Effect of adenosine receptor antagonists on LPS-induced oxidative stress in rat striatum

K Gołembiowska, K Noworyta-Sokołowska, A Górska, K Kamińska. Institute of Pharmacology PAS, Pharmacology 31-343 Kraków 12 Smętna Street, Poland

The contribution of inflammation to the progression of neurodegenerative diseases such as Parkinson’s or Alzheimer’s diseases is poorly understood. Brain inflammation is characterized by a glial activation and release of proinflammatory and cytotoxic factors. Several in vitro studies have shown that oxidative stress may be critical in causing neuronal loss in inflamed brain.

In recent years, adenosine A2A receptor antagonists have emerged as a new non-dopaminergic therapy for Parkinson’s disease (PD). Moreover, epidemiological and animal studies suggest that A2A adenosine receptors may contribute to degeneration of nigrostriatal dopaminergic neurons, and A2A antagonists may constitute dual motor/protective benefit for PD. The activation of A2A receptors on glial cells (microglial and astrocytes) is implicated in excitotoxicity through its effects on he release and uptake of GLU from astrocytes and adenosine from overactive microglia.

In this study, we investigated whether production of reactive oxygen species was increased in lipopolysaccharide (LPS) animal in vivo model of brain inflammation. Furthermore, the effect of LPS on dopamine (DA), glutamate (GLU) and adenosine (ADN) in rat striatum was studied. Male Wistar rats (280-300 g weight) received unilateral stereotaxic injection of LPS (10 µg/4 µl) dissolved in PBS over 4 min under general anaesthesia (ketamine 75 mg/kg and xylazine 10 mg/kg im) at the following coordinates: anteroposterior 0.1 mm; mediolateral 2.0 mm; dorsoventral - 6 mm. The production of hydroxyl radical was measured with in vivo microdialysis using salicylic acid as free radical trap. Additionally, the extracellular striatal levels of DA, GLU and ADN were estimated by HPLC with electrochemical, fluorescence and VIS detection, respectively. Caffeine (20 mg/kg), a nonselective adenosine A1/A2A receptor antagonist and KW60002 (3 mg/kg) as selective A2A receptor antagonist were given ip once daily for 6 days and 2 hr before and 4 hr after intrastriatal injection of LPS. The statistical significance of differences between groups (n = 6 - 8 rats) was calculated using a one-way ANOVA for repeated measures, followed by Tukey’s post hoc test. LPS decreased extracellular DA level and its tissue content 24 and 72 hrs after administration. At the same time an increase in extracellular level of GLU and ADN and production of hydroxyl radical was observed 24 hrs after LPS administration. Similarly, LPS enhanced hydroxyl radical tissue level 72 hr after injection. Caffeine and KW6002 reversed LPS-induced decrease in extracellular level and DA striatal content. Both adenosine receptor antagonists normalized extracellular concentration of GLU and ADN in striatal dialysates and attenuated production of hydroxyl radical.

Our findings indicate that LPS-induced inflammation is related to glial activation as shown by increased release of GLU, ADN and hydroxyl radical generation and may lead to degeneration of DA neurons. Reversal of these changes by caffeine and KW6002 suggests that blockade of striatal adenosine A2A receptors provides rationale for understanding neuroprotective mechanism of A2A adenosine receptor antagonists in PD.

Acknowledgement: This study was financially supported by grant NN 405 272337 from the Ministry of Science and Higher Education.