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
Rat brains were removed 4 days after ischemia and the 3.1. Neuroprotection of Bay x 3702 demonstrated in a
tissue of hippocampus and striatum was quickly taken and rat model of transient forebrain ischemia
stored at 2708C. DNA from the hippocampus and striatum was individually extracted using the QIAamp Tissue Kit
The histology of the slices demonstrated severe damage Qiagen, Hilden, Germany according to the manufactur-
of pyramidal neurons in the CA1 subfield of the hippocam- er’s protocol. Same amounts of total DNA were loaded and
Table 1
separated in a 1.5 agarose gel containing silver-gold
Physiological parameters of Bay x 3702 40 mg kg, i.v. B and
using electrophoresis for a period of 2.5 h.
vehicle-treated animals C during the period of drug infusion after
In one series of experiments rats n54 were infused
a
transient forebrain ischemia
with Bay x 3702 4 mg kg or vehicle for 4 h directly after
Hours of infusion
ischemia. In another series of experiments animals n53
1 2
3 4
were divided into four groups. The first group received Bay x 3702 4 mg kg, i.v. for a period of 4 h directly after
Glucose mg dl C
110.6616.6 110.0612.3
109.3613.0 108.4614.3
ischemia, the second group received the vehicle only. The
B 113.8619.1
114.0619.1 113.8623.5
114.1623.0
third group received Way 100635 1 mg kg, i.p. 60 min before ischemia with a following infusion of vehicle for 4
PH
h. Animals of the fourth group were injected with Way
C 7.2460.11
7.2960.03 7.2860.04
7.2860.04
100635 intraperitoneally 1 mg kg 60 min before is-
B 7.3960.01
7.4160.03 7.4260.03
7.4160.04
chemia followed by an intravenous infusion of Bay x 3702
pO mmHg
2
4 mg kg, i.v. for 4 h.
C 131.7617.3
139.0627.1 105.766.1
110.0612.4 B
113.5626.4 95.0620.7
92.0618.5 95.1620.2
2.8. Statistics
pCO mmHg
2
C 46.065.7
40.062.8 43.365.2
42.565.5
The values for the neuronal damage are presented as
B 39.562.7
38.363.1 36.764.0
36.963.0
means6S.D of n experiments. Multiple comparisons were
a
made by one-way analysis of variance ANOVA with
Values are given as means6S.D. from n54 animals.
44 C
pus in both drug- and vehicle-treated animals. However, Infusion of 4 and 12 mg kg Bay x 3702 exhibited no
the histological quantification 7 days after global ischemia reduction in mean arterial blood pressure Fig. 2B.
resulted in a 10 reduction of damaged neurons in the Bay x 3702 4 mg kg, i.v.-treated group 72.469.2 com-
3.2. Bay x 3702 prevents DNA degradation measured by pared to the control group 82.966.3 Fig. 1. Animals
TUNEL-staining and gel electrophoresis treated with higher doses of Bay x 3702 40 and 12 mg kg,
i.v. showed no significant reduction in the CA1 hippocam- The results of TUNEL-staining were demonstrated in
pal damage 40 mg kg, 89.962.2 compared to control Fig. 3. Control slices from animals which were subjected
86.964.2; 12 mg kg, 80.164.3 compared to control to 10 min of forebrain ischemia n54 showed marked
82.966,3. During the period of Bay x 3702 40 mg kg TUNEL-staining 3 days after ischemia. Positively stained
infusion, blood glucose, pO and pCO levels were not
neurons were localized in the CA1 subfield of the hip-
2 2
changed compared with vehicle-treated animals Table 1. pocampus and in the dorsolateral region of the striatum
However, the mean arterial blood pressure was signifi- Fig. 4A,B. Staining with celestine and acid fuchsin
cantly reduced Fig. 2A during the whole period of 40 revealed that most of the dying neurons in the hippocam-
mg kg Bay x 3702 infusion P,0.01 and P,0.001. pus ¯90 were TUNEL-positive. The number of
Fig. 2. Mean arterial blood pressure MABP during infusion of Bay x 3702 for a period of 4 h. A Infusion of 40 mg kg Bay x 3702, B infusion of 12 and 4 mg kg Bay x 3702. Values are given as means1S.D. of four to five animals. P,0.01, P,0.001, compared with control Duncan test.
C . Schaper et al. Brain Research 883 2000 41 –50
45
extent of DNA degradation in rats which received WAY 100635, Bay x 3702 plus WAY 100635 or vehicle,
respectively.
4. Discussion
