218 B
In experiments with exogenous hydroxyl radical stimu- column 12533 mm, pre-column 533 mm filled with
lation the perfusion fluid was changed for 60 min to a fluid Nucleosil 120-3 C18 Knauer, Berlin, Germany. Data
additionally containing 0.2 or 2 nmol 2 ml min of 6- were calculated by an external standard calibration.
hydroxydopamine for reverse dialysis to deliver 6-OHDA The detection limit for 2,3-DHBA was approximately
into the surrounding tissue of the microdialysis probe. 0.1 nmol l and its in vitro recovery microdialysis probes
Then, the perfusion fluid was changed back again to the CMA 12 was 15–20. The values of 2,3-DHBA and
perfusion fluid of the baseline recording, which was dopamine are expressed as percentages of three predrug
without 6-OHDA. In control experiments modified Ringer dialysates and were not corrected for the recovery of the
solution was used instead of 6-OHDA. probes.
Samples were collected every 20 min in vials containing 10 ml 0.4 M perchloric acid and were directly analyzed by
HPLC or stored at 2708C until analysis.
3. Results
2.4. HPLC-analysis 3.1. In vitro studies
The HPLC system for determination of 2,3- and 2,5- Incubation of salicylic acid alone in the radical generat-
DHBA and dopamine consisted of a solvent delivery ing system resulted in a similar increase in 2,3-DHBA and
system Waters model 600S in combination with Waters 2,5-DHBA levels and to a lesser extent in an increase in
model 616, Millipore, Milford, USA, an autoinjector with catechol data not shown, reflecting an enhanced hydroxyl
cooling module set at 48C Waters model 717 plus, radical formation. In control experiments without drug
Millipore, Milford, USA, a column thermostat set at 228C incubation 2,3-DHBA levels mean6S.E.M. of 4.460.12
Gynkotek model STH 585, Germering, Germany, an mM were obtained and set as 100. Increasing con-
online degasser Knauer model A1050, Berlin, Germany, centrations of pramipexole 0.5–5 mM and S-PBN 0.5–5
a two channel electrochemical detector BAS LC 4C, mM led to a significant decrease in 2,3-DHBA levels
Bioanalytical Systems, West Lafayette, USA and a data Fig. 1. In each concentration tested, pramipexole was
collection and calculation unit controlled by Gynkosoft more effective than S-PBN P,0.001. The highest con-
software Gynkotek, Germering, Germany. The detector centration of pramipexole 5 mM reduced the increase of
potentials were set at 1750 mV using a glassy carbon 2,3-DHBA to about 3.5 of the control values, whereas
electrode and an Ag AgCl reference electrode with a range S-PBN 5 mM reduced the increase of 2,3-DHBA to
of 1–10 nA V. The mobile phase contained 0.14 g octane about 35 of the control values.
sulfonic acid sodium salt as an ion-pair reagent, 0.1 g disodium EDTA, 6 ml triethylamine and 35 ml acetonitrile
3.2. Effects of saline, pramipexole and pergolide on
in 1 l of millipore Q water adjusted to pH 2.95 with
basal 2,3-DHBA levels
concentrated phosphoric acid. The eluent was delivered with a flow rate of 0.5 ml min onto a reversed-phase
The perfusion with 5 mM salicylic acid produced stable
Fig. 1. In vitro effects of S-PBN and pramipexole on hydroxyl free radical levels in a Fenton system. 2,3-DHBA values indicate the alterations of hydroxyl free radical levels. Control values were obtained by incubating saline instead of pramipexole and were regarded as 100. Data are given as mean6S.E.M.
of n56 experiments. Statistical analysis was performed using Kruskal–Wallis H-test with subsequent Mann–Whitney U-test. P,0.05, P,0.01, P,0.001 was considered significant by comparing drug values with controls.
B . Ferger et al. Brain Research 883 2000 216 –223
219
basal 2,3-DHBA levels, which were not significantly OHDA 0.2 nmol 2 ml min via the probe led to an about
different between the experimental groups. After injection 14-fold increase in 2,3-DHBA levels in the saline treated
of saline, pramipexole 1 mg kg or pergolide 0.05 mg group and to an about 12-fold increase in the S-PBN
kg no significant changes in 2,3-DHBA levels were group. This reduction was not statistically significant. In
observed compared to the corresponding pre-injection contrast, in the pramipexole 2 and 10 nmol 2 ml min
basal values mean6S.E.M. 53.4463.5, 55.6463.9 and treated groups the 6-OHDA-induced elevation in 2,3-
57.9262.2 nmol l, respectively Fig. 2. DHBA levels was significantly attenuated to a 7.8- and
8.6-fold increase, respectively Fig. 4A. 3.3. Effects of saline, pramipexole and pergolide on 6-
OHDA-induced increase in 2,3-DHBA levels
3.5. Effects of local application of saline, S-PBN and pramipexole on
6-OHDA-induced increase in dopamine Before onset of hydroxyl free radical stimulation with
levels 6-OHDA 2,3-DHBA mean basal levels6S.E.M. in the
saline, pramipexole 0.05, 0.2 and 1 mg kg i.p. and The following dopamine mean basal levels6S.E.M.
pergolide 0.05 mg kg i.p. treated groups of 53.4266.3, were obtained in the saline, S-PBN 2 nmol 2 ml min and
50.5663.0, 52.4965.8, 54.7865.3 nmol l and 50.4466.0 pramipexole 2 and 10 nmol 2 ml min treated groups of
nmol l, respectively were obtained. Perfusion with 6- 2.3160.3, 2.4360.2, 2.3460.5 and 2.2160.4 nmol l,
OHDA 0.2 nmol 2 ml min via the probe led to an about respectively. Perfusion with 6-OHDA 0.2 nmol 2 ml
14-fold increase in 2,3-DHBA levels in the saline treated min led to an about 25.5-fold increase in dopamine levels
group, to an about 12–13-fold increase in the pramipexole in the saline treated group compared to mean basal levels.
treated groups and to an about 17-fold increase in the In the S-PBN treated group during 6-OHDA perfusion an
pergolide treated group. There were no significant differ- attenuation to an 11-fold increase was observed. In con-
ences between any of the experimental groups Fig. 3. trast, pramipexole 2 and 10 nmol 2 ml min pretreatment
significantly attenuated the 6-OHDA-induced increase in 3.4. Effects of local application of saline, S-PBN and
dopamine levels to a 6.9- and 4.6-fold elevation, respec- pramipexole on
6-OHDA-induced increase of 2,3-DHBA tively Fig. 4B.
levels Before onset of hydroxyl free radical stimulation with
4. Discussion
