F . Mauler et al. Brain Research 888 2001 150 –157
151
glutamate receptor antagonists investigated in clinical trials Krebs–Ringer solution and were kept at 378C for 30 min.
revealed no therapeutic efficacy due to unfavorable risk– Slices were then transferred sequentially, 8 min each, to
benefit ratio or lack of efficacy [13,28]. However, new Eppendorf tubes containing Krebs–Ringer solution basal
compounds acting via modulatory sites at the NMDA release, Krebs–Ringer solution containing 75 mM KCl
receptor like the glycine receptor antagonist GV 150526 do stimulated release, Krebs–Ringer solution wash-out,
not produce neuronal vacuolization or cognitive distur- Krebs–Ringer solution containing BAY x 3702 effect of
bances. In addition, clinical studies revealed that this BAY x 3702 on basal release, and Krebs–Ringer solution,
compound is well tolerated, even at plasma levels, which containing both BAY x 3702 and 75 mM KCl effect of
displayed neuroprotective efficacy in preclinical models of BAY x 3702 on stimulated release. Controls without
stroke. drugs were handled similar. The hippocampal slices were
Alternative strategies to attenuate glutamate-mediated then dissolved in 2 N NaOH and protein content was
toxicity have been set up. These approaches include determined according to a modification of the Lowry
inhibition of second messenger cascades involved in method
[21]. Glutamate
content was
determined glutamatergic signaling and blockade of ion channels
fluorimetrically using the NAD glutamate dehydrogenase which may counteract excessive ischemia-induced neuro-
reduction assay. In brief, 100 ml of each sample were nal depolarization. 5-HT
receptor agonists have been added to 1 ml of an aqueous solution of 0.5 M glycine, 0.4
1A 1
tested in various models of ischemic damage and have M hydrazine. Finally, 20 ml of NAD
6.64 mg ml were revealed some neuroprotective efficacy in vivo and in vitro
added and fluorescence measured at ex. 340 nm em. 440 [14,34,35]. However, all compounds investigated so far
nm. The formation of NADH was induced by addition of showed only restricted 5-HT
receptor selectivity, affinity 10 ml glutamate dehydrogenase 10 mg ml and was
1A
and or intrinsic
activity. Recently,
the amino-
determined 5 min after reaction start. Fluorescence was methylchroman derivative BAY x 3702 was described as a
converted into mmol glutamate by means of a respective novel highly potent 5-HT
receptor full agonist with standard curve.
1A
pronounced neuroprotective properties in various animal models of ischemic brain injury [1,14–16,23,24,41].
2.2.2. Permanent middle cerebral artery occlusion In the present study we investigated the effects of BAY
pMCA-O x 3702 on glutamate release both in vitro and in vivo in
order to evaluate whether modulation of glutamatergic 2.2.2.1. Animals. Male Long–Evans rats 300–350 g,
neurotransmission may be involved in the neuroprotective Mollegaards Breeding Center Ltd, Li Skensved, Denmark
properties of this compound. were allowed to adapt to housing conditions for at least 1
week before they were subjected to the study. They were housed in groups of 3–5 individuals in macrolon cages
2. Material and methods type III, Ehret, Emmendingen, FRG bedded with saw-
dust. The animal housing room and the laboratory for 2.1. Chemicals and reagents
surgery were climate controlled and continuously illumi- nated from 7.00 a.m. until 7.00 p.m. Room temperature
All chemicals were of highest commercially available was 21.561.58C, relative humidity was 40–60. Food
purity and were purchased from E. Merck, Darmstadt, Altromin
1324, Altomin Spezialfutterwerk GmbH, Lage, FRG. BAY x 3702 R-2-2-h4-[chroman-2-ylmethyl-
FRG and water were available ad libitum. amino]-butylj-1,1-dioxo-benzo[d]isothiazolone hydrochlo-
ride and WAY 100635 N-[2-[4-2-methoxyphenyl-1- 2.2.2.2. Occlusion. Under general anesthesia Isoflurane
piperazinyl]ethyl]-N2-pyridinylcyclohexane carboxamide 4–1.5 mixed with air as inhalational anesthetics the
trihydrochloride were obtained from the Chemistry De- middle cerebral artery MCA was occluded unilaterally
partment of Bayer AG, Wuppertal, FRG. Isoflurane was based on the surgical procedure of Bederson and co-
obtained from Abbot GmbH, Wiesbaden, FRG. workers [4]. Briefly, the left temporal–parietal region of
the head was shaved, the skin was disinfected and opened 2.2. Methods
between the orbit and the external ear canal. Following a midline incision, the temporal muscle was divided and
2.2.1. Determination of glutamate release from pulled aside with surgical hooks to make the lateral aspect
hippocampus slices of the skull free. The facial nerve, major facial arteries and
Hippocampi were dissected from 6-week-old male Wis- veins, the lateral eye muscles, the intra- and extraorbital
tar rats and slices of 350 mm in thickness were prepared by lacrimal glands and the zygomatic bone were left intact.
means of a tissue chopper. Slices were then transferred to Under operating microscope a small burr hole was drilled
small containers sealed by a nylon mesh at the bottom directly under the zygomatic arc 1–2 mm rostrally to its
three slices container. For equilibration, these were caudal origin. After careful opening of the dura, the
placed into Eppendorf tubes containing pre-warmed exposed MCA and its branches were permanently occluded
152 F
between the olfactory tract and the inferior cerebral vein software Perkin Elmer, Friedrichshafen, FRG; Version 3,
by microbipolar electrocoagulation Bipolator 50, Fischer and stored electronically and as hardcopy.
MET GmbH, Freiburg, FRG. To avoid recanalization the occluded vessels were removed. The operation area was
2.2.5. Statistical analysis covered with a small piece of sterile absorbable gelatin
Statistical analysis of hippocampal glutamate release
sponge Marbagelan , Behringwerke AG, Marburg, FRG. data was performed by ANOVA followed by Fisher’s LSD
Muscle and skin wounds were closed with tissue glue post hoc analysis. P-values 0.05 were considered to be
Histoacryl , B. Braun Melsungen, Melsungen, FRG. statistically
significant. Statistical
analysis for
mi- During surgery and microdialysis the body temperature
crodialysis probes was performed by repeated-measures was monitored and maintained between 36.5–37.58C with
ANOVA with pairwise multiple comparison test using the a heating plate.
LSD test SAS version 6.12, SAS Institute Inc., USA. Significance was accepted at the P,0.05 level.
2.2.3. Microdialysis Three hours before occlusion of the MCA the mi-
2.2.6. Drug application crodialysis probes CMA10, 4 mm, Carnegie Medicine,
The lyophilized formulation of BAY x 3702 was recon- Stockholm, Sweden were implanted under general anes-
stituted by adding of physiological saline. The drug-solu- thesia into the cortex interaural line coordinates frontal
tion or vehicle was administered intravenously i.v. as a 17.7, lateral 15.8, horizontal 16.8 [33]. Microdialysis
bolus injection directly after pMCA-O. Application vol- probes were connected to a perfusion pump CMA100,
ume was 2 ml kg body-weight. Control groups received Carnegie Medicine, Stockholm, Sweden and to a fraction
physiological saline only. collector
CMA140, Carnegie
Medicine, Stockholm,
Sweden. Flow was set to 2 ml min. The dialysis fibers were perfused immediately after implantation with Krebs–
3. Results
