42 C
2H - 1 - benzopyran - 2 - ylmethyl]amino] - butyl] - 1, 2 - ben - removed, the blood was reinfused and NaHCO 50 mg
3
zisothiazol-32H-one 1,1-dioxide
monohydrochloride kg, i.v. was administered. Arterial pH, pCO , pO and
2 2
[15,16]. blood pressure were measured before and after ischemia.
The aim of this study was to investigate whether the The experiments were approved by the government ethics
5-HT agonist Bay x 3702 could cause an anti-apoptotic
committee. All efforts were made to reduce the suffering
1A
effect in vivo. In addition, we attempted to find out, of the animals during the experiments.
whether this anti-apoptotic effect could be blocked by the selective 5-HT
receptor antagonist WAY 100635 N-[2- 2.4. Drug administration
1A
[4-methoxyphenyl-piperazinyl]ethyl]-N-2-piperidinyl cy- clohexane carboxamide [20]. Electrophoresis and staining
Bay x 3702 40, 12 and 4 mg kg or saline were infused with terminal deoxynucleotidyl transferase TdT-mediated
i.v. with a perfusor Secura, Braun, Melsungen, Germany dUTP nick end labeling TUNEL were used to measure
immediately after ischemia for a period of 4 h. The DNA laddering and to detect internucleosomal DNA
infusion was given into the tail vein with a constant flow fragmentation in situ.
rate of 1 ml h. WAY 100635 1 mg kg was injected i.p. 60 min before ischemia. The body temperature was
maintained at 3760.58C with a heating lamp during the
2. Material and methods period of infusion and up to 6 h after infusion.
2.1. Animals 2.5. Histological evaluation of the neuronal damage
Male Wistar rats Charles River, Germany weighing For evaluation of the neuronal damage in the CA1
300–350 g were used. The animals had free access to food subfield of the hippocampus the rats n58–12 were
Altromin, Lage, Germany and water and were kept under anesthetized with chloral hydrate 400 mg kg, i.p.. They
standardized environmental conditions 12-h light dark were transcardially perfused with saline followed by 4
cycle, 23618C and 5561 relative humidity. There were phosphate-buffered formalin. The brains were removed
no significant differences between the mean body weight and embedded in paraffin. Coronal brain slices 5 mM,
of treated and untreated animals. 23.6 mm to bregma were taken and stained with 1
celestine blue and 1 acid fuchsin for histological evalua- 2.2. Drugs
tion. Damaged neurons appeared red and shrunken, where- as intact neurons appeared blue with round morphological
The 5-HT agonist Bay x 3702 2-R-2-[4-[[3,4-
features. Neuronal injury was blindly evaluated in the CA1
1A
dihydro-2H-1-benzopyran- 2 -ylmethyl]amino]-butyl]-1,2 - subfield of the hippocampus 7 days after ischemia by
benzisothiazol-32H-one 1,1-dioxide monohydrochloride counting all damaged and viable neurons. Neuronal injury
was obtained from Bayer Cologne, Germany. WAY was given as the percentage of the total number of
100635 N-[2-[4-methoxyphenyl-piperazinyl]ethyl]-N-2- neurons.
piperidinyl cyclohexane carboxamide, a selective 5-HT
1A
¨ receptor antagonist, was a gift from Wyeth Munster,
2.6. TUNEL-staining Germany.
Male Wistar rats n54 were treated with either Bay x 2.3. Model of transient forebrain ischemia
3702 4 mg kg, i.v. or vehicle n54 immediately after ischemia. Brains were removed 3 days after ischemia. For
Forebrain ischemia was produced in male Wistar rats detection of DNA-fragmentation TUNEL staining was
300–350 g, Charles River, Germany with some modi- performed in hippocampal and striatal coronal paraffin
fications as described previously [51]. The rats were fasted sections 23.6 and 1 mm to bregma, respectively using
overnight with free access to water. They were anes- the Apop Tag Kit Oncor, USA. Briefly, after deparaffini-
thetized using a mixture of N O O 70 30 with
sation slices were incubated with proteinase K 20 mg ml
2 2
halothane 1.5. During the surgery, body and skull for 15 min at room temperature. Endogenous peroxidase
temperature was maintained between 37.0 and 37.58C by a activity was quenched with 2 H O . After treatment with
2 2
temperature control feedback system TSE, Bad Homburg, equilibration-buffer, slices were incubated with terminal
Germany. The skull temperature was monitored with a transferase for 1 h at 378C followed by incubation with
thermocouple placed subcutaneously over the midline of stop wash buffer. After washing with phosphate-buffered
the skull, thereby supplying a stable brain temperature saline slices were treated with antidigoxigenin-peroxidase
[13]. Ischemia was induced for 10 min by clamping both for 30 min, equilibrated in 0.2 H O and then exposed to
2 2
common carotid arteries and reduction of the blood freshly prepared staining solution of 0.05 diaminoben-
pressure to 40 mmHg. Hypotension was induced with zidine in 0.02 H O . Counterstaining was performed
2 2
trimethaphan camphor sulfonate 5 mg kg, i.v. and with- with methyl green. The cells showing TUNEL-positive
drawal of blood. After 10 min of ischemia, the clips were staining with shrunken cell body and condensed chromatin
C . Schaper et al. Brain Research 883 2000 41 –50
43
Fig. 1. Effect of Bay x 3702 on neuronal damage in the CA1 subfield of the hippocampus 7 days after transient forebrain ischemia. Rats were treated with Bay x 3702 40, 12 and 4 mg kg, i.v. immediately after ischemia by infusion for 4 h. The control group received vehicle in the same way. The values are
given as means1S.D. of n58–12 animals. P,0.01 compared with control Duncan test.
were considered as TUNEL-positive cells. TUNEL-posi- subsequent Duncan test. Homogeneity of errors was de-
tive cells were calculated as a percentage of the total termined using Bartlett’s test. P values of ,0.05 were
number of neurons in the CA1 subfield of the hippocam- considered to be significant.
pus. 2.7. Analysis of DNA fragmentation
3. Results
