., Response of the medullary respiratory network of the cat to hypoxia, J. Physiol., 443 (1991) 231-256. 15 Sch~ifer, T., Morin-Surun, M.P., Denavit-Saubi6, M. and Naquet, R., Deoxyglucose uptake and oxygen partial pressure in the isolated perfused brainstem of adult guinea pig, Soe. Neurosci. Abstr., Vol. 18, Part 1 (1992) p.827. 16 Speckmann, E.-J., Bingmann, D., Lehmenkfihler, A. and Lipinski, H.G., Changes of the bioelectrical activity and extracellular micromilieu in the central nervous system during variations of local oxygen pressure. In H. Acker (Ed.), Oxygen Sensing in Tissues, Springer, Berlin, 1988, pp. 181-191. 17 Suzue, T., Respiratory rhythm generation in the in vitro brain stem - spinal cord preparation of the neonatal rat, J. Physiol. (London), 354 (1984) 173-183.
Brain Research, 618 (1993) 246-250 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19072
Oxygen supply and respiratory-like activity in the isolated perfused brainstem of the adult guinea pig Thorsten Sch~ifer
b, Marie-Pierre
Morin-Surun a and Monique Denavit-Saubi6
a
" Biologie Fonctionnelle du Neurone, Institut Alfred Fessard, CNRS, Gif-sur-Yvette (France) and b Department of Applied Physiology, Ruhr-Unicersity, Bochum (Germany) (Accepted 2 March 1993)
Key words: In vitro; Respiration; Hypoglossal nerve; Brain tissue oxygen; Hypoxia; Ischemia
In isolated brainstem preparations of mature guinea pigs the respiratory network remains functional only if perfused internally via the basilar artery with Krebs solution equilibrated with 95% O 2 / 5 % CO 2. In order to determine the oxygen availability in this preparation, we measured tissue partial oxygen pressures at the level of respiratory-related neurons using oxygen-sensitive microelectrodes. To estimate oxygen consumption we studied the effects of ischemia and cyanide-induced blockade of oxidative metabolism in relation to the respiratory-like rhythmic activity recorded from the hypoglossal nerve. The pO 2 profiles obtained from 9 stepwise measurements from the ventral to the dorsal surface decreased from 423+_32 (SE) mmHg on the ventral surface to 219±64 mmHg at 1900 ~m and stabilized near this value up to a depth of 5000/zm. In the superfused preparation without internal perfusion pO 2 was 0 mmHg within the first 500/Lm. An interruption of perfusion resulted in a rapid (less than 2 rain) decrease of tissue PO2 to 0 mmHg. During the ischemic period, respiratory-like neural activity exhibited first an increase in frequency and tonic discharge, followed by a marked decrease in both parameters. Cyanide added to the perfusate caused an immediate increase of tissue p O 2 and the drop of tissue p O 2 associated with ischemia was abolished. We conclude that there is a considerable oxygen consumption but no hypoxic or anoxic core in the isolated perfused brainstem at the level of the respiratory-related neurons.
INTRODUCTION
T h i s s t u d y was p e r f o r m e d to e x a m i n e t h e c o n d i t i o n s o f o x y g e n s u p p l y in t h e i n t e r n a l l y p e r f u s e d in v i t r o e n
I n v i t r o p r e p a r a t i o n s o f n e u r o n a l tissue h a v e b e e n
b l o c b r a i n s t e m o f t h e m a t u r e g u i n e a pig a n d to d e t e r -
w i d e l y u s e d for n e u r o p h y s i o l o g i c a l a n d p h a r m a c o l o g i -
mine whether tissue hypoxia might be a contributing
cal studies. T h e
f a c t o r to t h e s l o w e r r e s p i r a t o r y - l i k e f r e q u e n c y o b s e r v e d
o x y g e n t e n s i o n i n s i d e t h e slices o r
tissue blocks, h o w e v e r , s i g n i f i c a n t l y a f f e c t s t h e p e r f o r -
in this p r e p a r a t i o n
m a n c e o f s i n g l e n e u r o n s a n d t h e n e u r o n a l c i r c u i t r y 6.
P r e l i m i n a r y r e s u l t s o f this w o r k h a v e b e e n p u b l i s h e d in a b s t r a c t f o r m tS.
Therefore,
knowledge
of the
tissue
oxygen
profile
compared
to in v i v o c o n d i t i o n s .
w i t h i n t h e s e i s o l a t e d p r e p a r a t i o n s is e s s e n t i a l in o r d e r to i n t e r p r e t t h e r e s u l t s c o r r e c t l y .
MATERIALS AND METHODS
The perfused isolated brainstem preparation of the a d u l t g u i n e a pig has b e e n s h o w n to b e a u s e f u l t o o l to s t u d y r h y t h m i c n e u r o n a l d i s c h a r g e t°. P r o v i d e d t h a t this in v i t r o p r e p a r a t i o n
is p e r f u s e d w i t h a n o x y g e n a t e d
K r e b s s o l u t i o n via t h e b a s i l a r a r t e r y , s p o n t a n e o u s respiratory-like
activity c a n b e
recorded
from neurons
w i t h i n t h e r e s p i r a t o r y a r e a s , as w e l l as f r o m t h e r o o t l e t s o f t h e h y p o g l o s s a l n e r v e s for u p to 8 h 11. A s w e will d e m o n s t r a t e , t h e l a c k o f i n t e r n a l p e r f u s i o n r e s u l t s in a loss o f r e s p i r a t o r y - l i k e activity in t h e h y p o g l o s s a l n e r v e .
Ten adult male guinea pigs (body weight 180-350g), were obtained from an authorized supplier. The surgical procedure was performed as described elsewhere 1°. In short, the animals were anesthetized with sodium pentobarbital (30-40 mg/kg i.p.), and decapitated. The brainstem and cerebellum were rapidly removed, mounted in a recording chamber with the ventral surface upwards, and continuously superfused with Krebs solution equilibrated with 5% CO2-95% 0 2 at 26_+ I°C at a flow rate of 2.0-2.5 ml/min. The rostral end of the basilar artery was cannulated and retrogradely perfused with Krebs solution equilibrated with the same gas mixture. The flow rate of the internal perfusion was 1.5-1.8 ml/min. The superfusion and perfusion medium consisted of (in mM): NaCl 124,
Correspondence: M.-P. Morin-Surun, Institut Alfred Fessard, C.N.R.S., Biologie Fonctionelle du Neurone, 1 av. de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France. Fax: (33X1)69070538.
247 followed by a decrease as the electrode approached the cerebellum, which is not perfused by the retrograde basilar perfusion. In the superfused preparation without internal perfusion tissue p O 2 was zero 500 /~m below the VLMS (Fig. 1B), and no respiratory-like activity could be recorded from the hypoglossal nerve rootlets. The interruption of perfusion caused an immediate and rapid drop of tissue p O 2 reaching zero within 2 min (Fig. 2). Under these 'ischemic' conditions, respiratory-like nerve activity first showed an increase in tonic discharge and a concomitant acceleration of the burst frequency, followed by a slowing of the burst frequency and a decrease in tonic activity below preischemic values. Changes in the respiratory burst amplitude were less pronounced. After resuming the perfusion, partial oxygen pressures quickly recovered, and respiratory-like activity returned to baseline values (Fig. 2). If ischemia was induced in the later portion of the experiment (e.g. 4-6 h after decapitation), the loss of neural activity was irreversible. The time course of 'ischemia'-induced changes in tissue p O 2 were similar in different experiments (n = 9): after 30 s of ischemia, p O 2 dropped to 50.2 + 7.9 (S.E.)%, after 60 s to 22.4 + 6.6%, and after 120 s to 1.7 + 3.2% of the initial value. Blockade of oxidative metabolism with the addition of 10 mM sodium cyanide to the internal perfusate caused a rapid increase of tissue p O 2 (Fig. 3A) from 100 to 360 mmHg and an irreversible arrest of respiratory-like hypoglossal nerve activity. The interruption of perfusion during cyanide administration no longer caused a drop of tissue p O 2 (Fig. 3B). The postischemic increase in p O 2 in the presence of cyanide might indicate that the blockade of oxidative
KCI 5, MgSO 4 2.2, K H 2 P O 4 1.2, N a H C O 3 26, CaCI 2 2, glucose 10, H E P E S 20, without addition of an oxygen carrier. Respiratory-like neural activity was recorded from the hypoglossal roots using suction electrodes, full wave rectified and integrated using a passive R C integrating circuit with a time constant of 20 ms. Tissue p O 2 m e a s u r e m e n t s were performed in steps of 100 ~ m from the ventral to the dorsal medullary surface at the level of the ventral respiratory group ( V R G ) at 1.5-2.5 m m rostral to the obex and 1.5-2.5 m m lateral to the midline 13 using an electrically driven micromanipulator. Two types of oxygen-sensitive microelectrodes (Clark type, tip 20/~m, and combined needle electrodes, cathode 25 tzm, D i a m o n d Electro-Tech Inc., A n n Arbor, MI, U.S.A.) with time constants < 3 s for a 90% response and a stirring sensitivity less than 1% relative to unstirred m e d i u m , connected to an Oxygen Meter (Model 781, Strathkelvin Instruments, Glasgow, U.K.), were used. Electrodes were allowed to stabilize for at least 4 h prior to use and calibrated in Krebs solutions equilibrated with 100% N 2 and air at the same temperature as the bathing solution before and after the experiments to control for probable drifts. A profile of tissue oxygen was constructed with 9 traces obtained from 9 animals. In each trace p O 2 m e a s u r e m e n t s were made at 100 /xm intervals from the surface to a depth of 6 m m below the ventrolateral medullary surface (VLMS). To study the effect of 'ischemia' the electrode was then placed at depths between 1 and 3 m m and the internal perfusion was stopped for 2 min in these 9 experiments. O n e trace was obtained from a superfused preparation without internal perfusion of the arterial system. In one experiment oxidative metabolism was finally blocked by the addition of 10 m M sodium cyanide (NaCN) to the perfusate. All signals were displayed on an oscilloscope and recorded on digital tape and a thermal recorder.
RESULTS External superfusion and internal perfusion of the isolated brainstem preparation with oxygenated Krebs solution provide high tissue partial oxygen pressures as demonstrated in Fig. 1A. A m e a n p O 2 of 522_+ 33 (SE) mmHg in the bath above the ventrolateral medulla decreased to 423 _+ 32 mmHg on the surface, dropped further to 219 + 64 mmHg at 1900 /~m below the VLMS and remained stable to a depth of 5000 tzm,
i 500
i
~
DRG i A
B
~~
soo
3oo •
%300 ~' 200
200
100
0 -1000
0
I000
2000
3000
Depth below VLMS [~m]
4000
5000
6000
0 -1000
0
1000
~.000
Depth below V L M S # z m ]
Fig. 1. A: tissue p O 2 profile (means and standard errors) obtained from 9 traces beginning on the ventrolateral medullary surface (VLMS) to 6 m m below VLMS in 9 brainstem preparations at the level of the ventral respiratory group. The value at - 5 0 0 /xm indicates bath p O 2. The shaded bars show the sites of the ventral ( V R G ) and dorsal ( D R G ) respiratory groups. B: tissue p O 2 profile in a superfused preparation without internal perfusion.
248 Stop of Perfusion ~
A
mmHg
metabolism was not complete at the onset of ischemia, which caused a further reduction of oxygen consumption in this experiment.
150(~-J~I rain
0j
DISCUSSION
100
75 50
~ 25 ~
0
~, -25 -50 -75 -100 -1
0
1
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4
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6
Time [0.5 rain Intervals] Fig. 2. A: original recording of tissue p O 2 obtained 2.2 m m below the ventrolateral medullary surface. With onset of 'ischemia', p O 2 rapidly drops to 0 m m H g accompanied by a dramatic change in respiratory-like activity. Interruption of perfusion for 2 min is indicated by the horizontal bar. N.XII: hypoglossal nerve activity, int. N.XlI.: full wave integrated hypoglossal nerve activity (time constant 20 ms). B: relative changes in respiratory-like minute activity (mean burst amplitudes of integrated N.XI! activity x respiratory frequency in bins of 0.5 min) before, during (hatched area) and after ischemia (same experiment as A).
A
Our data show that oxygen supply is sufficient to maintain oxidative metabolism in the internally perfused brainstem preparation without the use of an oxygen carrier, and that the slower respiratory rhythm is not due to tissue hypoxia at the level of respiratoryrelated neurons. Polarographic oxygenxxxxx microelectrodes 2 have been widely used to determine the oxygen supply to different tissues. Direct measurements of tissue oxygen in cortical slices of guinea pigs 5, maintained in a solution bubbled with 95% 0 2 and 5% CO 2, revealed an anoxic core in the middle layers of the slice when the thickness exceeded 430 izm. Similar results were found in brain slices from adult rats. However, in slices from neonatal tissue, p O 2 values were significantly higher and an anoxic layer was only found if the tissue thickness exceeded 1500 izm 8. The isolated en bloc preparation of the brainstem and spinal cord from neonatal rata ~7, superfused with a bathing solution equilibrated with 95% O z and 5% CO 2, was shown to have an anoxic core beginning at a depth of about 700 /xm below the ventral surface 4. In addition, tissue p H decreases at a rate of 0.1 p H units/100 Ixm, reaching values of 6.9 in the center of the preparation ~2. Nevertheless, in this neonatal in vitro preparation the essential part of the respiratory rhythm generating network is reputedly under aerobic conditions 4.
B 5°°]
400-
s,o.
350-
4°°1
300-
/
I
250200-
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150-
Control
100-
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50
tNaCN lOmM r
0 -2
-,
'o )
2
Time [mini
j
'4
F
0
i
-2
-1
0
1
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3
4
Time [min]
Fig. 3. A: blockade of oxidative metabolism with the addition of 10 m M NaCN to the internal perfusate causes a rapid increase of tissue p O 2 at 2.5 m m below VLMS. Time course of one experiment. B: during NaCN application (upper trace) internal perfusion arrest (indicated by the shaded area) no longer causes a drop in tissue p O 2 as compared to control conditions (lower trace). The post-ischemic increase in pO 2 might reflect an incomplete blockade of oxidative metabolism at the onset of ischemia in this experiment.
249 The present study provides data to characterize the oxygen supply in the isolated perfused brainstem preparation of adult guinea pigs. The oxygen profile of the non-perfused tissue bloc is very similar to the profile of the neonatal rat brainstem-spinal cord preparation 4 with an anoxic core beginning at a depth of 500 tzm. In the perfused preparation no anoxic region could be detected. A p O 2 gradient of 99 mmHg was found in the bath when approaching the brain surface. This gradient could be the combined result of the oxygen consumption of the tissue and an unstirred liquid layer around the preparation which would restrict the delivery of oxygenated Krebs solution. Furthermore a small stirring artefact resulting from the flow above the preparation might have led to a slight overestimation of the bath pO~. However, the stirring sensitivity of the electrodes was negligible. The p O 2 gradient arising between the surface and 2000 /zm below can be attributed to oxygen consumption and not to incomplete perfusion or diffusion limitations, because cyanide blockade of oxidative metabolism immediately increased tissue p O 2 levels in this area. The small but consistent p O 2 increase at about 2000 t~m from the VLMS might reflect an improved oxygenation near the perforant artery that extends from the VLMS to the dorsomedial medulla. Ischemia produced a rapid drop of p O 2 to 0 mmHg with similar time courses at different recording depths. The concomitant biphasic response of the respiratorylike hypoglossal nerve activity was similar to the effects of hypoxia on respiratory related neurons and phrenic nerve activity in vivo TM, where hypoxia as well as ischemia induces an initial augmentation of respiratory activity followed by a secondary depression. The same observations have recently been reported from the perfused brainstem preparation ef adult rats, whereas the neonatal brainstem-spinal cord preparation showed a much higher hypoxia tolerance ~. During arterial occlusions in air-breathing cats 9 cortical tissue p O 2 values fall to below 5 mmHg within less than 10 s. Tissue p O 2 values in this preparation exceed those reported in vivo. For example, the average tissue oxygen tension in the cerebral cortex of rats breathing 21% 0 2 is about 31.8 mmHg 7. This hyperoxia is probably the result of bubbling the perfusate with 95% 0 2 and decreasing the oxygen consumption of the tissue by maintaining the preparation at lower temperatures than in vivo. In anesthetized rats and cats elevations of partial oxygen pressures above normal values elicited discrete deviations in neocortical DC potentials 16. These effects, however, are at least in part attributed to an associated increase of the carbon dioxide pres-
sure resulting from higher concentrations of oxygenated hemoglobin in the venous blood (Bohr effect). The elevation of tissue pO 2 in hippocampal slices from 150 to 300 mmHg led to depolarization and an increase in the membrane input resistance 3. Similar effects in our en bloc preparation cannot be ruled out, but oxygen pressures for the most part were below this critical value. Furthermore, the discharge frequency of respiratory-related neurons in vitro is comparable to that found in vivo u. In conclusion no hypoxic or anoxic core was revealed in the isolated brainstem preparation of adult guinea pigs when perfused with an oxygenated Krebs solution via the basilar artery. Average pO 2 values exceeded in vivo values without use of special oxygen carrier molecules added to the perfusate. As proof of considerable oxygen consumption, the arrest of perfusion led to an immediate drop in tissue pO 2 to 0 mmHg paralleled with a transient increase of tonic hypoglossal nerve activity and an increased respiratory-like burst frequency. This was followed by a decrease in tonic activity and a marked reduction of the respiratory-like burst frequency. Acknowledgements.
The authors wish to thank Dr. P. Lacombe for his helpful advice in oxygen measurements. T. Sch~ifer was supported by an European Neuroscience Programme Short-Term Fellowship of the European Science Foundation. This work was supported by CNRS.
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