Journal of Neurochemistry Raven Press, Ltd., New York Q I992 International Society for Neurochemistry

Correlation Between Electroencephalogram Isoelectric Time and Hippocampal Norepinephrine Levels, Measured by

Microdialysis, During Ischemia in Rats

C. Perego, S. Gatti, G. C. Vetrugno, *F. Marzatico, and S. Algeri

Istituto di Ricerche Farmacologiche Mario Negri, Milan, and *Facoltd di Scienze, Universitd di Pavia, Pavia, Italy

Abstract: It is suggested that norepinephrine (NE) plays a role during transient forebrain ischemia. NE may have a protective action against neuronal cell death in the hippo- campus, or it may be one of the causes of injurious ischemic effects. We used the microdialysis technique to study extra- cellular NE levels in the rat hippocampus before, during, and after 30 min of transient incomplete forebrain ischemia (induced by four-vessel occlusion) to describe the time course of NE in this condition. There was a maximal in- crease (fivefold) in extracellular NE after 10 min of reflow only when the electroencephalogram was isoelectnc. NE

levels returned to baseline 40 min after release of the carotid clamps and remained constant for the next 80 min. Thus there appears to be a transient NE overflow in the hippo- campus during ischemia, closely related to the complete loss of brain electrical activity. Key Words: Norepinephrine- Microdialysis-Ischemia-Hippocampus-Electroenceph- alogram. Perego C. et al. Correlation between electroen- cephalogram isoelectric time and hippocampal norepineph- rine levels, measured by microdialysis, during ischemia in rats. J. Neurochem. 59, 1257-1262 (1992).

During ischemia the concentrations of many neuro- transmitters, including y-aminobutyric acid, seroto- nin, and dopamine, rise dramatically in the extracel- lular compartment (Globus et al., 1988; Damsma et al., 1990; Sarna et al., 1990). The roles of these differ- ent neurotransmitters in ischemia are still under in- vestigation. There is ample experimental evidence that dopamine is involved in ischemic striatal injury, because prior lesioning cif the wbstantia nigra has a strong protective effect against postischemic striatal neuronal death (Globus et al., 1987). Also, massive glutamate and aspartate overflow can be observed in the hippocampus and striaturn during ischemic or hy- poglycemic coma (Benveniste et al., 1984; Hagberg et al., 1986; Sandberg et al., 1986; Globus et al., 1988). This rise of excitatory amino acid levels seems to be important in hippocampal and striatal neuronal dam- age after ischemia (Choi and Ruthman, 1990).

Under physiological conditions the locus ceruleus noradrenergic system, which innervates most regions of the brain (Nal and Bloom, 1974), modulates h ip pocampal postsynaptic glutamate activity (Nicoll et al., 1987). In vitro, norepinephrine (NE) reduces syn-

aptic inhibition in the hippocampal pyramidal cells, facilitating excitatory synaptic responses (Madison and Nicoll, 1982) and stimulation of the N-methyl-B aspartate receptor channel complex enhanced depo- larization-evoked NE release from hippocampal slices (Vezzani et al., 1987; Zhao et al., 1990). These results suggest an interaction between glutamatergic and nor- adrenergic systems that could be important during an ischemic episode. The NE system appears 2lso to be involved in the development of ischemic brain dam- age (Blomqvist et al., 1985; Miyauchi et al., 1989) and Globus et al. (1989) showed that extracellular NE lev- els in the hippocampus increase during ischemia. In spite of the amount of information available about neurotransmitter overflow during ischemia, it is not yet clear whether this is a nonspecific event secondary to the neuronal energy crisis.

To clarify the relationship between the lack of en- ergy metabolism and the alteration of the noradrener- gic system activity during ischemia, we simulta- neously measured cortical electrical activity and the response of the NE system during and after an isch- emic coma. Electroencephalogram (EEG) activity

Received September 17, 1991; revised manuscript received F e b ruaq 20, 1992; accepted March 24, 1992.

Address correspondence and reprint requests to Dr. C. Perego at Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20 I57 Milan, Italy.

Abbreviufions used: EEG, electroencephalogram; MABP, mean arterial blood pressure; NE, norepinephrine.

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I258 C. PEREGO ET AL.

can in fact be considered a means of monitoring cere- bral function, as shown by the relation between EEG suppression after cerebral vessel occlusion and the im- mediate ischemic loss of high-energy cellular sub- stances (Naruse et al., 1984; Raffin et al., 1991).

We chose the hippocampal area because dorsal h i p pocampus, particularly the CA I subfield, are very vul- nerable to brief episodes of ischemia, showing thetypi- cal delayed neuronal death (Kirino, 1982). The disap pearancc of pyramidal neurons in the CAI subfield is the last event in a chain that starts immediately after the onset of ischemia, with cellular energy depletion (Lowry et al., 1964; Arai et al., 1986; Raley and Lip ton, 1990).

The aim of this study was to establish the possible correlation between energy crisis, evaluated by the lack of cortical electrical activity, and NE overflow in dorsal hippocampus during ischemia. We measured hippocampal extracellular NE levels applying the technique of cerebral microdialysis b e h e , during, and after an incomplete forebrain ischemic episode under continuous EEG monitoring.

MATERIALS AND METHODS Animals and experimental groups

Male Sprague-Dawley rats (CD-COBS, 400-450 g, Charles River, Calco, Italy) were used. The animals were housed under 12 h light/dark cycles with constant tempera- turc (23°C) and humidity (50%), and were fasted overnight before the induction of ischemia.

Animals were divided into two experimental groups: 30 min of ischemia and sham-operated. Animals with a post- ischemic survival time of 96 h were utilized for histopatho- logical analysis.

Dialysis probe implantation and perfusion The rats were anesthetized with equitensin (pentobarbital

9.72 g/L; chloral hydrate 42.5 g/L; MgSO, 2 1.28 g/L; propyl- enc glycol 396 ml/L; ethanol 100 ml/L) and placed in a stereotaxic frame. The dialysis tubing (220 pm internal di- ameter, 3 10 p m external diameter; AN 69 membrane, HO- SPAL Spa, Meyzieu, France), with a molecular cut-off of > 15,000, was inserted transversally through the dorsal hip- pocampus. This was covered with Super Epoxy glue except for the part corresponding to the areas of interest (7 mm). The procedure used to insert the probe was essentially as previously described by Imperato and Di Chiara ( 1984) and Zettcrstrom and Ungerstedt (1 984). The coordinates for im- planting the probe were: AP -3.6 mm from bregma, V -3.7 mm from temporal bone (Konig and Klippel, 1963).

Recovery through the dialysis probe, prepared in the same manner as the one implanted in the hippocampus, was measured in vitro. Dialysis tubes were placed in a vial filled with Ringer’s solution containing NE in a final con- centration of 2 nM and perfused with the same solution ( 2 pl/min) at room temperature. The average NE recovery (1 1%) was established in 10-min samples collected over a 120-min perfusion period, as a percentage of the NE con- centration in the solution outside the probe.

On the day after implantation the dialysis probe was eon- nected to a CMA/ 100 microinfusion pump (Carnegie Medi- cin AB, Sweden) by polyethylene tubing and perfused at a

constant flow rate of 2 pl/min with Ringer’s solution (147 mM NaCl, 3.4 m M CaClz, and 4 mM KCl, pH 6.3 with NaOH 0.005 M). The perfusate was collected every 10 min in the presence of 10 pl of 0. I Mperchloric acid. At the end ofthe collection the perfusate was immediately injected into an HPLC system.

NE analysis The HPLC system consisted of a Spectra Physics SP 88 10

precision isocratic pump (Spectra Physics, San Joe, CA, U.S.A.), a reversed-phase analytical column, Waters pBon- dapak C,8 3.9 mm X 300 mm (Waters, Millipore, Milford, MA, U.S.A.), and an electrochemical detector ESA Coulo- chem 5 100 A equipped with an analytical cell model 501 1 (ESA, Bedford, MA, U.S.A.). The detection limit of the as- say was about 10 fmol/sample.

The mobile phase consisted of citric acid 5.25 g/L; so- dium acetate 3.3 g/L; EDTA 37 mg/L; sodium octyl sulfate I26 mg/L; and methanol 150 ml/L. The final pH was 4.2. All reagents used were analytical grade.

Induction of ischemia Transient forebrain ischemia was induced for 30 min

by four-vessel occlusion (Pulsinelli and Brierley, 1979; Schmidt-Kastner ct al., 1989). On the first day, the rats were anesthetized with equitensin, the microdialysis probe was inserted, and both vertebral arteries were cauterized with a monopolar coagulator (Della Matta, Milan, Italy). One day after the surgical procedure, animals were anesthetized with 4% halothane (Huotane, ICI) for 3 min, rapidly intubated, connected to a small animal respirator (Basile, Comerio, VA, Italy), and ventilated (Ohmeda, Tre7zano sul Naviglio, Italy) with 0.75% halothane in a 2:l NzO/OZ mixture throughout the operation. Pancuronium bromide (75 pg/kg i.v. every 20 min) (Pavulon, Organon) was used as muscle relaxant. A polyethylene catheter was inserted 10 mm into a tail artery, connected to a blood pressure transducer (Grass, Quincy, MA, U.S.A.) and used to take the blood samples for blood gas analysis (Instrumentation Laboratory, Ascoli Pi- ceno, Italy) and plasma glucose assays (Test Combination Glucose GOD-Perid, Boehringer Mannheim) before, dur- ing, and after ischemia.

To avoid bleeding problems, the animals were not hcpa- rinized and the catheters were kept open by replacing the contents with heparin (100 IU/ml) after sampling.

EEG signal and blood pressure were continuously moni- tored by a multichanncl recorder (Grass, Quincy, MA, U.S.A.) throughout the experiment. A rectal thermometer was inserted and the body temperature was kept at 37-38°C with a heating lamp.

Ischemia was induced by clamping the common carotid arteries. The EEG revealed a complete loss of activity within about 100 s after placement of the clips.

EEG recording The EEG was used to monitor the depth of anesthesia

during the preparatory phase, to assess the depth of isch- emia (isoelectric time) and to examine neurophysiological recovery. During the surgical procedure steel screws were inserted bilaterally into the temporal bone and the inter- hemispheric EEG was monitored by a multichannel re- corder (Grass, Quincy, MA, U.S.A.).

Microdialysis Microdialysis samples (perfusion rate, 2 pl/min) were col-

lected every 10 rnin starting 120 rnin before ischemia (stabi-

J. Neurochem., Vol. 59. No. 4, 1992

EEG ISOELECTRIC TIME AND HIPPOCAMPAL NE LEVELS 1259

FIG. 1. Coronal slice showing the position of the transversal microdialysis probe. The fiber was stereotaxically implanted in the dorsal hippocampus with these coordinates: AP -3.6 mm from bregma, V -3.7 mm from temporal bone. The dialysis tubing was covered with Super Epoxy glue except for the part that corresponded to the areas of interest.

lization phase), then during the 30 rnin of ischemia and the 2 h of recirculation.

Histological examination Rats surviving 96 h after surgery were anesthetizcd with

equitensin and brain tissue was fixed by transcardiac perfu- sion of4% formaldehyde in phosphate buffer(pH 7.3). Coro- nal slices (20 pm) were Nissl stained for histological exami- nation.

RESULTS Rats with cortical electrical activity completely de-

pressed and without corneal reflex were classified as “ischemic”; animals not showing one of these condi- tions were classified as “oligoemic.”

Histological examination showed no tissue damage due to surgical manipulation; the probe position is indicated in Fig. I .

During the experiment pH, Po,, Pco,, and temper- ature remained under standard conditions. The mean arterial blood pressure (MABP) was higher in the isch- emic group during the first 10 rnin of ischemia; plasma glucose slightly decreased in the ischemic group (Table 1). During the stabilization phase (halo- thane 0.75%) the EEG amplitude decreased and the frequency spectrum increased.

In ischemic rats the EEG flattened by 100 s after carotid clamping and remained flat during the entire

period of occlusion, and during the first 30-40 min of reflow. Recovery of EEG was difficult and in fact it was still altered 2 h after the restoration of cerebral blood flow; no additional pancuronium bromide was needed during and after ischemia.

In oligoemic rats the EEG flattened 0-600 s after carotid clamping, but did not remain flat throughout occlusion; it quickly restartcd after carotid opening. In this group the rats needed additional doses of pan- curonium bromide about every 20 rnin during the recovery period.

Figure 2 illustrates the changes in dialysate NE con- tent produced by 30 rnin of ischemia. NE levels re- mained constant during the last hour before carotid occlusion. Basal NE release in these conditions was 0.047 k 0.001 pmol/lO min. The NE content started to rise 10 rnin after carotid clamping, and reached a maximum (0.2 * 0.04 pmol/lO min) 10 rnin after the end of the ischemic episode. NE levels came back to baseline after 40 min of recirculation and remained unchanged until the end of the experiment (2 h of reflow).

Under those experimental conditions NE overflow was observed when the cortical activity was com- pletely depressed and the animals showed complete loss of corneal reflex, whereas in oligoemic and sham- operated rats NE levels were unchanged (Fig. 3).

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1260 C. PEREGO ET AL.

0.3

TABLE 1. Physiological parameters in ruts before, during, and ujier ischemia

Before" Duringh Aftef

Variables Ischemia Sham Ischemia Sham Ischemia Sham

Po2 (mm Hg) 104.8 5 13 98.2 f 5 102.8 f 3 103.7 f 9 107.4 f 9 96.3 t 7 P a 2 (mm Hg) 33.9 f 2 38.2 f 3 33.4 f 3 39.1 f 4 27.8 f 4 42.1 +- 3 PH 7.43 -+ 0.02 7.43 t 0.01 7.42 f 0.02 7.41 _+ 0.03 7.43 ? 0.02 7.38 t 0.01 Glucose (mg/dl) 68+- I 1 68.5 +_ 5 54 * 4 80.5 f 5 6 3 k 10 80.5 -t 0.5 MABPd (mm Hg) 81 - t 9 75.0 _+ 8 149 _+ 9 75 f 1 82 ? 9 72 +- 3 Temperature 37.7 f 0.4 37.5 f 0.3 37.2 f 0.2 37 k 0.2 37.5 -c 0.3 37.2 t 0.1

r

Data are expressed as means -+ SEM. For ischemia n = 7; for sham n = 5. 'Before: 10 rnin before ischemia.

During: 10 min after carotid occlusion. After: 1 h of reflow. Only MABP was significantly higher 0, s 0.01) in the ischemic group during the first minutes of ischemia.

DISCUSSION

Hippocampal NE release measured by microdiaiy- sis perfusion was characterized in pharmacological and behavioral studies as suggested by Abercrombie et al. (1988). In brain sections from rats with im- planted transversal dialysis tubing the blood-brain barrier was intact in the hippocampus 1 day after sur- gery (Benveniste et al., 1984). No acute barrier changes were documented after ischemia (Petito, 1982) and no peak corresponding to epinephrine was found in our chromatographic assay. This means that NE concentrations measured in the dialysate reflect the extracellular NE concentrations near the dialysis tubing.

NE levels measured during ischemia were different from those reported by Globus et al. (1989); this au- thor, using the microdialysis technique, found a max- imal increase of 18 times in NE levels, whereas we obtained only a fivefold maximal increase during isch-

1

emia. This is probably a consequence of the different ischemia model and anesthesia.

in our ischemia mociei the N'E overflow was cioseiy related to the ischemic coma conditions. No changes in NE release were seen in hippocampus in animals with corneal reflex or without the isoelectric EEG (oli- goemic rats) (Fig. 3). The same relation between coma conditions and NE increase was seen in rats dialyzed during hypoglycemic coma. NE started to rise only when the EEG was flattening (data not shown).

In our ischemia model the EEG recording flattened about lo0 s after carotid occlusion, whereas NE levels started to rise only 10 rnin later. This delay (not seen during hypoglycemic coma) is probably thc result of residual blood flow to the hippocampal area during the first minutes of ischemia (Schmidt-Kastner et al., 1989), suggesting a slow decrease in the cellular en- ergy level in the hippocampus during ischemic coma. In fact, as reported by de la Torre et a]. ( I 99 l), 31P magnetic resonance spectroscopy ofhigh-energy phos-

FIG. 2. Hippocampal NE levels before. during, and after 30 min of transient forebrain ischemia in rats (n = 7). H ip pocampal dialysis samples were col- lected every 10 min beginning 60 min before ischemia. during 30 min of isch- emia, and during 2 h of reflow. The NE content was analyzed by HPLC with mulometric electrochemical detection, and the data are means k SEM (bars). Statistical significance was assessed by one-way analysis of variance fol- lowed by Dunnett's test. All animals showed a significant increase in NA lev- els during ischemia and in the early re- circulation period("p < 0.01 versus ba- sal).

o . o l . 1 . 1 . I . , . , . I . , . , . ! . , . , . , . I . I . 1 . I . I . I . I . I . 1

time ( min ) 0 2 0 40 6 0 80 100 120 1 4 0 160 180 200

J. Neurochem.. Val. 59. NO. 4, 1992

EEG ISOELECTRIC TIME AND HIPPOCAMPAL NE LEVELS 1261

FIG. 3. Simultaneous determination of hippo- campal extracellular NE levels and EEG of (A) an ischemic rat; and (B) an oligoemic rat. A The EEG started to be isoe(echic 100 s after carotid clamping. NE levels started to rise 20 min after carotid clamping, from the basal level of 0.049 pmol/lO rnin to a maximum of 0.29 pmol/lO rnin. The animals showed a complete lack of corneal reflex during ischemia and re- circulation periods. B: No changes were ob- served in NE levels. The EEG sgnal started to be isoelectric 15 min after carotid clamping. After opening the carotids the dectrmrtical activity started up sooner than in ischemic ani- mals. The intrahemispheric bipolar EEG was recorded by three steel screws placed on the temporal bone.

I basal t isch rellow - time (min)

10 mln

O'' 1 6

time (min) 004.. , , . . _ , . ! , _ . ! ...-~-.... . . . - . - . 1 0 2 0 4 0 60 10 1 0 0 120 140 160 1DO ZOO

10 rnin

phate metabolites in vivo showed loss of phospho- creatine and @-ATP signals after 6 rnin of brain isch- emia. This is also in agreement with Raffin et al. ( 199 l), who assume that EEG suppression spares en- ergy for other functional activities such as ion trans- port, whereas only later, during ischemia, anoxic de- polarization accelerates the decline in tissue oxygen and energy stores. This suggests that the enhancement of NE might be secondary to the energy crisis caused by the interruption of blood circulation.

NE levels returned to the baseline 120 rnin after

carotid clamping and were constant for the remaining observation time. This is in apparent contrast with Miyauchi et al. (1989), who reported an increase in NE turnover I h after reflow. However, in vivo deter- mination of overflow gives an indication of the change in overflow almost at the same time as it oc- curs, whereas metabolite concentrations may reflect changes produced before but still present in the tissue at the sampling time.

The EEG signal showed no recovery after recircula- tion and remained flat for about 40 rnin of reflow, in

J. Neurochem.. Vol. 59, No. 4, 1992

1262 C. PEREGO ET AL.

contrast to the return of basal NE levels and the rapid recovery of the energy metabolism after recirculation described by Naruse et al. (1 984). ATP levels seem to recover much sooner than cerebral function after re- flow in cerebral ischemia (Naruse et al., 1984) and this might help explain the delay between NE return to the basal levels and EEG recovery.

However, it remains to be established whether the marked increase of extracellular NE in ischemia af- fects the extent of ischemic hippocampal damage, chiefly in relation to the simultaneous increases in extracellular glutamate and aspartate during the isch- emic period. In any case, the direct relation between the lack of brain electrical activity and NE overflow during the early phase of ischemia is evident.

Acknowledgment: The authors thank the Lions Club S. Remo Matutia. This work was partially supported by CNR (National Research Council, Rome, Italy) grant 9 1.00460.PF40.

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